# Ist2 is a phospholipid scramblase that links lipid transport at the ER to organelle homeostasis

Heitor Gobbi Sebinelli, Camille Syska, Hafez Razmazma, Véronique Albanèse, Ana Rita Dias Araujo, Cecile Hilpert, Cédric Montigny, Christine Jaxel, Manuella Tchamba, Karolina Belingar, et al.

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Heitor Gobbi Sebinelli, Camille Syska, Hafez Razmazma, Véronique Albanèse, Ana Rita Dias Araujo, et al.. Ist2 is a phospholipid scramblase that links lipid transport at the ER to organelle homeostasis. Journal of Cell Biology, 2026, 225 (2), 10.1083/jcb.202502112. hal-05527912

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Ist2 is a phospholipid scramblase that links lipid transport at the ER to organelle homeostasis

Heitor Gobbi Sebinelli¹*, Camille Syska²*, Hafez Razmazma³*, Véronique Albanès², Ana Rita Dias Araujo⁵, Cecile Hilpert³, Cédric Montigny¹, Christine Jaxel¹, Manuella Tchamba¹, Karolina Belingar², Juan Martín D'Ambrosio², Luca Monticelli³‡, Guillaume Lenoir²‡ and Alenka Čopič¹‡

¹Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France

²Centre de Recherche en Biologie Cellulaire de Montpellier (CRBM), Université de Montpellier, CNRS, Montpellier, France

³Molecular Microbiology and Structural Biochemistry (MMSB), UMR 5086 CNRS & Université Claude Bernard Lyon 1, Lyon, France

⁴Institut Jacques Monod, Université de Paris, CNRS, Paris, France

⁵Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Université Côte d'Azur, CNRS and Inserm, Sophia Antipolis, France

(*) These authors contributed equally to this work.

(‡)To whom correspondence should be addressed:

luca.monticelli@inserm.fr

guillaume.lenoir@i2bc.paris-saclay.fr

alenka.copic@crbm.cnrs.fr

Running title: Phospholipid scrambling by Ist2

Abstract

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one leaflet, scramblase activity should be essential for equilibrated membrane growth. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane (PM) and participates in the transfer of phosphatidylserine. We show both in vitro and in silico that the ER domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity that is not regulated by Ca²⁺. In cells, over-expression or deletion of the ER domain of Ist2 affects ER-related processes including COPII-mediated vesicular transport, lipid droplet homeostasis and general phospholipid transport, with a specific contribution of residues implicated in lipid scrambling. The weak phenotypes can be augmented by the deletion of another putative scramblase, the protein insertase Get1, suggesting that combined action of different proteins supports lipid scrambling at the ER.

Keywords: Lipid scrambling, TMEM16, membrane contact sites, transbilayer asymmetry, lipid transfer proteins, molecular dynamics simulations
48 Introduction

A hallmark of eukaryotic cells is their highly diversified membrane system, which is essential for the organization and compartmentalization of cellular functions. Membrane diversity is reflected by large differences in their bulk lipid composition, driven by localized lipid synthesis and by the action of cytosolic lipid transfer proteins (LTPs), which mediate selective transport of lipids between different organelles (Holthuis and Menon, 2014; Lenoir et al., 2021). For example, phosphatidylserine (PS) and cholesterol are both enriched at the plasma membrane (PM) through the action of specific LTPs (Drin, 2023). Diversity in lipid composition can also be observed within the same membrane either as lateral inhomogeneity or as difference between the two leaflets (trans-bilayer asymmetry).

Membrane trans-bilayer asymmetry is most striking and well-studied in the case of the PM. The cytosolic leaflet of the PM is highly negatively charged due to an abundance of anionic phospholipids, such as PS, whereas the exoplasmic leaflet is almost devoid of these phospholipids but enriched in sphingolipids (Bretscher and Raff, 1975; Lorent et al., 2020). It is also well established that, in contrast to the PM, the cytosolic leaflet of the ER is poorly charged (Bigay and Antonny, 2013; Yeung et al., 2008). However, when we zoom in on the ER membrane, the data on its precise lipid organization is scarce and often conflicting. Taking again PS as an example, Fairn and coworkers have shown that it is largely confined to the luminal ER leaflet (Fairn et al., 2011). In contrast, a subsequent study suggests that PS concentration may be much more equilibrated between the two ER leaflets, or possibly even reversed (Tsuji et al., 2019). While much more data is clearly needed, these conflicting observations may reflect the highly dynamic and multifunctional nature of the ER, which hosts many processes such as protein and lipid synthesis, membrane transport and lipid transport via LTPs, and the biogenesis of organelles such as lipid droplets (LDs) and autophagosomes.

Three mechanisms are essential for the establishment of bilayer asymmetry: (1) localized phospholipid synthesis (Bell et al., 1981; Long et al., 2024; Vance et al., 1977); (2) slow rate of spontaneous phospholipid exchange (flip-flop) between the two leaflets (Bretscher and Raff, 1975), and (3), the action of phospholipid flippases, which use ATP hydrolysis to promote phospholipid flow from the extracellular/luminal towards the cytosolic membrane leaflet (Sakuragi and Nagata, 2023). In opposition to these mechanisms, phospholipid scramblases enable flow of phospholipids down their concentration gradient, disrupting bilayer asymmetry (Sebinelli et al., 2024). A well-studied example is the TMEM16 protein family, which includes several Ca²⁺-activated PM lipid scramblases (Brunner et al., 2014; Falzone et al., 2019; Kalienkova et al., 2021; Yang et al., 2012). Mutations in TMEM16F are associated with Scott Syndrome, a bleeding disorder resulting from defective PS exposure in activated platelets (Jan and Jan, 2024; Suzuki et al., 2010). An increase in cytosolic Ca²⁺ concentration induces a conformational change that leads to the opening of a hydrophilic lipid permeation pathway, enabling the passage of phospholipid headgroups via the so-called ‘credit-card’ model (Pomorski and Menon, 2006).

Because most phospholipids are synthesized in one or the other ER leaflet, scramblase activity at the ER would be essential for equilibrated membrane growth (Chauhan et al., 2016). However, specific ER-localized lipid scramblases only recently began to be identified. Two mammalian ER proteins of the DedA family, TMEM41B and VMP1, scramble phospholipids *in vitro* and the scramblase activity of TMEM41B has also been assessed in cells (Ghanbarpour et al., 2021; Huang et al., 2021; Li et al., 2021). Furthermore, TMEM16K, a TMEM16 family scramblase which localizes to the ER in mammalian cells, has also been shown to facilitate lipid scrambling in a Ca²⁺-dependent manner (Bushell et al., 2019; Tsuji et al., 2019). The activities of
these proteins are often coupled to other lipid/membrane transport pathways (Hama et al., 1982). TMEM41B and VMP1 function in autophagy, where they may work in concert with the LTP ATG2 and the autophagosomal scramblase ATG9 to promote autophagosomal membrane expansion (Ghanbarpour et al., 2021; Matoba et al., 2020; Van Vliet et al., 2022). TMEM41B scramblase activity has been implicated in the production of lipoprotein particles and its deletion is often associated with accumulation of cytosolic LDs (Huang et al., 2021; Moretti et al., 2018; Morita et al., 2018; Wu et al., 2024). TMEM16K has been shown to affect endo-lysosomal transport pathway and localizes at ER-endosome contact sites, whereas TMEM16H functions at ER-PM contact sites (Jha et al., 2019; Petkovic et al., 2020). In addition to these recently identified specific ER scramblases, other ER proteins may moonlight as lipid scramblases. G-protein coupled receptors (GPCRs) have been proposed to function as constitutive scramblases in the ER while en route to their final destination at the PM, where they would be inhibited by cholesterol (Goren et al., 2014; Morra et al., 2022). Very recently, it has also been proposed that all ER membrane insertases involved in the synthesis of ER membrane proteins possess additional lipid scramblase activity (Li et al., 2024). Therefore, the field has recently witnessed a sharp turn from too few known ER scramblases to a host of different ER scramblases, raising the question of coordination of the various processes at the ER.

In budding yeast, a striking example of a protein machinery that could combine different lipid transport activities at the ER is provided by the Ist2-Osh6 complex. Ist2 localizes to ER-PM membrane contact sites and is composed of two distinctive parts: an ER-embedded transmembrane domain (TMD) that is preceded by a short cytosolic N-terminus (N-ter), and a largely disordered C-terminal (C-ter) tail that tethers the ER to the PM (Collado et al., 2019; Hoffmann et al., 2019; Kralt et al., 2014; Manford et al., 2012; Wolf et al., 2012). The cytosolic LTPs Osh6 and Osh7 (Filseck et al., 2015; Maeda et al., 2013) bind to the cytosolic tail of Ist2, enabling their localization and efficient PS transfer from the ER to the PM (D'Ambrosio et al., 2020; Wong et al., 2021). The TMD of Ist2 contains 10 putative transmembrane helices and shows sequence similarity with the TMEM16 protein family, with conservation of some residues previously identified as critical for scrambling (Fig 1A, B) (Brunner et al., 2014; Falzone et al., 2019; Kalienkova et al., 2019). This suggests that Ist2 could be a phospholipid scramblase in the ER, although an initial attempt using purified and reconstituted Ist2 failed to reveal any flip-flop activity for this protein (Malvezzi et al., 2013). In the present work, we investigate whether Ist2 is a phospholipid scramblase and how this activity at the ER might impact PS transfer and other ER functions.

We show using biochemical reconstitution and molecular dynamics (MD) simulations in an ER-like bilayer that Ist2 can scramble phospholipids in a non-selective and Ca²⁺-independent manner. In yeast, deletion of the ER domain of Ist2 does not have a strong impact on cellular phospholipid levels or their steady-state distribution. However, we find that deleting this domain is lethal in the context of the PS-synthesis mutant *cho1Δ*. We further show that the TMD of Ist2 affects the ER vesicular export pathway as well as LD abundance and composition, and that these phenotypes are rescued by mutations in conserved residues predicted to be critical for phospholipid scrambling. Overall, our results point to the inter-connectedness of ER functions and underline the importance of the regulation of ER trans-bilayer lipid distribution.

## 144 Results

Ist2 functions as a phospholipid scramblase *in vitro*
To get insights into the function of the ER-embedded TMD of Ist2, we first performed structural homology modelling and sequence alignment. For homology modelling, we used the portion of the *Saccharomyces cerevisiae* Ist2 sequence homologous to TMEM16 proteins, i.e. from residue 1 to 600 (Ist2ΔC), excluding the disordered C-tail, which is unique to yeasts (D'Ambrosio et al., 2020). Comparison of Ist2ΔC with the top ten homologous proteins identified by I-TASSER revealed that the two closest Ist2 orthologs belong to the TMEM16 family, nhTMEM16 from *Nectria haematococca* (Brunner et al., 2014; Kalienkova et al., 2019) and afTMEM16 from *Aspergillus fumigatus* (Falzone et al., 2019), whose structures have been experimentally determined (Fig. S1A). Both nhTMEM16 and afTMEM16, as well as other TMEM16 family proteins, form homodimers, suggesting that Ist2 TMD also dimerizes (Fig. 1A). These proteins have been shown to function as Ca²⁺-activated phospholipid scramblases and/or as Ca²⁺-activated chloride channels (Brunner et al., 2014; Falzone et al., 2019; Jiang et al., 2017; Malvezzi et al., 2013). Sequence alignment shows a strong conservation of residues critical for lipid scrambling between nhTMEM16, afTMEM16 and Ist2, whereas residues involved in Ca²⁺-binding are mostly not conserved in Ist2 (Fig. 1B).

To test whether Ist2 scrambles phospholipids, we used a well-established fluorescence-based dithionite assay (Ploier and Menon, 2016). We first purified Ist2 fused to a biotin-acceptor domain following its overexpression in budding yeast, using a previously established procedure (Azouaoui et al., 2014; Dieudonné et al., 2022). In addition, mCherry was fused to Ist2 to facilitate its detection (Fig. S1B). Both full-length Ist2 and Ist2ΔC could be efficiently purified upon extraction with n-dodecyl-β-D-maltoside (DDM) from yeast membranes (Fig. S1C). Next, Ist2 was reconstituted into proteoliposomes (PLs) containing a small percentage of nitrobenzoxadiazole (NBD)-labeled lipid. Despite some protein loss during the reconstitution process, Ist2 was effectively recovered after detergent removal with Bio-beads (Fig. S1D) and unilamellar PLs were generated, as assessed by cryo-electron microscopy (Fig. 1C). The average diameter deduced from the observation of cryo-electron micrographs was ~80 nm for Ist2 PLs and 100 nm for control liposomes.

In the dithionite assay, the emission of the NBD-labeled lipid is irreversibly quenched due to the reduction of NBD into a non-fluorescent ABD moiety by dithionite (Fig. 1D). In control liposomes, 50% of the initial fluorescence is expected to be bleached as only NBD-lipids in the outer leaflet are accessible, whereas in PLs containing a scramblase that exchanges lipids from one leaflet to the other, all NBD lipids should be accessible and fluorescence reduction should in principle be complete. As shown in Fig. 1E and 1F, dithionite addition to Ist2-containing PLs induced a significantly larger fluorescence reduction than for control liposomes. Fluorescence reduction by dithionite of protein-free liposomes reached ~60%, which is consistent with the relatively small size of the PLs. When a control, ATP-dependent Drs2-Cdc50 lipid flippase complex was reconstituted in PLs (Timcenko et al., 2019), fluorescence reduction in the absence of ATP was much less pronounced than for Ist2 PLs (Fig. 1E,F). The difference between control liposomes and Drs2-Cdc50 PLs may be due to an uneven distribution of NBD-PS between the PL leaflets during reconstitution. We also verified that the presence of an mCherry tag on Ist2 did not affect fluorescence reduction by dithionite (Fig. S1E).

To exclude the possibility that the increased fluorescence reduction upon addition of dithionite is due to the preferential incorporation of NBD-PS in the outer leaflet of Ist2 PLs, we used iodide collisional quenching of the NBD fluorophore (Goren et al., 2014). In this assay, membrane-impermeable iodide ions reversibly quench the fluorescence of the NBD-PS located in the outer leaflet of the proteoliposomes, reporting on its steady-state distribution. Since scramblases continuously exchange lipids from one leaflet of the PL to the other, iodide treatment should result in 50% quenching. Analysis of the quenching experiment revealed that NBD-PS is
symmetrically distributed in both liposomes and proteoliposomes (Fig. 1G). Furthermore, the observed 50% protection of NBD-PS demonstrates that Ist2 PLs are tight to iodide ions. Further purification of PLs on a sucrose gradient confirmed the successful reconstitution of Ist2 into vesicles, as Ist2 co-sedimented with NBD-lipids (Fig. 1H, S1F). The top fraction (F1) contained pure lipid vesicles whereas the fluorescent ring observed in a lower fraction (F6) mostly corresponded to PLs. Consistent with this fractionation, fluorescence reduction was almost complete when dithionite was added to the PL-enriched fraction (F6, Fig. 1I). In contrast, little change was observed for the Drs2-Cdc50 control sample (Fig. 1J). We also asked whether the disordered C-ter tail of Ist2 contributes to lipid scrambling mediated by Ist2. Ist2ΔC reconstituted similarly to full-length Ist2, and the truncation of the C-ter tail did not alter the transbilayer movement of NBD-PS (Fig. 1K, Fig. S1G). Finally, we also performed a bovine serum albumin (BSA) back-extraction assay, in which BSA is used to extract and quench the fluorescence of short-chain NBD-lipids (Li et al., 2024). Since BSA cannot penetrate inside the proteoliposomes, it serves as a selective probe for externally exposed NBD-lipids, providing a useful comparison to dithionite-based quenching. Upon BSA addition, a greater fluorescence decrease was observed for Ist2-containing proteoliposomes than for control liposomes, confirming that Ist2 catalyzes the transport of NBD-PS across the bilayer leaflets (Fig. S1H,I). Altogether, these data indicate that the TMD of Ist2 promotes lipid scrambling after its purification and reconstitution in proteoliposomes.

## Ist2 dimer scrambles phospholipids in a Ca²⁺-independent manner with some selectivity

To further characterize the properties of Ist2 TMD, we verified experimentally whether it forms homodimers, as predicted by homology modelling with TMEM16 proteins. For this, we used flow-induced dispersion analysis (FIDA), which evaluates the size of proteins and protein complexes, including detergent-solubilized membrane proteins, by measuring their hydrodynamic radius ($R_h$). We used Ist2ΔC, since the long and disordered C-ter tail reduced the accuracy of the measure. FIDA measurements indicated the presence of a predominant species with a residence time of 1.85 min, corresponding to an experimental $R_h$ of 6.11 ± 0.23 nm for Ist2ΔC (Fig. 2A). To relate the $R_h$ value to the size of Ist2ΔC, we used CHARMM-GUI and AlphaFold3 models of monomeric and dimeric Ist2ΔC as templates to build protein/detergent complexes. CHARMM-GUI was able to accommodate a maximum of 160 and 240 DDM molecules around the monomer and the dimer, respectively, leading to complexes with theoretical $R_h$ of 5.18 nm and 6.18 nm (Fig. 2A). This strongly suggests that purified Ist2ΔC is a dimer.

The TMEM16 proteins characterized so far have been shown to be stimulated by Ca²⁺. Upon Ca²⁺ binding, a lateral groove called the subunit cavity opens, thereby providing a pathway large enough for lipids to cross the membrane (Falzone et al., 2019; Kalienkova et al., 2019). In contrast to other TMEM16 members, Ist2 was essentially insensitive to Ca²⁺, as the addition of Ca²⁺ to reconstituted Ist2 did not alter its ability to transport NBD-PS (Fig. 2C). Guided by the homology between Ist2 and proteins of the TMEM16 superfamily (Fig. 1A,B), we built models for the TMD of Ist2 using AlphaFold2 (Jumper et al., 2021) and trRosetta (Du et al., 2021). AlphaFold provided model structures of homodimers very similar to the nhTMEM16 X-ray structure (PDB ID: 4WIS) (Brunner et al., 2014), with an open subunit cavity; trRosetta, instead, provided (in addition to models in the open state) also model structures similar to the conformation of nhTMEM16 in a closed state (PDB ID: 6QMB) (Kalienkova et al., 2019) (Fig. 1A). Two Ca²⁺ ions were initially inserted in both the open and closed structural model, then the proteins were embedded in lipid bilayer membranes and we performed MD simulations at the all-atom level to assess the stability of calcium binding. In all simulations, in the absence of restraints, Ca²⁺ ions dissociated spontaneously and very rapidly from the protein, suggesting
248 that calcium binding is weak (Fig. 2D). This finding is consistent with our *in vitro* measurements
249 and with the observation that most of TMEM16 residues involved in calcium binding are not
250 conserved in Ist2 (Fig. 1B and Fig. 2B).
251

252 Since our initial investigations into Ist2-mediated lipid transport were conducted in a simple
253 PC/PS lipid composition, we also measured transport in a more physiologically relevant
254 environment. For this, we prepared Ist2 PLs using liposomes with an ER-like composition
255 containing a complex mix of phospholipids and ergosterol (see Methods). NBD-PS scrambling in
256 these PLs was robust, although somewhat less efficient than in simple PC/PS liposomes (Fig.
257 2E,F).
258

259 We further assessed lipid scrambling by Ist2 using MD simulations. All-atom simulations were
260 used to fine-tune coarse-grained (CG) models for the protein, using both the open and closed
261 conformation; we did not include Ca²⁺ in the CG simulations. For each conformation we
262 performed multiple long time-scale (20 µs) CG MD simulations using both a simple lipid mixture
263 and a more complex one, mimicking the composition of the ER membrane (Reinhard et al.,
264 2024) (see Table S3 for the lipid composition used in MD simulations, Table S4 for a full list of
265 simulations, and the Methods section for the details of the simulation setup). Simulations
266 provided a clear and reproducible result: a high number of flip-flop events were recorded in
267 simulations of the open Ist2 conformation, and close to none for the closed conformation,
268 irrespective of the membrane composition (Fig. 2G, Table 1-4 and Supplemental Movie 1). The
269 rate of phospholipid flip-flop was in the same range as what has been observed for other
270 TMEM16 scramblases *in silico* (Stephens et al., 2025). By contrast, flip-flop of ergosterol, which
271 does not require scramblases, was fast and not affected by Ist2 conformation (Table 2, 4). In
272 both simple and complex ER-like membranes, we observed some selectivity for different
273 phospholipids, with a preference for PC and PS, and the following over-all ranking:
274 PC>PS>PE>PI>PA (Table 1-2). Interestingly, the probability of flip-flop also depended on acyl
275 chains, with shorter chains (16:1) promoting flip-flop (Table 2). This is consistent with
276 observations of lipid scrambling by TMEM16K *in vitro*, which showed faster scrambling rates in
277 thinner membranes (containing shorter acyl chains) (Bushell et al., 2019). Similar to the results
278 of the dithionite scrambling assays, the rate of phospholipid flip-flop was higher in simple
279 compared to complex lipid mixture, which could be explained by the combined acyl chain and
280 headgroup preference, as well as some increase in the thickness of the complex bilayer.
281 However, no strong phospholipid preference was observed in the *in vitro* dithionite assay, in
282 which NBD-PS, NBD-PC and NBD-PE where consistently transported (Fig. 2H,I). This difference
283 with *in silico* results may at least in part be due to lower temporal resolution of the dithionite
284 assay, which is rate-limited by the relatively slow chemical reduction of NBD by dithionite
285 (Malvezzi et al, 2019), making it less sensitive to differences in the scrambling rates.
286

287 We next identified the pathway for lipid transport by calculating protein-lipid contacts during the
288 simulations. In all of the flip-flop events, lipids passed through the subunit cavity (between TM3-
289 6) facing the lipid bilayer on both sides of the dimer, as previously observed for other TMEM16
290 proteins (Fig. 3A). This cavity is closed in the closed conformation (Fig. 3B). Importantly, several
291 studies have reported that TMEM16 can induce membrane distortion, reducing membrane
292 thickness in an area near the subunit cavity and thereby lowering the energetic cost for
293 phospholipid movement from one leaflet to the other (Bethel and Grabe, 2016; Falzone et al.,
294 2022; Kalienkova et al., 2019). By calculating the local membrane thickness, we saw that both
295 conformations of the protein induced some membrane thinning in the immediate proximity
296 (within 1 nm) of the protein, and the thinning was more pronounced in the case of the open
297 conformation (Fig. 3C). For the latter, two thinner regions were observed around each protomer,
298 one in the proximity of each subunit cavity, between TM4 and TM6, and the other at the
interface between the protomers. In summary, our MD simulations support the scramblase activity of Ist2, and show an inter-relationship between Ist2 activity and the physico-chemical properties of the bilayer.

## Influence of the scramblase domain of Ist2 on cellular lipid homeostasis

Having established that Ist2 can scramble phospholipids, we next investigated whether this activity affects lipid homeostasis in yeast. The cytosolic C-ter tail of Ist2 mediates the transport of PS from the ER to the PM via its interaction with the PS-transfer proteins Osh6 and its homolog Osh7 (D'Ambrosio et al., 2020; Wong et al., 2021). Mutations in the C-ter tail of Ist2 that prevent interaction with Osh6/7 mimic the phenotypes associated with the deletion of Osh6 and Osh7. To assess the defects associated with the deletion of the N-ter TMD of Ist2 independently of the function of its cytosolic tail, we constructed chromosomal yeast mutants expressing truncated versions of Ist2. We used GFP-tagged variants to verify their intracellular localization: a mutant lacking the majority of the TMD except for the last two TM helices, GFP-ΔNist2, retains the localization at ER-PM contact sites (Manford et al., 2012), whereas Ist2ΔC lacking the C-ter cytosolic tail localizes throughout the ER (Fig. 4A).

We previously showed that despite their impairment in PS transfer from the ER to the PM, *ist2Δ* or *osh6Δ osh7Δ* mutants do not show a strong defect in the steady-state cellular distribution of PS, evaluated with the PS probe C2*Lact*-GFP (D'Ambrosio et al., 2020). We did not observe any defect in C2*Lact*-GFP distribution in Δ*Nist2* mutant cells, and a slight defect in *ist2ΔC* truncation mutants (Fig. S2A,B). We then used thin-layer chromatography (TLC) to analyze the impact of Ist2 truncations on the total yeast lipidome. As previously shown, the deletion of *IST2* results in a decrease in total cellular PS levels by about 50%, similar to the defect observed in *osh6Δ osh7Δ* or in strains with mutations in the Ist2-Cter cytosolic tail (Fig. 4B,C) (D'Ambrosio et al., 2020). This reduction is likely due to the feedback inhibition of the sole yeast PS synthase, Cho1, caused by impaired PS export from the ER. As expected, no PS can be detected in *cho1Δ* cells. Furthermore, *cho1Δ* cells display a decrease in phosphatidylethanolamine (PE), which can be generated from PS by two PS decarboxylases, Psd1 and Psd2 (Fig. 4B-D) (Lenoir et al., 2021; Storey et al., 2001). Some other lipids are also highly affected in *cho1Δ* cells, especially ceramide (~4-fold increase) and sterol ester (SE; 30% increase), whereas this is not the case in *osh6Δ osh7Δ* or *ist2Δ* strains (Fig. 4B,C). In contrast to *cho1Δ*, these strains also show normal PE levels, as previously reported (D'Ambrosio et al., 2020).

We then compared the lipidomes of *ist2Δ* and Δ*Nist2* cells. In contrast to *ist2Δ*, no reduction in cellular PS levels was observed in Δ*Nist2*. The total levels of other major phospholipids were also not affected (Fig. 4E,F). A separate analysis of more hydrophobic lipids revealed a small but consistent increase (~15%) in SE in both *ist2Δ* and Δ*Nist2*, as well as a slight decrease in ergosterol levels (Fig. 4G,H). This observation led us to evaluate the distribution of sterols in these strains using the fluorescent sterol probe GFP-D4H (Encinar Del Dedo et al., 2021). A polarized PM-localized GFP-D4H signal was observed in WT as well as in *ist2* mutant strains, in contrast to two control strains, *cho1Δ* and *sac1Δ*, where the GFP-D4H signal was highly perturbed (Fig. S2C). Overall, these results show that deletion of the N-ter scramblase domain of Ist2 does not lead to any gross perturbations of the cellular lipidome.

A more direct way to check for defects in PS export from the ER is using an *in vivo* PS transport assay (D'Ambrosio et al., 2019; Filseck et al., 2015; Maeda et al., 2013). This assay requires the use of *cho1Δ* cells, in which PS production can be restored via acylation at the ER level of exogenously added lyso-PS. However, we were not able to evaluate whether Δ*Nist2* affects PS
export because we could not construct a Δ*Nist2 cho1Δ* strain, likely due to a negative genetic interaction between *CHO1* and *IST2*. This interaction is difficult to verify due to the growth and mating defects of *cho1Δ* cells, but could be observed when *CHO1* expression is under the control of a galactose (Gal)-inducible promotor: growth of *ist2Δ Gal-CHO1* and of Δ*Nist2 Gal-CHO1* cells is repressed on glucose-containing plates (YPD), whereas all strains grow well when Gal is used as the carbon source (Fig. 4I). Of note, these synthetic growth defects show a strong temperature dependence, being more pronounced at lower temperature (22°C).

Another assay to evaluate the function of Ist2 in Osh6-mediated PS transport is by genetic interaction with *psd1Δ* (Wong et al., 2021). The growth of *ist2Δ psd1Δ* cells is strongly inhibited in synthetic media lacking ethanolamine whereby direct synthesis of PE via the Kennedy pathway is blocked (Storey et al., 2001), because supply of PS to the other PS decarboxylase, Psd2, is also diminished (Fig. 4D,J and Fig. S2D). Growth in this strain is rescued by the expression of WT Ist2, but not by the expression of an Ist2 mutant with randomized C-ter tail (Ist2-RL), which cannot interact with Osh6 (D'Ambrosio et al., 2020; Kralt et al., 2014) (Fig. 4J). In contrast, expression of Δ*Ist2* rescues growth as efficiently as the WT plasmid, suggesting that the TMD of Ist2 is not implicated in this pathway. Finally, we considered the possibility that Ist2 could function as an anion channel, because this activity is shared by a number of other TMEM16 proteins instead or in addition to their scramblase activity (Jan and Jan, 2024; Malvezzi et al., 2013). It has been suggested that the increased sodium tolerance (ist) phenotype of *ist2Δ* cells (Entian et al., 1999) is linked to a Ca²⁺-activated Cl⁻ channel activity (Kunzelmann et al., 2016). However, this phenotype, revealed on YPD plates supplied with 1 M NaCl, is weak and likewise not dependent on the TMD of Ist2, as it can be observed in *ist2Δ* but not in *ist2ΔC* cells (Fig. S2E). In summary, our results so far do not reveal a direct effect of Ist2 TMD on the cellular lipidome and do not show that the scramblase activity of Ist2 is required to promote efficient PS export via Osh6.

**Genes required for vesicular budding from the ER display negative interaction with Ist2 scrambling mutants**

Defects in lipid transport pathways are often difficult to detect (Peter et al., 2022; Toulmay et al., 2022). We decided to evaluate the effect of Ist2 TMD on ER processes that may be directly impacted by trans-bilayer lipid distribution in the ER or by localized changes in the bilayer composition. One such process is the formation of COPII vesicles, which occurs at discrete ER-exit sites where the COPII coat assembles following the activation of the small GTPase Sar1 by the ER-resident GTP-exchange factor Sec12 (Fig. 5A) (Hutchings et al., 2021). Vesicle formation requires deformation of the ER bilayer. It was shown that lyso-phospholipids, which have a conical shape, can promote vesicle formation by inducing membrane curvature, and membrane protein asymmetry can oppose it (Copic et al., 2012; Derganc et al., 2013; Melero et al., 2018). Furthermore, COPII binding to membranes *in vitro* is affected by phospholipid composition (Matsuoka et al., 1998). The activity of an ER scramblase could therefore negatively impact the formation of COPII vesicle by reducing the amount of asymmetry between the two leaflets, or it could have a positive or negative feedback by changing the phospholipid composition of the cytosolic leaflet.

To test these possibilities, we assayed for genetic interactions between *ist2* and COPII temperature sensitive (*ts*) mutants (Schekman and Novick, 2004). We could observe a genetic interaction between *sec12-4* and *ist2Δ*. Interestingly, this interaction was temperature-dependent, negative at 25°C and slightly positive at 30°C (Fig. 5B and Fig. S3A). By contrast, *ist2Δ* had no effect on the growth of *sec17-1*, which acts downstream of COPII, blocking vesicle fusion (Fig. S3B). We used another COPII *ts* allele, *sec23-1*, but we had difficulties in combining
this mutation with *ist2Δ*. We therefore decided to test the opposite phenotype, i.e., the effect of over-expression of *IST2*. Over-expression of Ist2 TMD (*Ist2ΔC*) negatively affects the growth of *sec23-1* cells at 30°C in a dose-dependent manner (Fig. 5C), as does the expression of full-length Ist2 (Fig. S3C).

We used the over-expression phenotype in the *sec23-1* background to compare the effect of mutations in Ist2 TMD that could impair its scrambling activity. Six residues of interest in TM3 (L229, E240), TM4 (T267), and TM6 (R359, Q363, Y366) were selected based on their conservation in afTMEM16 and nhTMEM16, where they were shown to be important for phospholipid scrambling (Fig. 1B and Fig. 5D) (Lee et al., 2018). Among these residues, L229, T267 and Y366 were also identified by our MD simulations to be in contact with the lipid headgroup during flip-flop (Fig. 3A). Over-expression of single point mutants E240R, N380V, T267A, L229Q, or the double mutant K276E_Y277A resulted in improved growth of *sec23-1* cells at 30°C, compared to cells over-expressing WT Ist2ΔC, with the E240R mutation showing the strongest rescue (Fig. 5E). The rescue was even stronger when all six of these residues were mutated to alanine (*Ist2ΔC_6A* and *Ist2_6A*) (Fig. 5F, S3C), or to tryptophan (*Ist2ΔC_6W*, Fig 5F). Addition of a C-ter tag did not impact on the toxicity of Ist2ΔC in *sec23-1* (Fig. S3D). We used mScarlet- or Halo-tagged Ist2ΔC over-expression plasmids to verify that mutating the six selected residues does not change protein levels (Fig. S3E-F). Furthermore, the localization of Ist2_6A-GFP, expressed at the endogenous *IST2* locus, or of over-expressed Ist2ΔC_6A-mScarlet was indistinguishable from WT (Fig. 5G, S3G). For the strongest 6W substitution, we observed a slight tendency in some cells towards clustering in larger puncti (Fig S3G); for this reason, we preferentially used the 6A mutant in the subsequent cellular assays.

To verify our assumption that these point mutations in Ist2 TMD decrease its phospholipid scrambling activity, we performed MD simulations with open conformations of the 6A and 6W Ist2ΔC mutants embedded in ER-like membranes. Indeed, we observed a significant decrease in the scrambling activity of the strongest, 6W mutant (9.3 ± 2.1 scrambling events in 20 µs, averaged over 3 simulations, compared to 31 ± 10 in WT). By contrast, the 6A mutant scrambled at a similar rate as WT Ist2 (35 ± 18 events over 20 µs) (Table 5 and 6). We also purified the Ist2_6W, 6A and the Ist2_E240R and N380V single mutants from yeast and reconstituted them into PLs to assess their scrambling activity in the dithionite assay. Although all mutants were efficiently purified (Fig. S4A), we could not observe any effect of these mutations on Ist2 scrambling *in vitro*. This discrepancy could again be due to the limited temporal resolution of the dithionite assay and will require further investigation.

Next, we wanted to assess if mutations in Ist2 TMD directly impact the COPII pathway. We tested the effect of Ist2 TMD over-expression in strains depleted of COPII coat proteins Sec23, Sec24 and Sec31 using the auxin-inducible degron system (AID) (Gamerio et al., 2024). We optimized auxin concentration to observe a mild growth defect in *SEC23-*, *SEC24-* and *SEC31-AID* strains expressing an empty plasmid (Fig. 5H). In all three strains, the growth defect was exacerbated in yeast over-expressing Ist2ΔC, but not when they were over-expressing the Ist2ΔC_6A mutant. By contrast, we did not observe and Ist2-specific effect in WT yeast. We then evaluated the effect of Ist2-TMD over-expression on the distribution of ER exit sites, where COPII vesicles form, in WT cells expressing the ER exit site marker Sec16-sfGFP (Fig. 5I) (Gomez-Navarro et al., 2020). In cells expressing Ist2ΔC, we frequently observed large Sec16 clusters, whereas clusters were absent from control cells and were smaller and less frequent in cells expressing the Ist2ΔC_6A mutant (Fig. 5J). We also examined the rate of COPII cargo protein secretion by monitoring the maturation of the vacuolar protease carboxypeptidase Y (CPY) in *sec23-1* cells at 30°C using a pulse-chase assay (Stevens et al., 1982). Over-expression of Ist2ΔC caused a slight but reproducible delay in the maturation of CPY from
its precursor (p) ER form to its mature (m) vacuolar form, indicating a delay in ER-to-Golgi transport compared to the control strain. This delay was decreased in cells over-expressing the Ist2ΔC_6A mutant (Fig. 5K). Together, our results suggest that over-expression of the Ist2 scramblase domain in the ER directly impacts the formation of COPII vesicles from the ER membrane.

## Ist2 scrambling activity affects LDs and other lipid pathways at the ER

Another process that requires ER membrane asymmetry is the formation of LDs that bud from the ER (Chorlay et al., 2019; Huang et al., 2021; Nieto et al., 2023). In agreement, it has been shown in mammalian cells that downregulation or loss of the ER scramblases TMEM41B and VMP1 results in accumulation of cytosolic LDs (Huang et al., 2021; Li et al., 2021; Moretti et al., 2018). Our whole-cell lipid analyses suggested an increase in neutral lipids in *ist2Δ* and in Δ*Nist2* mutants (Fig. 4). In agreement, we saw a small but significant increase in the number of LDs present in *ist2Δ* (+22%), Δ*Nist2* (+26%) *ist2_6A* (+18%), and in *ist2_6W* mutant cells (+25%), but not in the Ist2-tail mutant *ist2ΔC* (Fig. 6A,B). Furthermore, given that phospholipid synthesis is restricted to a single ER leaflet, we reasoned that a decrease in ER scrambling activity could affect the phospholipid composition of the LD monolayer. To probe for changes in LD surface composition, we decided to use an amphipathic helix (AH) derived from the human LD protein perilipin 4 (PLIN4), which targets LDs in yeast. Previously, we generated a series of PLIN4 AH mutants with subtle changes in their amino acid composition that impacted LD targeting (Copic et al., 2018). One of these AH variants, [2Q, 2V], composed of 4 x 33 aa stretches of the same sequence, interacts weakly but specifically with LDs in yeast and is overall negatively-charged (Fig. 6C) (Copic et al., 2018). We reasoned that the weak LD affinity and negative charge of PLIN4-AH[2Q,2V] should make it sensitive to changes in LD surface composition, in particular to changes in electrostatics resulting from an increase or decrease in charged lipids such as PS. In WT cells, this probe localized to LDs in a fraction of cells. Interestingly, its localization to LDs increased in TMD mutants *ist2Δ*, Δ*Nist2* and *ist2_6A*, whereas the localization to LDs decreased in the tail mutant *ist2ΔC* and in *osh6Δ osh7Δ* cells (Fig. 6D,E). These changes could reflect a decrease or an increase in LD surface charge, respectively. Overall, these results suggest that Ist2 TMD activity influences the formation and composition of LDs.

Finally, we explored whether Ist2 activity might influence other lipid transport pathways. Scramblases have been shown to play an important role in the formation of autophagosomes, where their activity is coupled to lipid transfer by channel-like LTPs of the Vps13 family, such as Atg2. However, we did not detect any defect in general autophagy in *ist2Δ* cells, in agreement with a recent report (Fig. S5A,B) (Liu et al., 2024). We then focused on CSF1, which encodes another Vps13-like LTP that can channel phospholipids between the ER and other compartments (Peter et al., 2022; Toulmay et al., 2022). As suggested by a genome-wide screen (Costanzo et al., 2016), we could observe a negative genetic interaction between *ist2Δ* and a null allele of CSF1, *csf1ΔC* (Peter et al., 2022), which was cold-temperature dependent (Fig. 6F). In contrast, *ist2ΔC* had no effect on the growth of *csf1ΔC*, whereas the TMD mutants Δ*Nist2* and *ist2_6A* had some effect, but less than *ist2Δ*, again showing non-additive effects between deletions of the two subdomains of Ist2. Together, our data suggest that Ist2 TMD impacts several processes originating from the ER, and these effects correlate with mutations in residues implicated in phospholipid scrambling (Fig. 6G). We therefore propose that phospholipid scrambling by Ist2 affects lipid pathways at the ER.

## The LD phenotype of *ist2Δ* can be potentiated by the deletion of another putative ER scramblase
500 Whereas we could observe defects in several ER-linked pathways associated with a potential increase or decrease in phospholipid scrambling by Ist2, these defects were invariably weak.
501
502 One possible reason for this may be the presence of other proteins in the ER that can mediate
503 phospholipid scrambling, therefore masking the defects associated with mutations in Ist2 TMD.
504
505 While no dedicated ER scramblase has so far been identified in yeast, many proteins have been
506 suggested to moonlight as scramblases, including GPCRs and many different ER protein
507 insertases (Goren et al., 2014; Li et al., 2024). To check if other scramblases might be acting in
508 parallel with Ist2, we first searched the SGD database for candidate scramblase genes that are
509 non-essential, well-documented in the literature, and whose genetic networks might overlap with
510 that of IST2. We found no overlap of genetic interaction networks between IST2 and STE3,
511 encoding a GPCR that can scramble lipids in vitro and in silico (Li et al., 2024), 4% with AIM25,
512 encoding a predicted mitochondrial scramblase, 6% with TVP38 encoding a DedA domain
513 protein homologous to TMEM41B and VMP1, 7% with ATG9, encoding the well-characterized
514 autophagosomal scramblase (Matoba et al., 2020), and 16% with VDAC1, which encodes a
515 mitochondrial porin that can scramble phospholipids (Jahn et al., 2023). Interestingly, we found
516 a strong overlap in genetic interactions between IST2 and GET1 (43%) or GET2 (42%) (Fig. 7A).
517
518 These two genes encode a protein complex required for insertion of tail-anchored proteins into
519 the ER, and Get1, but not Get2, has been shown to possess scramblase activity in vitro and in
520 silico (Li et al., 2024; Schuldiner et al., 2008). We observed a negative genetic interaction
521 between ist2Δ and get1Δ, evident at 20°C and at 37°C, but not between ist2Δ and get2Δ (Fig.
522
523 Whereas we could detect some increase in LD number in get1Δ (Fig. S5C), we observed a more
524 striking defect when we evaluated LD size using the LD marker Erg6-GFP (Fig. 7C,D). LDs size
525 was significantly increased in ist2Δ, even more in get1Δ, and ist2Δ get1Δ cells showed an
526 additive effect. Interestingly, LDs in get1Δ cells showed large cell-to-cell variability. While get1Δ
527 cells display pleiotropic phenotypes (Joshi et al., 2016; Schuldiner et al., 2008), in line with their
528 implication in the biogenesis of tail-anchored proteins, the LD phenotype has not been reported
529 so far. It could suggest that Get1 also functions as a scramblase in cells, acting in parallel with
530 Ist2.

529

530 Discussion

531 We show that the budding yeast protein Ist2 possesses phospholipid scrambling activity in its
532 ER-localized TMD (Fig. 6G). This is not surprising, given the conservation between this domain
533 and the TMEM16 protein family, in particular its closest fungal orthologs nhTMEM16 and
534 afTMEM16, which have both been demonstrated to act as phospholipid scramblases (Brunner et
535 al., 2014; Falzone et al., 2019). Ist2 is the only TMEM16-like protein present in budding yeast. Its
536 scramblase activity has recently been independently confirmed and is supported by its structure
537 determined by cryo-EM (Arndt et al., 2025). Our evidence is based on the reconstitution of Ist2
538 that was expressed and purified from its native host using mild detergent to preserve its function.
539 We were able to detect robust and rapid lipid scrambling both with full-length Ist2 and with
540 Ist2ΔC, lacking the C-ter cytosolic tail, which is unique to yeasts. These results were confirmed
541 by MD simulations. Importantly, similar to TMEM16 and other scramblases evaluated so far
542 (Sebinelli et al., 2024), Ist2 can scramble a variety of different phospholipid substrates. Both in
543 vitro and in silico, Ist2 scrambling activity was higher in simple compared to complex, ER-like
544 bilayers. This could at least in part be explained by a possible substrate preference detected in
545 silico, where Ist2-mediated scrambling of PC, PS and PE was faster than that of PI and PA, and
546 we could also observe faster scrambling of phospholipids with shorter acyl chains. Similar
547 results have been reported for mammalian ER-localized TMEM16K (Bushell et al., 2019).

548
549 Both *in vitro* and *in silico*, phospholipid scrambling by Ist2 was not sensitive to Ca²⁺. This
550 deviates from the activity of other TMEM16 scramblases, which are all activated by Ca²⁺ (Bushell
551 et al., 2019; Falzone et al., 2019; Kalienkova et al., 2019; Malvezzi et al., 2013), but is in
552 accordance with the lack of conservation of Ca²⁺-binding residues in the Ist2 sequence. It is
553 possible that Ist2 is a constitutive ER scramblase, or it could be regulated by a different
554 mechanism. Our predicted structural models suggested the possible occurrence of both closed
555 and open conformations for the Ist2 subunit cavity in the absence of Ca²⁺, pointing to alternative
556 mechanisms to Ca²⁺ binding for the control of phospholipid scrambling. These questions will be
557 the subject of further investigations.
558

559 The TMEM16 scramblases are suggested to function via a 'credit card' mechanism (Pomorski
560 and Menon, 2006), whereby the phospholipid headgroups sliding through the lipid bilayer utilize
561 a hydrophilic slit between TM helices 3-7 that form the subunit cavity. This mechanism was
562 observed in our MD simulations, and mutagenesis of some of the residues lining this pathway
563 resulted in a reversal of the phenotypes in yeast and *in silico*. Alternative or additional
564 mechanisms of lipid scrambling have also been proposed (Jan and Jan, 2024). Both afTMEM16
565 and nhTMEM16 can induce membrane thinning (Bethel and Grabe, 2016; Falzone et al., 2022;
566 Kalienkova et al., 2019), which would facilitate the passage of phospholipids through the
567 membrane; we have likewise observed membrane thinning in our MD simulations, particularly
568 with the 'open' (active) model of Ist2. Furthermore, 'out-of-the-groove' scrambling has also been
569 observed for both of these proteins, whereby phospholipids utilize an alternative pathway that
570 does not require Ca²⁺ activation (Falzone et al., 2022; Feng et al., 2024). Out-of-the-groove
571 scrambling of nhTMEM16 is highly dependent on membrane lipid composition (Feng et al.,
572 2024). Unlike Ist2, afTMEM16 and nhTMEM16 both function at the PM, therefore in a very
573 different lipid environment. These considerations are all important when evaluating the activity of
574 Ist2 in comparison to these highly-studied scramblases, and can guide further design of Ist2-
575 scrambling mutants.
576

577 In cells, direct evidence of scramblase activity is difficult to obtain (Tsuji et al., 2019). The
578 limitation of bulk cellular lipid assays performed at steady state is that they cannot detect
579 localized effects and that a defect in one pathway can be compensated by other pathways or by
580 other lipids (Bao et al., 2021; Peter et al., 2022). Furthermore, many proteins have been
581 suggested to contribute to the scramblase activity of the ER, such as GPCRs (Goren et al.,
582 2014) or all ER protein insertases (Li et al., 2024). Nevertheless, the phenotypes that we could
583 observe upon deletion or over-expression of the Ist2 TMD strongly support a scramblase activity
584 for this domain (Fig. 6G). Our strongest phenotype is the effect of Ist2 TMD over-expression on
585 the COPII vesicular pathway. The propensity of a cellular membrane to be deformed by a coat
586 machinery depends on the efficient recruitment of the coat components and on physical factors
587 including membrane bending modulus, membrane tension, and protein crowding. In the case of
588 the COPII coat, lipid unsaturation and negative charge have been shown to facilitate the
589 recruitment of the small G protein Sar1 and of the Sec23/24 complex, respectively (Matsuoka et
590 al., 1998), whereas cargo crowding on the luminal side has been shown to oppose the
591 mechanical effect of the coat (Copic et al., 2012; Derganc et al., 2013). However, the difficulty in
592 making liposomes with asymmetric lipid composition has hampered testing the role of lipid
593 distribution between the two leaflets on coat-induced membrane budding. Nevertheless, indirect
594 evidence suggests an impact of interleaflet lipid distribution on membrane deformation. In
595 Drosophila, high scramblase activity leads to a symmetric and highly deformable PM (Shiomi et
596 al., 2021). Furthermore, a recent study in mammalian cells has shown that TMEM41B influences
597 export of GPI-anchored protein from the ER (Cao et al., 2023). Finally, MD simulations suggest
598 that polyunsaturated lipids, when present in the convex side, facilitate membrane deformation
599 (Pinot et al., 2014; Tiberti et al., 2020). By contrast, recent biophysical measurements indicate
600 that asymmetric membranes containing PE and PS in one leaflet are rather stiff (Frewein et al.,
601 2023). Clearly, the link between membrane asymmetry and coat-induced membrane
deformation is a challenging issue for future studies.
602
603

604 More well-documented is the influence of mammalian ER scramblases on the production of LDs
605 or lipoprotein particles that bud into the ER lumen, which appears particularly pronounced in
606 strongly-secretory liver cells or tissue (Huang et al., 2021; Wu et al., 2024). In agreement with
607 these observations, deletion or targeted mutagenesis of Ist2 TMD both affect LD abundance and
608 composition to a similar extent, whereas deletion of the C-terminal domain does not.
609 Interestingly, the effect on LD abundance could be potentiated by simultaneous deletion of the
610 ER protein insertase Get1, which has recently been proposed to moonlight as a lipid
611 scramblase. These results should be treated with caution, because Get1 is required for insertion
612 of tail-anchored proteins into the ER, and can therefore affect lipid homeostasis in an indirect
613 way (Farkas et al., 2022; Schuldiner et al., 2008). Nevertheless, the overlap in LD phenotypes
614 between get1Δ and ist2Δ suggest that they may function in parallel and supports the model that
615 many proteins may contribute to phospholipid scrambling in the ER. Another important clue is
616 that a number of our cellular assays showed a strong dependence on temperature, suggestive
617 of changes in membrane composition upon Ist2 TMD deletion and a homeoviscous membrane
618 adaptation (Klose et al., 2012; Renne and Kroon, 2017). Together, these observations support
619 the implication of Ist2 TMD domain in the regulation of the ER lipid homeostasis via its
620 phospholipid scramblase activity, in agreement with our in vitro and in silico data. A scramblase
621 activity of Ist2 TMD can also explain other phenotypes that have been reported for ist2Δ cells,
622 such as osmotic sensitivity (Smith et al., 2024) and an effect on ER-phagy (Liu et al., 2024).
623

624 An unusual feature of Ist2 is the presence of a long cytosolic tail that interacts with the PM,
625 localizing Ist2 to ER-PM contact sites (Fig. 6G). Although this tail is unique to yeasts, two other
626 mammalian TMEM16 proteins, TMEM16H and TMEM16K, have been shown to localize to the
627 ER and engage in contacts with the PM and the endosomes, respectively (Jha et al., 2019;
628 Petkovic et al., 2020). TMEM16K has also been shown to act as a scramblase. These
629 observations raise the obvious question of the advantage of localizing an ER lipid scramblase to
630 a membrane contact site. We showed previously that the Ist2 tail interacts with the PS-specific
631 LTP Osh6 and is required for efficient transport of PS from the ER to the PM (D'Ambrosio et al.,
632 2020). Mutations in the tail or deletion of Osh6 and its homolog Osh7 result in a decrease in the
633 total levels of PS, which can be explained by a feedback inhibition of the PS synthase Cho1 at
634 the ER (Kannan et al., 2017; Sohn et al., 2016). Cho1 has generally been assumed to
635 synthesize PS in the cytosolic leaflet of the ER (Chauhan et al., 2016), although this has been
636 recently put under question by the structural characterization of the human PS synthase PSS1,
637 whose active site is positioned in the ER lumen (Long et al., 2024). Under either scenario, the
638 scramblase activity would affect the total pool of PS available for transfer by Osh6, and thereby
639 would be expected to modulate the efficiency of PS export from the ER. The intrinsic set-up of
640 Ist2 would allow for direct coupling between two lipid transport activities, lipid flip-flop and lipid
641 transfer between compartments, enabling more precise regulation of the two processes and a
642 faster and non-linear response (feed-back mechanisms). However, we have not detected any
643 impact of the deletion of the TMD of Ist2 on the cellular PS levels or distribution, in contrast to
644 the deletion of the C-ter tail. One issue may be the sensitivity of our assays, especially since we
645 could not perform the cellular PS transport assays due to the synthetic interaction between
646 cho1Δ and ΔNist2. Of note, the TMD domain of Ist2 promotes PS transfer by Osh6 between
647 liposomes in vitro, demonstrating coupling between the two lipid transport activities (Fabbre et
al., 2025). While we so far do not have direct evidence of such coupling in cells, several of our cellular assays revealed non-additive effects between the ΔN and ΔC deletions of Ist2, most prominently the genetic interaction between *ist2* mutants and *CSF1*, which encodes a bridge-like lipid transport protein. How the two lipid transport activities of Ist2 are coordinated and regulated in the cell will require further investigation.

653

654

## 655 Acknowledgments

We wish to acknowledge Sophie Dupré, Anne Claude Gavin, Elizabeth Miller, Simonetta Piatti Elizabeth Venkoff and Anton Khmelinskii for gift of strains and plasmids, Daniel Lévy and Aurélie Di Cicco for help with reconstitution in proteoliposomes, Juliette Martin for support with the MODELLER software, Cyril Moulin, Ivan Fayol and Maxime Bello for help with strain construction and phenotype testing, Clément Benedetti for assistance with image analysis, and Thibaud Dieudonné, Guillaume Drin, and Bruno Antonny for helpful discussions and comments on the manuscript. We thank Isabelle Goiffon and the joint IGMM-CRBM “yeast media and technologies service”. This work benefited from the CryoEM platform of I2BC, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-05], the MRI imaging facility, a member of the national infrastructure France-BioImaging, supported by the French National Research Agency (ANR-10-INBS-04, Investissements d'avenir). All MD simulations were performed at French supercomputing centers (CINES and TGCC) supported by Grand Équipement National de Calcul Intensif (GENCI, grant number A0120710138, A0140710138). We acknowledge CC-IN2P3 (https://cc.in2p3.fr) for computing services (data storage and backup). LM acknowledges funding by the Institut National de la Santé et de la Recherche Médicale (INSERM). This work was supported by the Agence Nationale de la Recherche (ANR-20-CE13-0030-02, ANR-21-CE11-0015-01 and ANR-23-CE44-0026).

673

674

## 675 Materials and Methods

### 676 Plasmid construction

All plasmids used in this study are listed in Table S1. The Gibson Assembly Hifi Kit (Ozyme) was used to generate the following plasmids: pADH-Ist2(N), pGPD-Ist2(N), pGPD-Ist2, and their corresponding tagged versions (mScarlet or Halo tag amplified from Addgene 179067 and 188929 plasmids, respectively) (Bean et al., 2022). Site-directed mutagenesis kit (Agilent) was used for generating point mutations in plasmids. Briefly, plasmids were amplified using overlapping oligonucleotides containing the desired mutations, designed with the most frequent codon usage. All constructs were verified by DNA sequencing. For overexpression of Ist2 in S. cerevisiae, the tagged genes were cloned into the pYeDP60 plasmid (Pompon et al., 1996). The preparation of these plasmids was performed as follows. The gene encoding Ist2 was amplified from yeast genomic DNA. The *IST2* gene was inserted into the shuttle pJET1.2 plasmid (Thermo Fisher Scientific) to obtain the pJET-*IST2* plasmid. For the cloning of various PCR fragments into the pYeDP60 vectors, the ‘in-fusion’ reaction (Takara) was used. All the inserted fragments were checked by DNA sequencing.

690

### 691 Reagents for purification and *in vitro* reconstitution

Products for yeast and bacteria cultures were purchased from Difco (BD Biosciences) and Sigma. n-dodecyl-β-d-maltopyranoside (DDM, D310) was purchased from Anatrace. Streptavidin-sepharose resin was purchased from GE/Cytiva (17511301). 1,2-dioleoyl-sn-
695 glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)
696 (DOPS) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L-α-phosphatidylinositol (Soy)
697 (sodium salt) (SoyPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-
698 oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-
699 yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (ammonium salt) (C6-NBD-PS), 1-palmitoyl-2-
700 {12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine
701 (ammonium salt) (C12-NBD-PS), 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-
702 yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine (C12-NBD-PC), 1-palmitoyl-2-{12-[(7-nitro-2-
703 1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoethanolamine (C12-NBD-PE)
704 were purchased from Avanti Polar lipids. mCherry was detected using a rabbit anti-mCherry
705 antibody from Invitrogen (PA5-34974). Goat anti-rabbit HRP-coupled IgG antibody (1706515),
706 Bio-Beads® SM-2 Adsorbent (152-3920), and Precision Plus Protein Standards (1610393) were
707 purchased from Biorad. EDTA-free protease inhibitor cocktail (S8830) was purchased from
708 Sigma.
709

710 Expression of Ist2 in yeast membranes
711 W303.1b/GAL4 (a, leu2-3, his3-11, trp1-1:TRP1-GAL10-GAL4, ura3-1, ade2-1, can r, cir +)
712 yeast strain was transformed by the lithium-acetate method. Yeast cultures and recombinant
713 protein expression were performed as previously described (Dieudonné et al., 2022). Yeast cells
714 were then harvested by centrifugation, washed with ice-cold deionized H2O, then with ice-cold
715 TEKS buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1 M KCl, 0.6 M sorbitol), and resuspended
716 in TES buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.6 M sorbitol) supplemented with protease
717 inhibitors (SIGMAFAST EDTA-free protease inhibitor cocktail) and 1 mM PMSF. The cells were
718 subsequently broken with 0.5 mm glass beads using a 'Pulverisette 6' planetary mill (Fritsch).
719 The crude extract was spun down at 1,000 g for 20 min at 4°C to remove cell debris and nuclei.
720 The resulting supernatant was centrifuged at 20,000 g for 20 min at 4 °C, yielding S2
721 supernatant and P2 pellet. The S2 supernatant was then ultracentrifuged at 125,000 g for 1 hr at
722 4 °C. The resulting P2 and P3 pellets were then resuspended at about 30–50 mg.mL-1 of total
723 protein in TES buffer.
724

725 Purification of Ist2 from yeast
726 Membranes obtained after expression of Ist2 (P2 or P3) were diluted to 5 mg.mL⁻¹ of total
727 protein in ice-cold buffer A (50 mM MOPS-Tris at pH 7.0, 500 mM NaCl, 20% (w/v) glycerol),
728 supplemented with 1 mM PMSF and protease inhibitors. n-dodecyl-β-D-maltoside (DDM) was
729 then added to 15 mg.mL⁻¹, resulting in a DDM/protein ratio of 3/1 (w/w). The suspension was
730 then stirred gently on a wheel for 1 hr at 4 °C. Insoluble material was pelleted by centrifugation
731 at 100,000 g for 1 hr at 4 °C. The supernatant was applied onto streptavidin-sepharose resin (1
732 mL per 3 mg Ist2) and incubated for 2 hours at 6 °C to allow binding of BAD-tagged Ist2mC to
733 the resin. The resin was then washed thrice with three resin volumes of ice-cold buffer A
734 supplemented with 0.5 mg.mL⁻¹ DDM. Elution was performed by addition of 60 µg of purified
735 TEV per mL of resin and overnight incubation at 6 °C. Before reconstitution, the eluted fraction
736 was concentrated to 0.3-0.4 mg.mL⁻¹ using a Vivaspin unit (100 kDa MWCO).
737

738 Liposome preparation and protein reconstitution
739 Two lipid compositions were used to prepare liposomes: a 9:1 molar ratio of POPC:POPS, and
740 an ER-like complex mixture composed of DOPC:DOPS:DOPE:SoyPI:DOPA:ergosterol in an
741 8:4:4:2:1:1 molar ratio. For POPC:POPS liposomes, lipids were dissolved in 5 mL of chloroform
742 at a concentration of 10 mg.mL⁻¹. For the ER-like mixture, lipids were also dissolved at 10
743 mg.mL⁻¹ but in a 2:1 chloroform:methanol solution. Each lipid solution was transferred to a glass
744 round-bottom flask and dried using a rotary evaporator under reduced pressure (~30 minutes) to
745 form a thin lipid film. Residual solvents were removed by placing the lipid films in a vacuum
746 desiccator for at least 1 hour. The dried lipids were then rehydrated in 12.5 mL of buffer R (200 mM NaCl, 50 mM MOPS/Tris pH 7.0) to a final concentration of 4 mg·mL⁻¹. The suspension was vortexed every 10 minutes for 1 hour to facilitate hydration, yielding multilamellar vesicles. The vesicles were aliquoted, flash-frozen in liquid nitrogen, and stored at -80 °C. Immediately before use, 400-µL aliquots were thawed and allowed to equilibrate at room temperature. The vesicles were solubilized by adding Triton X-100 (TX-100) under magnetic stirring for 15 minutes at room temperature. The detergent-to-lipid ratios were as follows: 2.5:1 (w/w) for POPC:POPS and 3:1 (w/w) for the ER-like mixture, resulting in final concentrations of 7 and 8 mg·mL⁻¹ TX-100, respectively, and ~2.7 mg/mL lipids. Next, 0.3 mol% of NBD-labeled lipids—C12-NBD-PS, C12-NBD-PE, or C12-NBD-PC (for dithionite assay), or C6-NBD-PS (for BSA back-extraction)—resuspended in 0.5 mg/mL⁻¹ DDM, were added to the lipid/TW-100 mixture, along with 1 mM EGTA. The purified protein was added to the detergent/lipid mixture at a 14.4 mg·mmol⁻¹ protein-to-lipid molar ratio (~0.05 mg·mL⁻¹ final protein concentration) and incubated for 30 minutes at room temperature. Detergent removal was performed by adding pre-washed SM-2 Bio-Beads (Bio-Rad) in three sequential steps (Rigaud and Lévy, 2003): first, the sample was incubated with 10 mg Bio-Beads/mg TX-100 for 2 hours, then a second of Bio-beads was added (10 mg/mg TX-100) and the sample was incubated for another 2-hours. Lastly, Bio-beads from the first two steps were removed and an additional 20 mg Bio-beads/mg TX-100 was added for 1 hour to the sample. The resulting proteoliposomes were separated from the beads and stored at 4 °C for up to one week.

766
767 Sucrose gradient fractionation
768 Reconstitution was assessed using a discontinuous sucrose gradient, prepared with five sucrose solutions (5, 10, 20, 30 and 40%) prepared in buffer R. Each solution was layered in 2 mL increments in a centrifuge tube. Then, 350 µL of samples were carefully placed on top of the 5% sucrose layer and spun down at 160,000 g for 20 hours. After centrifugation, 500 µl fractions were collected from the top to evaluate lipid, protein and sucrose concentration. Sucrose concentration was measured with a hand refractometer, protein incorporation checked by Western blotting using an anti-mCherry antibody and lipids were tracked by NBD-fluorescence. For the dithionite transport assay of proteoliposomes separated on a sucrose gradient, 250 µL fractions were collected from the top of the gradient and submitted to dithionite quenching.

771
772
773
774
775
776
777
778 Cryo-EM of proteoliposomes
779 Samples were prepared with 3 µL of liposomes or proteoliposomes diluted 2-fold in buffer R, spotted onto Lacey grids and vitrified by plunge-freezing performed with an in-house guillotine. Blotting time and force were adjusted on a per sample basis. Images were acquired with a Tecnai Spirit 120 kV LaB6 FEI microscope operated at 120 kV equipped with a K2 Base camera (Gatan).

781
782
783
784
785 Dithionite scramblase assay
786 A 20-µL aliquot of either liposomes or proteoliposomes was diluted to a final volume of 2 mL in buffer R supplemented with either 1 mM EGTA (8 nM free Ca²⁺ concentration) or without EGTA and with 1 mM CaCl₂. Then, the fluorescence of NBD lipids was monitored over time in a quartz cuvette, using excitation at 470 nm and emission at 530 nm. After 200 seconds, freshly prepared sodium dithionite was introduced at a final concentration of 20 mM and the bleaching of NBD-lipids was followed for ~800 s. At t=900 s, 0.1% (w/w) TX-100 was added to solubilize the vesicles and to allow complete reduction of the NBD. Data acquisition was done using a Fluorolog®-3 Spectrofluorometer (HORIBA Instruments Incorporated) packed with FluorEssence™ software. All fluorescent points were normalized (Fₙorm) to the background fluorescence using the following formula: Fₙorm = (F - Fₐend)/(Fₐstart - Fₐend) × 100, where F is the
796 intensity at each time point, Fstart is the intensity just before dithionite addition and Fend is the
797 intensity after TX-100 addition.

798
799 BSA back extraction assay
800 For BSA back-extraction, C6-NBD-PS was used during protein reconstitution. When bound to
801 BSA, C6-NBD-PS exhibits a fluorescent quantum yield of 0.55 compared to its fluorescence in a
802 liposomal lipid environment. The BSA back-extraction was performed in the same set-up as the
803 dithionite assay, using a Fluorolog®-3 Spectrofluorometer (HORIBA Instruments Incorporated)
804 packed with FluorEssence™ software. However, the NBD fluorescence was monitored for 1800
805 s and instead of dithionite, fatty acid-free BSA (Sigma A7030) at final concentration of 0.25
806 mg.mL⁻¹ was introduced after 200 seconds of equilibration. The extraction of the NBD-labelled
807 phospholipids was continuously followed for 1200 s and 20 µL of 0.1 % (w/w) TX-100 and 40 µL
808 of 1 M sodium dithionite were finally added to bleach NBD fluorescence. As for the dithionite
809 assay, the fluorescent points were normalized (Fnorm) to the background fluorescence using the
810 following formula: Fnorm = (F - Fend)/(Fstart - Fend) × 100, where F is the intensity at each time
811 point, Fstart is the intensity just before BSA addition and Fend is the intensity after TX-100 and
812 dithionite addition.

813
814
815
816 Iodide collisional quenching
817 A 10-µL sample of liposomes or Ist2 proteoliposomes was diluted to a final volume of 2 mL in
818 Buffer R supplemented with 40 mM Na₂S₂O₃ (to preserve iodide ions in the solution) and
819 concentrations of KI ranging from 0 to 0.2 M. KCl was used to adjust the ionic strength to 0.2 M.
820 NBD-fluorescence was followed by setting excitation and emission wavelengths at 470 nm and
821 530 nm, respectively. The mean fluorescence value recorded over the first 5 minutes was taken
822 and used to plot ΔF (F₀-F). The data were analysed according to the modified Stern-Volmer
823 equation (Vehring et al, 2007; Goren et al, 2014): F₀/ΔF = (1/fₐ · K[Q]) + (1/fₐ) where F₀ is the
824 fluorescence intensity in the absence of the quencher, ΔF is the fluorescence intensity in the
825 presence of the quencher at concentration [Q] subtracted from F₀, fₐ represents the fraction of
826 fluorescence accessible to iodide ions and K is the Stern-Volmer quenching constant.
827
828
829 Determination of the oligomeric state of purified Ist2ΔC by Flow-Induced Dispersion
830 Analysis (FIDA)
831 For FIDA, a construct where the disordered C-ter tail of Ist2 is truncated (Ist2ΔC) and with
832 Ist2ΔC fused to eGFP and a Twin-Strep tag (TwS) was used (Ist2-ΔC-eGFP-TwS). Purified
833 Ist2ΔC-eGFP-TwS was diluted to 50 nM in buffer A (MOPS-Tris 50 mM pH 7, KCl 100 mM,
834 MgCl₂ 1 mM) supplemented with 0.5 mg/mL DDM. The FIDA was conducted on a capillary
835 electrophoresis instrument (Pedersen et al., 2019). The samples ran on a standard uncoated
836 glass capillary treated beforehand with FIDAbio Hydrophilic Solution (HS) to prevent stickiness.
837 Treated capillary was then equilibrated with buffer A supplemented with DDM. The capillary and
838 the samples were temperature-controlled to 25 °C inside the PrinCE instrument. Fluorescence
839 was detected after excitation at 488 nm. The Taylorsgrams were processed using FIDA data
840 analysis software (version 3.0), assuming the presence of at least 2 components, and with
841 viscosity correction set at 1.8, in order to calculate experimental hydrodynamic radius (Rh) of the
842 major species. Experiments were repeated thrice. The CHARMM-GUI micelle builder online tool
843 was used to construct protein/detergent complexes, using an AlphaFold 3 (AF3) model of Ist2ΔC
844 as a template (Abramson et al., 2024): AlphaFold3 models of Ist2ΔC-eGFP-TwS were made,
845 considering both monomeric and homodimeric forms. To achieve micelle construction around
846 membrane protein structural models, we resorted to the micelle builder package from CHARMM-
847 GUI (Cheng et al., 2013). The protein/DDM complexes were built through trial and error to

848
849 Determination of the oligomeric state of purified Ist2ΔC by Flow-Induced Dispersion
850 Analysis (FIDA)
851 For FIDA, a construct where the disordered C-ter tail of Ist2 is truncated (Ist2ΔC) and with
852 Ist2ΔC fused to eGFP and a Twin-Strep tag (TwS) was used (Ist2-ΔC-eGFP-TwS). Purified
853 Ist2ΔC-eGFP-TwS was diluted to 50 nM in buffer A (MOPS-Tris 50 mM pH 7, KCl 100 mM,
854 MgCl2 1 mM) supplemented with 0.5 mg/mL DDM. The FIDA was conducted on a capillary
855 electrophoresis instrument (Pedersen et al., 2019). The samples ran on a standard uncoated
856 glass capillary treated beforehand with FIDAbio Hydrophilic Solution (HS) to prevent stickiness.
857 Treated capillary was then equilibrated with buffer A supplemented with DDM. The capillary and
858 the samples were temperature-controlled to 25 °C inside the PrinCE instrument. Fluorescence
859 was detected after excitation at 488 nm. The Taylorsgrams were processed using FIDA data
860 analysis software (version 3.0), assuming the presence of at least 2 components, and with
861 viscosity correction set at 1.8, in order to calculate experimental hydrodynamic radius (Rh) of the
862 major species. Experiments were repeated thrice. The CHARMM-GUI micelle builder online tool
863 was used to construct protein/detergent complexes, using an AlphaFold 3 (AF3) model of Ist2ΔC
864 as a template (Abramson et al., 2024): AlphaFold3 models of Ist2ΔC-eGFP-TwS were made,
865 considering both monomeric and homodimeric forms. To achieve micelle construction around
866 membrane protein structural models, we resorted to the micelle builder package from CHARMM-
867 GUI (Cheng et al., 2013). The protein/DDM complexes were built through trial and error to
847 determine the maximum number of detergent molecules needed to accommodate the
848 transmembrane domains of monomeric or dimeric Ist2. Note that CHARMM-GUI returns an error
849 message if the amount of detergent is excessive. FIDA PBD correlator tool was then used to
850 calculate theoretical Rh from the models. Theoretical Rh were then compared to experimental
851 Rh obtained by FIDA.

852
853 **Molecular models of the protein**
854 Since there is no crystallography or cryo-EM structure for the yeast Ist2 protein in the protein
855 data bank (PDB), homology modeling techniques were employed to predict 3D models for the
856 transmembrane domain (TMD) of Ist2. HHpred (pairwise comparison of profile hidden Markov
857 models (HMMs)), an online enactment of HHsearch at the Tuebingen toolkit (Gabler et al.,
858 2020), was used for profile-profile searches for TMD of Ist2 homologs in "PDB_mmCIF70"
859 database, using default parameters. HHpred revealed 25%, 24%, 20%, and 16% identities
860 between the TMD amino acid sequence of Ist2 and afTMEM16 (PDB ID: 6E1O), nhTMEM16
861 (PDB ID: 4WIS), hTMEM16K (PDB ID: 5OC9), and mTMEM16A (PDB ID: 5OYB), respectively.

862 We used locally installed AlphaFold2 (Jumper et al., 2021) with default structure prediction
863 pipeline, where three different Multiple Sequence Alignments (MSAs) generated by searching
864 the Big Fantastic Database (BFD), MGnify and Uniref90 with jackhmmer from HMMER3.3.2
865 (Eddy, 2011). During modelling, relaxation was turned off. The locally installed AlphaFold2 gave
866 us homodimer of TMD with open conformation, similar to the crystal structure of nhTMEM16
867 (PDB ID: 4WIS). In order to sample alternative conformations of TMD, ColabFold with different
868 MSA sizes were used by modifying the "max_msa clusters" and "max_extra_msa" parameters.
869 We obtained different conformations of TMD, but none of them represented the closed state of
870 TMD.

871 Then we modelled Ist2 TMD using I-TASSER and trRosetta web servers, using fully automated
872 protein structure prediction pipeline. Among all generated models, only models provided by
873 trRosetta tend to be very similar to the closed state conformation. Each model's similarity to the
874 experimentally open (PDB ID: 4WIS) and closed (PDB ID: 6QMB) conformation were quantified
875 using TM-score. MODELER v10.1 (Webb and Sali, 2016) was then used to build the homodimer
876 model of the closed state using the PDB ID: 6QMB as template. Two trRosetta made promoters
877 were superimposed onto 6QMB structure (RMSD=1.101 Å) using the UCSF Chimera software
878 (Pettersen et al., 2021) (MatchMaker tools) and used as input for the MODELER program. One
879 hundred models were generated, and the one with the lowest discrete optimized protein energy
880 (DOPE) score was adopted for MD simulations. In conclusion, two TMD models were built,
881 representing the "closed" obtained from trRosetta and the "open" state obtained from
882 AlphaFold2. Both models were used in subsequent MD simulations.

883
884 **All-atom molecular dynamics simulations**
885 Prior to MD simulations, PROPKA 3.2 (Olsson et al., 2011) was used to determine the
886 protonation states of ionizable residues. As a result, standard protonation states at neutral pH
887 were assigned to all residues, except for protonated H506, which are located at the dimer
888 interface and form salt bridges with residues E505 of the other protomer. For both the open and
889 closed structural model, we built a Ca²⁺-bound and a Ca²⁺ unbound form, hence 4 systems were
890 built. Each protein model was embedded into two different lipid mixtures mimicking the ER
891 composition using the membrane builder module of CHARMM-GUI server (Lee et al., 2016).
892 Lipids were symmetrically distributed between the leaflets. For details of bilayer compositions,
893 see Suppl. Table 1. The TIP3P water model was used to solvate the system, and 0.15 M KCl
894 were added to neutralize the system. The resulting systems contained ~707,000 atoms in total
895 and extended by 21 nm*21 nm*18 nm in XYZ directions.

896 We carried out MD simulations using the GROMACS 2019.2 molecular dynamics program
897 package with the CHARMM36m all-atom force field. Periodic boundary conditions were applied
in all directions. Non-bonded interactions were calculated using the Verlet cutoff scheme, and a buffer tolerance of 0.005 kJ/mol; a cut-off of 12 Å was applied for non-bonded interactions. The particle mesh Ewald (PME) approach was employed for long-range electrostatic interactions, with a Fourier grid spacing of 1.2 Å. The LINCS algorithm was used to constrain bond lengths involving the hydrogen atoms.

After standard energy minimization using the steepest descent algorithm, the resultant systems were pre-equilibrated for 250 ns in isothermal-isobaric ensemble (NPT), in this step backbone atoms of protein were kept fixed with a harmonic positional restraints potential of 2000 kJ mol⁻¹ nm⁻² which allowing other molecules to adjust with protein. Following pre-equilibration, all the systems were equilibrated for 250 ns with a harmonic positional restraints potential of 50 kJ mol⁻¹ nm² (in the same NPT ensemble). The temperature was held at 298 K with a v-rescale thermostat (Bussi et al., 2007), using separate temperature coupling for the solvent and solute (protein and membrane). The time constant for temperature coupling was set to 1 ps. During equilibration, the pressure was kept constant using the semi-isotropic Berendsen barostat (Bernetti and Bussi, 2020)] set to 1 bar using a 10 ps coupling constant and compressibility 4.5 × 10⁻⁵ bar⁻¹. The equations of motion were integrated using the leap-frog algorithm, with an integration time step of 2 fs.

Production runs were performed for 1 µs with the same parameters as the equilibration described above, except that the pressure was maintained at 1 bar using the semi-isotropic Parrinello-Rahman barostat (τP = 10 ps and compressibility = 4.5 × 10⁻⁵ bar⁻¹). In addition, cylindrical (in parallel to the z-axis) flat-bottomed restraints were applied on protein atoms in lipid bilayer, using a force constant of 2000 kJ mol⁻¹ nm⁻², and a cylinder radius of 9.8 nm, to prevent the protein from crossing the periodic boundary and move out of the simulation box. This setup allowed completely free internal motion of the protein, as well as free translation within a radius of 9.8 nm from the center of mass of the protein (the radius of the protein is about 5.6 nm), which simplified the analysis of the simulations.

## Coarse-grained (CG) MD simulations

CG models were built using the predicted structure for the open and closed states of Ist2, after energy minimization. The minimized atomistic structures were mapped to a CG representation using the Martinize2 (Kroon et al., 2024) with Martini force field (v3.0) (Souza et al., 2021). DSSP (https://github.com/cmbi/dssp) was used to determine the secondary structure. To stabilize the protein's tertiary and higher-order structures, we applied a network of Gō potentials between the backbone (BB) beads using the GōMartini3 approach (Souza et al., 2024). Contact maps were generated from http://pomalab.ippt.pan.pl/GoContactMap/ using the OV + rCSU variant, which accounts for both van der Waals (vdW) radii overlap (OV) and repulsive chemical structural units (rCSU), incorporating the chemical properties of the atoms in contact. The depth of the Lennard-Jones potential, in the Gō-like model, were ε = 12.0 kJ/mol and 11.0 kJ/mol in the open and closed states, respectively.

We used the insane.py python script to embed the CG protein into a lipid bilayer (Wassenaar et al., 2015). Systems were solvated using the standard Martini water model and neutralized with 0.15 M KCl. The box dimensions of the system were 20 × 20 × 17 nm³. Each system consisted of ~58,000 CG beads.

CG simulations of Ist2 (open and closed states; Ca²⁺-unbound form) were performed in 2 different lipid mixtures mimicking the ER composition (see table 1), for a total of 4 systems. Control systems of both membranes without Ist2 protein (membrane only system) were also prepared. The details of the simulations performed, with their system composition are summarized in Table 2.

All simulations were carried out with the Gromacs software (Abraham et al., 2015), version 2019.

Non-bonded interactions were calculated using the Verlet cutoff scheme, and a buffer tolerance of 0.005 kJ/mol; the interaction cutoff of 1.1 nm. The reaction field approach was used for long-
range electrostatic interactions, with a Coulomb cut off of 1.1 nm and a Potential shift Verlet modifier, as implemented in Gromacs.

Energy minimization was carried out using the steepest descent algorithm for 50,000 steps, followed by two 250-ns equilibrations, during which the backbone atoms from the protein structure were positionally restrained ($k = 1000 \text{ kJ mol}^{-1} \text{ nm}^{-2}$ and $100 \text{ kJ mol}^{-1} \text{ nm}^{-2}$, respectively). The temperature and pressure were controlled with a velocity-rescale thermostat (Bussi et al., 2007) (reference temperature $T = 298 \text{ K}$, coupling constant $\tau_T = 1 \text{ ps}$) and a Parrinello-Rahman semi-isotropic barostat ($p = 1 \text{ bar}$, $\tau_p = 12 \text{ ps}$, compressibility $\beta = 3 \times 10^{-4} \text{ bar}^{-1}$), respectively. Solvent and solute (lipids and protein) beads were coupled separately to the temperature bath. The leap-frog integrator was used, with a time step of 20 fs.

After these two equilibration steps, production runs were carried out in 3 independent replicas, with different initial velocities. Each run was carried out for 20 µs, resulting in a total of 60 µs per system, and 360 µs in total (2 protein systems and one membrane-only as a control). Simulation parameters were as above, except for the absence of position restraints on the protein. Coordinates were saved every 500 ps, resulting in 100,000 snapshots per trajectory for subsequent analysis.

To investigate the effect of mutations on the scrambling function of Ist2, we generated two mutants, Ist2_6A and Ist2_6W, by introducing six point mutations on transmembrane helices TM3 (L229A/W, E240A/W), TM4 (T267A/W), and TM6 (R359A/W, Q363A/W, Y366A/W). The all-atom structures of the mutants were predicted using AlphaFold3 (AF3) (Abramson et al., 2024). AF3 predicted an open, dimeric conformation of Ist2 for both the 6A and 6W mutants. These structures were then converted into CG representations using Martinize2 with the Martini 3.0 force field. Gō-like potentials were applied using the GōMartini3 approach, as in the wild-type (WT) simulations (Souza et al., 2021). Mutant simulations were performed only in the complex ER-like lipid mixture. The system setup, including membrane embedding, equilibration protocols, and production runs, followed the same procedure as for the WT systems. Each system was simulated in three independent replicas of 20 µs, resulting in a total of 60 µs per mutant.

### Simulation analysis

Prior to analysis, trajectories were translationally and rotationally fitted to the protein backbone (BB beads in Martini proteins, see (Monticelli et al., 2008) using Gromacs tools (gmx trjconv).

For protein-lipid contact analysis we used the Prolint (Sejdiu and Tieleman, 2021) package. For scrambling analysis, we used the MDanalysis library (Gowers et al., 2016), the LiPyphilic tool (Smith and Lorenz, 2021), and in-house Python scripts. To quantify lipid flip-flop events, we monitored the z-coordinate (direction of the membrane normal) of the PO4 group of each lipid over the entire simulation trajectory. An individual lipid molecule was determined to belong to the upper (lower) leaflet if the z coordinate of its PO4 bead (representing the phosphodiester group in phospholipids) at $t = 0$ of the production run was higher (lower) than the z coordinate of the membrane center of mass. A complete scrambling event was defined as the movement of a PO4 group from one leaflet to the opposite leaflet, followed by its sustained presence there for a duration of 5 nanoseconds. Statistical uncertainties are reported in tables 1-4 as the standard error on each metric based on 3 independent samples (i.e., the 3 independent simulation replicas obtained for each system).

The bilayer thickness over both leaflets was calculated by means of the g_lomepro tool (Gapsys et al., 2013). The distances between the PO4 beads of lipids located in the upper and lower leaflets with respect to the z-axis were considered as references, using a grid of 100 × 100 points to define their location. Visual Molecular Dynamics (VMD) version 1.9.3 (Humphrey et al., 1996) was used for visual inspection.

### Yeast strain construction and manipulation
1000 Yeast strains are listed in Supplemental Table S2. Yeast were grown in rich medium (YPD) or in
1001 synthetic dextrose (SD) medium containing 2% (wt/vol) glucose and appropriate amino acid
1002 drop-out mix (MP Biomedicals). Transformations were performed using the standard lithium
1003 acetate/polyethylene glycol procedure. SD medium was supplemented with 1 mM ethanolamine
1004 when growing *cho1Δ* strain. Deletion and C-ter truncation strains were generated by
1005 homologous recombination using appropriate marker (Janke et al., 2004), as indicated. To
1006 generate the strains *ist2ΔN*, *ist2ΔC*, *ist2-6A*, and *ist2-6W* in *ist2Δ* and in *csf1ΔC* *ist2Δ*
1007 background, we employed a marker-less CRISPR-Cas9 genomic editing system targeting the
1008 *HygMX* antibiotic cassette (Soreanu et al., 2018) inserted at the *IST2* locus, retaining promotor
1009 and 3'UTR regions. Hygromycin-sensitive colonies were verified by PCR and sequencing.
1010 Chromosomal C-terminal GFP-tagging of Ist2 WT and mutant versions, as well as of Erg6, was
1011 performed by homologous recombination using a *GFP::HIS3* cassette in frame with the coding
1012 sequence of the target protein. Gal-induced *CHO1* deletion strains were generated by replacing
1013 the *CHO1* locus with a GALpr-*CHO1::NatMX* cassette via homologous recombination. Strains
1014 used in Figure 7 were generated by crossing *ist2Δ* Erg6-GFP (BY4741) with *get1Δ::NatMX*
1015 (BY4742) or *ist2Δ* Erg6-GFP (BY4742) with *get2Δ::kanMX* (BY4741), followed by sporulation
1016 and tetrad dissection.
1017

1018 **Reagents and standards for lipid analysis**
1019

1020 Lipid standards (reference, abbreviation) were acquired from Sigma and Avanti Polar Lipids: Egg
1021 and 17:1 L-α-lyso-phosphatidylcholine (830071, 855677 LPC), egg or 16:0-18:1 L-α-
1022 phosphatidylcholine (840051, 850457 PC), brain or 16:0-18:1 L-α-phosphatidylserine (840032,
1023 840034 PS), liver or 16:0-18:1 L-α-phosphatidylinositol (840042, 850142 PI), heart cardiolipin
1024 (840012, CL), egg or 16:0-18:1 L-α-phosphatidic acid (840101, 840857 PA), liver or 16:0-18:1 L-
1025 α-phosphatidylethanolamine (840026, 850757 PE), egg L-α-phosphatidylglycerol (841138, PG),
1026 N-24:0 (2S-OH) Phytosphingosine C24 (860925, phytoSp), egg ceramide (860051, Cer), 5-
1027 cholesten-3-β-ol (C8667, Cho), cholest-5-en-3β-yl heptadecanoate (700186, SE), cholesteryl
1028 formate (S448532, ChoF), arachidonic acid (A3611, FFA), 1-oleoyl-rac-glycerol (330724, MG),
1029 1,3-di(cis-9-octadecenoyl)-glycerol (D3627, DG1.3), 1,2-dipalmitoyl-sn-glycerol (800816,
1030 DG1.2), 1,2,3-tri-(9Z-octadecenoyl)-glycerol (T7140, TG or TG18:1), 1,2,3-
1031 tritetradecanoylglycerol (T5141, TG14:0). Chloroform (CHCl₃), methanol (MeOH) and hexane
1032 (Hex) were HPLC grade; toluene (Sigma, 244511); ethyl acetate (VWR, 23880.290, EtAc). Other
1033 reagents: butylhydroxytoluene (Sigma, 34750, BHT), ammonium molybdate tetrahydrate (Sigma,
1034 A7302), ascorbic acid (Sigma, 33034), sodium dihydrogen phosphate dihydrate (28011.291),
1035 perchloric acid 65-71% (Carlo Erba, 409193), nitric acid 65% (RP Normapur), sulphuric acid
1036 95% (RP Normapur, 20700), phosphoric acid (Sigma, 1.00565), ammonium formate (99%,
1037 Acros Organics), copper(II) sulphate pentahydrate (Sigma, C8027).
1038

1038 **Cell growth, lipid extraction and phospholipid quantification**
1039

1040 Samples were prepared following the protocol described by (Klose et al., 2012), with a minor
1041 modification: cells were cultured in a 1:2.5 liquid-to-air ratio. Briefly, overnight cultures were
1042 adjusted to an OD₆₀₀=0.15 in synthetic complete medium using drop-out mix – Ura (MP
1043 biochemicals), supplemented with uracil. Cells were harvested by centrifugation in mid-
1044 logarithmic phase (OD₆₀₀=0.6), washed once and resuspended in 155 mM ammonium
1045 bicarbonate solution. Pellets were snap-frozen in liquid nitrogen, within 12 min from the first
1046 centrifugation. Lipid extraction was performed using the MTBE method (Matyash et al., 2008),
1047 with modifications. Briefly, 200 µL water was added to samples. After vortexing, samples were
1048 transferred into 2 mL plastic tubes with O-ring screw caps containing 0.5 mm glass beads,
1049 previously rinsed in MeOH. 300 - 400 µL of MeOH was added to samples and they were
1050 homogenized in a Precellys Evolution 2x 20s, 5000 rpm. Then, samples were transferred into
glass tubes and the homogenization tubes were rinsed with MeOH by vortexing. MTBE was
added to the glass tubes, followed by vortexing at 4°C for 30 min. All extraction solvents contained 50 µg/mL BHT prepared fresh. Extracted samples were kept dried at -20°C under Ar. Phospholipids were quantified using the (Rouser et al., 1970) method, with scaled-down volumes as described in (Dias Araújo et al., 2025).

### 1055 Lipid class separation by high performance thin layer chromatography (HPTLC)

For separation of lipid classes, HPTLC instrumentation was used and operated through visionCATS software version 3.0.20196.1. Unless mentioned, instrumentation parameters are as described in (Dias Araújo et al., 2025). PL were dissolved in chloroform as 2 µg/µL and applied (spray 150 nL/s) onto Merck HPTLC glass plates coated with silica gel 60 only (Fig. 4B, 4G) (20x10 cm, layer thickness 200 µm) or embedded with F254 (Fig. 4E) using an automatic TLC sampler 4 (ATS4, CAMAG). Standards were prepared at 1 µg/µL. 2 biological replicates were applied per condition.

To separate polar and non-polar lipids in a single plate, plates were eluted, dried, derivatized and imaged as described in (Dias Araújo et al., 2025). To separate neutral lipid classes only, the plate was washed in CHCl₃:MeOH (1:1, V:V) in an automated developing chamber (ADC2, CAMAG) up to 85 mm and activated before use (15 min, 100°C). After sample application, the plate was eluted in an automated multiple development chamber (AMD2, CAMAG) with 4 different solvent mixes (V:V) in the following order: 1) CHCl₃: Hexane: MeOH (45:52:3) up to 55 mm, with MeOH pre-conditioning, 2) Hexane: diethyl ether (80:20) up to 70 mm, 3) Hexane: diethyl ether: acetic acid (80:20:2) up to 72 mm, and 4) Hexane (100%) up to 80 mm. Mix 3 was prepared in one single bottle and used as 100%. Drying time between elutions was 3 min. Images of the 3 plates were treated in Fiji. The areas of the peaks of interest on the chromatograms generated by Fiji were manually delimited and were used to determine the relative lipid composition per lane.

### 1076 Live yeast cell imaging

Yeast cells were grown for 14–18 h in appropriate SD medium at 30°C, except for *sec23-1* where cells were grown at 25°C. Cells were diluted and harvested by centrifugation in mid-logarithmic phase (OD₆₀₀=0.6–0.8) and prepared for visualization on glass slides. For LD counting and size measurements, cells were grown to early stationary phase (1.2 < OD₆₀₀ < 2.2).

For the pKE41 probe localization, cells were grown for 24h prior to observation. When indicated, LD staining was obtained by incubating cells with 1 mg/mL of BODIPY® 493/503 (ThermoFisher) or with 0.1 mM of AUTODOT™ (Cliniscience) for 30 min. For rapamycin-induced autophagy, cells were grown to early exponential phase (OD₆₀₀=0.3) before being treated with 200 nM of rapamycin for 3h (Liu et al., 2024). Imaging was conducted at room temperature, using a Spinning disk SR system (Olympus), equipped with an oil immersion plan apochromat 60x objective NA 1.42, an sCMOS Fusion BT camera (Hamamatsu) and a spinning-disk confocal system CSU-W1 (Yokogawa). For LD counting and size measurements, as well as imaging GFP-tagged versions of Ist2, the SoRa spinning-disk module was employed. AUTODOT, BODIPY/GFP-tagged proteins and mScarlet-tagged proteins were visualized with DAPI (447/50), GFP (525/50), and mCherry (593/40) filters, respectively. Cells were imaged in 3 to 15 z-sections separated by 0.25 µm. Images were acquired using CellSens software (Olympus) and processed with Fiji (ImageJ).

### 1097 Image analysis

Images were processed with Fiji (ImageJ). For the rapamycin-induced autophagy experiment, the number of foci per cell was manually counted from stacks of 3–5 z-sections. LD counting, LD size measurements, and estimation of LD fluorescence-positive cells using the Plin4-AH probe were performed manually from stacks of 15 z-sections. Steady-state distribution of C2Lact-GFP
1102 was analyzed from stacks of 3 z-section of each cell. The quantification of peripheral peaks
1103 (mostly PM and cortical ER signal) and internal (mostly perinuclear ER) was performed by
1104 profiling cell signal intensity across a transversal line drawn in the cell with an automated macro.
1105 Intensities of the peaks were quantified and normalized relative to the total signal as previously
1106 described (D'Ambrosio et al., 2019, 2020).

1107
1108 Viability and kinetic assays

1109 Yeast cells were grown for 14–18 h at 30°C in appropriate medium (YPD or SD medium), except
1110 for *sec23-1*, which was cultured at 25°C. For drop-test assays, cells were adjusted to the lowest
1111 OD₆₀₀, serially diluted, and plated onto the appropriate agar media. Plates were incubated for 2-
1112 6 days at designated temperatures. For growth kinetic, cells were adjusted to an OD₆₀₀=0.1 in
1113 the appropriate SD medium, and grown in technical and in biological duplicates, in 96-well
1114 microplates using the Spark® microplate reader (Tecan). For the *ist2Δpsd1Δ* strain, cells were
1115 supplemented with 2mM of ethanolamine overnight before the kinetic, in presence or absence of
1116 ethanolamine. For *SEC-AID* strains (Gameiro et al., 2025), cells were grown for 4h prior to
1117 indole-3-acetic acid (IAA) addition. IAA was dissolved in DMSO and stored as single-use
1118 aliquots; the same volume of DMSO was added as a negative control. Different concentrations
1119 of IAA were tested, between 0 and 50 µM. Each kinetic cycle consisted of 9 min of shaking
1120 (2mm amplitude, 150 rpm) followed by a 1-min settling period before measuring the absorbance
1121 at 600nm. Measurements were taken every 10 min and continued until control cultures reached
1122 the plateau of the stationary phase.

1123
1124 Pulse-chase analysis

1125 Pulse-chase analysis of CPY maturation was performed essentially as described (Copic et al.,
1126 2012). Strains were grown in SD-Ura medium to midlog phase, starved for 15 min, pulsed for 5
1127 min with ³⁵S-methionine, then chased for 0 to 55 min with unlabelled amino acids. Samples were
1128 removed at indicated time-points and CPY was immuno-precipitated from cell lysates using a
1129 mouse monoclonal antibody (ThermoFisher clone 10A5B5, Cat# A-6428). The mature and
1130 precursor forms of CPY were separated by SDS-PAGE and detected on a phosphorimager
1131 screen using a Typhoon scanner (Cytiva).

1132
1133 Western blot analysis of total yeast extracts

1134 Proteins were extracted from one OD₆₀₀ equivalent of yeast cells grown to mid-logarithmic phase
1135 in appropriate SD medium. Protein samples were heated at 55°C for 10 min and 30 µL were
1136 loaded on SDS-PAGE (4–20% Mini-PROTEAN TGX Stain-Free, Bio-Rad for Supp Fig. 4C and
1137 10% homemade gel for Supp Fig. 4D) and analyzed by western blotting. Briefly, nitrocellulose
1138 membranes were saturated with 5% of milk, incubated ON at 4°C with primary antibody (1:1000,
1139 anti-Halo, Promega G9211, and anti-RFP, Chromotek 6G6), washed with TBS-Tween20,
1140 incubated 1h at RT with secondary antibody (Invitrogen™ AlexaFluor™ anti-mouse 568, or anti-
1141 rabbit 488 respectively), washed with TBS-Tween20 and revealed with an Odyssey imaging
1142 system.

1143
1144 Statistical analysis

1145 For cross-analysis a Shapiro test was used to control the normality of the values and a Bartlett
1146 test for the homogeneity of variances. Kruskal-Wallis test followed by Dunn's post-hoc test was
1147 then used to test the equality of ranks across conditions. The representation of p values is as
1148 follows: ns, non-significant *<0.05, **<0.01, ***<0.001, ****<0.0001. The nomenclature (N=x)
1149 refers to the number of biological replicates and (n=x) refers to the number of measurements per
1150 replicate.
1151

1152
<table><thead><tr><th colspan="2">Lipid composition</th><th colspan="3">Total events / simulation (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage</th><th>R1</th><th>R2</th><th>R3</th><th>average ±<br>SD</th><th>Flip-flops per<br>ms per Ist2<br>dimer</th><th>Phospholipid<br>preference<br>index<sup>(1)</sup></th></tr></thead><tbody><tr><td>DYPC</td><td>50%</td><td>67</td><td>39</td><td>39</td><td>48 ± 16</td><td>2417 ± 808</td><td>1.3</td></tr><tr><td>DYPE</td><td>25%</td><td>13</td><td>6</td><td>6</td><td>8 ± 4</td><td>417 ± 202</td><td>0.5</td></tr><tr><td>DYPS</td><td>25%</td><td>21</td><td>12</td><td>12</td><td>15 ± 5</td><td>750 ± 260</td><td>0.8</td></tr><tr><td>Phospholipids</td><td>100%</td><td>101</td><td>57</td><td>57</td><td>72 ± 25</td><td>3583 ± 1270</td><td>1.0</td></tr></tbody></table>

<table><thead><tr><th colspan="2">Lipid composition</th><th colspan="3">Total events / simulation (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage<br>(phospholipids<br>only)</th><th>R1</th><th>R2</th><th>R3</th><th>average ±<br>SD</th><th>Flip-flops per<br>ms per Ist2<br>dimer</th><th>Phospholipid<br>preference<br>index<sup>(1)</sup></th></tr></thead><tbody><tr><td>DYPC</td><td>21%</td><td>17</td><td>18</td><td>10</td><td>15 ± 4.4</td><td>750 ± 218</td><td>2.3</td></tr><tr><td>YOPC</td><td>21%</td><td>9</td><td>9</td><td>3</td><td>7.0 ± 3.5</td><td>350 ± 173</td><td>1.1</td></tr><tr><td>DYPE</td><td>11%</td><td>4</td><td>1</td><td>2</td><td>2.3 ± 1.5</td><td>117 ± 76</td><td>0.7</td></tr><tr><td>YOPE</td><td>11%</td><td>2</td><td>0</td><td>2</td><td>1.3 ± 1.2</td><td>67 ± 58</td><td>0.4</td></tr><tr><td>YOPS</td><td>11%</td><td>5</td><td>3</td><td>3</td><td>3.7 ± 1.2</td><td>183 ± 58</td><td>1.1</td></tr><tr><td>POPI</td><td>21%</td><td>3</td><td>1</td><td>1</td><td>1.7 ± 1.2</td><td>83 ± 58</td><td>0.25</td></tr><tr><td>YOPA</td><td>5%</td><td>1</td><td>0</td><td>0</td><td>0.3 ± 0.6</td><td>16 ± 29</td><td>0.2</td></tr><tr><td>Phospholipids</td><td>100%</td><td>41</td><td>32</td><td>21</td><td>31 ± 10</td><td>1567 ± 501</td><td>1.0</td></tr><tr><td>ERGO</td><td>5%</td><td>34704</td><td>34878</td><td>35062</td><td>34 881 ±<br>180</td><td>1 744 070 ±<br>8950</td><td>n/a</td></tr></tbody></table>

<table><thead><tr><th colspan="2">Lipid composition</th><th colspan="3">Total events / simulation (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage</th><th>R1</th><th>R2</th><th>R3</th><th>average ±<br>SD</th><th>Flip-flops per<br>ms per Ist2<br>dimer</th><th>Phospholipid<br>preference<br>index</th></tr></thead><tbody><tr><td>DYPC</td><td>50%</td><td>2</td><td>1</td><td>2</td><td>1.7 ± 0.6</td><td>83 ± 29</td><td>n/a</td></tr><tr><td>DYPE</td><td>25%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>DYPS</td><td>25%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>Phospholipids</td><td>100%</td><td>2</td><td>1</td><td>2</td><td>1.7 ± 0.6</td><td>83 ± 29</td><td>n/a</td></tr></tbody></table>

<footnote>(1) Calculated by multiplying the average # of flip-flops with % of phospholipid species, divided by total # of flip-flop events.</footnote>

<footnote>(1) Calculated by multiplying the average # of flip-flops with % of phospholipid species, divided by total # of flip-flop events.</footnote>
<table><caption>Table 4. Scramblase activity of Ist2 TMD (closed state) in the complex lipid mixture.</caption><thead><tr><th colspan="2">Lipid composition</th><th colspan="4">Total events / simulation (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage<br>(Phospholipids<br>only)</th><th>R1</th><th>R2</th><th>R3</th><th>average ±<br>SD</th><th>Flip-flops per<br>ms per Ist2<br>dimer</th><th>Phospholipid<br>preference<br>index</th></tr></thead><tbody><tr><td>DYPC</td><td>21%</td><td>0</td><td>0</td><td>1</td><td>0.3 ± 0.6</td><td>17 ± 29</td><td>n/a</td></tr><tr><td>YOPC</td><td>21%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>DYPE</td><td>11%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>YOPE</td><td>11%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>YOPS</td><td>11%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>POPI</td><td>21%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>YOPA</td><td>5%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>Phospholipids</td><td>100%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>n/a</td></tr><tr><td>ERGO</td><td>5%</td><td>33156</td><td>33493</td><td>33269</td><td>33306 ± 172</td><td>1 665 300 ± 8576</td><td>n/a</td></tr></tbody></table>

<table><caption>Table 5. Scramblase activity of Ist2 TMD 6W (open state) in the complex lipid mixture.</caption><thead><tr><th colspan="2">Lipid composition</th><th colspan="4">Total events / simulations (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage<br>(PL only)</th><th>R1</th><th>R2</th><th>R3</th><th>Average ±<br>SD</th><th>Flip-flops per ms<br>per Ist2 dimer</th><th></th></tr></thead><tbody><tr><td>DYPC</td><td>21%</td><td>2</td><td>9</td><td>7</td><td>6 ± 3.6</td><td>300 ± 180</td><td></td></tr><tr><td>YOPC</td><td>21%</td><td>4</td><td>5</td><td>2</td><td>3.7 ± 1.5</td><td>183 ± 76</td><td></td></tr><tr><td>DYPE</td><td>11%</td><td>1</td><td>0</td><td>0</td><td>0.3 ± 0.6</td><td>17 ± 29</td><td></td></tr><tr><td>YOPE</td><td>11%</td><td>0</td><td>1</td><td>1</td><td>0.7 ± 0.6</td><td>33 ± 29</td><td></td></tr><tr><td>YOPS</td><td>11%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>POPI</td><td>21%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>YOPA</td><td>5%</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td></td></tr><tr><td>Phospholipids</td><td>100%</td><td>7</td><td>11</td><td>10</td><td>9.3 ± 2.1</td><td>467 ± 104</td><td></td></tr><tr><td>ERGO</td><td>5%</td><td>32192</td><td>31817</td><td>32223</td><td>32 077 ± 226</td><td>1 603 867 ±<br>11299</td><td></td></tr></tbody></table>

<table><caption>Table 6. Scramblase activity of Ist2 TMD 6A (open state) in the complex lipid mixture.</caption><thead><tr><th colspan="2">Lipid composition</th><th colspan="4">Total events / simulations (20 µs)</th><th colspan="2">Ist2 activity</th></tr><tr><th>Lipid type<br>(1200 lipids in<br>total)</th><th>Percentage<br>(PL only)</th><th>R1</th><th>R2</th><th>R3</th><th>Average ±<br>SD</th><th>Flip-flops per ms<br>per Ist2 dimer</th><th></th></tr></thead><tbody><tr><td>DYPC</td><td>21%</td><td>18</td><td>11</td><td>22</td><td>17 ± 5.6</td><td>850 ± 278</td><td></td></tr><tr><td>YOPC</td><td>21%</td><td>6</td><td>4</td><td>13</td><td>7 ± 4</td><td>383 ± 236</td><td></td></tr><tr><td>DYPE</td><td>11%</td><td>2</td><td>4</td><td>3</td><td>3 ± 1</td><td>150 ± 50</td><td></td></tr><tr><td>YOPE</td><td>11%</td><td>1</td><td>0</td><td>6</td><td>2 ± 3</td><td>117 ± 161</td><td></td></tr><tr><td>YOPS</td><td>11%</td><td>2</td><td>1</td><td>9</td><td>4 ± 4</td><td>200 ± 218</td><td></td></tr><tr><td>POPI</td><td>21%</td><td>2</td><td>0</td><td>1</td><td>1 ± 1</td><td>50 ± 50</td><td></td></tr><tr><td>YOPA</td><td>5%</td><td>0</td><td>0</td><td>1</td><td>0.3 ± 0.6</td><td>17 ± 29</td><td></td></tr><tr><td>Phospholipids</td><td>100%</td><td>31</td><td>20</td><td>55</td><td>35 ± 18</td><td>1767 ± 895</td><td></td></tr><tr><td>ERGO</td><td>5%</td><td>31974</td><td>32011</td><td>32574</td><td>32186 ± 336</td><td>1 609 317 ±<br>16812</td><td></td></tr></tbody></table>
Figure Legends
1172

**Figure 1. Reconstitution of Ist2 into proteoliposomes and dithionite scrambling assay. (A)**
Comparison of nhTMEM16 (4WIS) and afTMEM16 (6E1O) structures with that of Ist2 predicted by AlphaFold2. (B) Sequence alignment of Ist2 from *S. cerevisiae* with *N. haematococca* TMEM16 (nhTMEM16) and *A. fumigatus* TMEM16 (afTMEM16) (see also phylogenetic tree in Fig. S1A). Sequences were aligned using the MUSCLE server (EMBL-EBI). The predicted transmembrane helices (TM) of Ist2 based on its AlphaFold2 model are shown as grey cylinders. The residues critical for phospholipid scrambling in nhTMEM16 and the two prolines in TM4 serving as pivots for helix rearrangements, as well as the corresponding residues in afTMEM16 and Ist2, are highlighted in red and green, respectively (Jiang et al 2017; Lee et al, 2018; Kalienkova et al, 2019). Residues defining the Ca²⁺-binding sites in nhTMEM16 are highlighted in blue (Brunner et al, 2014). Conservation is shown below the sequences (brown – yellow = least – most conserved). Uniprot identifiers: Ist2 (P38250), nhTMEM16 (C7Z7K1), afTMEM16 (Q4WA18). (C) Cryo-electron micrographs of PLs containing Ist2 or the Drs2/Cdc50 lipid flippase complex. The average diameter is ~ 80-100 nm for both PLs Scale bar = 100 nm. (D) Schematic for the dithionite-based lipid transport assay. Unilamellar vesicles are formed in the presence of a trace amount of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-labeled phospholipid (NBD-phospholipid), which can be reduced by dithionite to a non-fluorescent 7-amino analog. In the case of lipid flip-flop, dithionite treatment of scramblase-reconstituted PLs should result in 100% fluorescence loss, whereas treatment of mock liposomes should result in 50% fluorescence loss as only NBD in the outer leaflet is available to react with dithionite. (E) NBD-PS scrambling assay. C12-NBD-PS-containing liposomes or PLs were stirred in a cuvette. The initial fluorescence was recorded for 200 s before dithionite addition. The shaded area around each trace represents the standard deviation for each fluorescent point. (F) Quantification of residual fluorescence in liposomes versus PLs measured 500s after dithionite addition (panel E). Data are a mean ± SD of 7 to 15 biological replicates (independent reconstitutions). (G) Collisional quenching of C12-NBD-PS fluorescence with iodide ions in liposomes and Ist2-containing PLs. The data are as modified Stern-Volmer plots (see Materials and Methods). F₀ is the fluorescence intensity of the sample in the absence of quencher, whereas F is the fluorescence intensity at a given iodide ion concentration. The fluorescence data points were fitted to a linear regression. The inverse of the y-intercept represents the fraction of C12-NBD-PS that is accessible to the quencher. Data are a mean ± SD of 3 biological replicates (independent reconstitutions) for both liposomes and Ist2-containing PLs. Some of the error bars are not visible because they are smaller than the symbol. The equation of the linear regression for liposomes is Y=0.5462X + 2.074, meaning that 48% of NBD-lipids are accessible to the quencher, whereas for Ist2-containing PLs, the equation is Y=0.2469X + 2.191, meaning that 46% of NBD-lipids are accessible to the quencher. (H) Western-blot analysis of fractions collected upon fractionation of Ist2-mCherry-containing PLs on a sucrose gradient. (I) NBD-PS quenching of fractions recovered upon fractionation of Ist2 PL on a sucrose gradient, compared to Ist2 PL and liposomes before fractionation. (J) NBD-PS quenching of fractions recovered upon fractionation of Drs2/Cdc50 PL on a sucrose gradient, compared to Drs2/Cdc50 PLs and liposomes before fractionation. (K) NBD-PS quenching of liposomes or proteoliposomes reconstituted with either full-length Ist2 or Ist2ΔC.

1216
1217
**Figure 2.** Lipid scrambling by the Ist2 dimer is not sensitive to Ca²⁺. (A) Ist2ΔC is purified
1218
1219 as a dimer. Top: Taylorsgram obtained with 50 nM Ist2ΔC-eGFP-TwS in the presence of 0.5
1220
1221 mg.mL⁻¹ DDM. FIDA measurements were performed in triplicate. Bottom: reconstruction of
protein/detergent micelles using CHARMM-GUI, taking either monomeric (left) or dimeric (right)
1222 Ist2ΔC-eGFP-TwS. Ist2 is colored yellow and GFP is in green. The detergent micelle is represented as an electrostatic surface. (B) Effect of Ca²⁺ on Ist2-mediated NBD-lipid scrambling. The effect of Ca²⁺ was assessed by adding 1 mM Ca²⁺ to both liposomes and Ist2-containing PLs. (C) Quantification of residual fluorescence in liposomes versus PLs upon dithionite quenching. The residual fluorescence measured in panel B at 500 s was plotted. Data are a mean ± SD of 5 biological replicates (independent reconstitutions). (D) Comparison of the region involved in Ca²⁺ binding between Ist2, afTMEM16, and nhTMEM16. (Only 2 out of 5 negatively charged residues involved in Ca²⁺ binding in TMEM16 are conserved in Ist2 – see Fig. 1B). (E) Scramblase activity of Ist2 in ER-like liposomes. Full-length Ist2 was reconstituted in liposomes made of DOPC (40 mol%), DOPS (20 mol%), DOPE (20 mol%), DOPA (5 mol%), Soy PI (10 mol%) and ergosterol (5 mol%). Normalized fluorescence time-courses upon dithionite bleaching of C12-NBD-PS. (F) Quantification of residual fluorescence in mock liposomes versus Ist2 PLs measured 500 s after dithionite addition. Data are mean ± SD from 3 independent reconstitutions. (G) Illustration of the mechanism of scrambling of a PS lipid by Ist2. The protein is shown in green, the position of the lipid membrane is shaded in yellow, and one lipid is shown in 4 different time frames, corresponding to 4 different positions along the scrambling path. The lipid is shown with a thick stick representation and the serine head group is represented as a larger sphere, to highlight the flip-flop about half-way through the scrambling path. For a dynamic representation see the movie in supporting information. (H) Scramblase activity of Ist2 in NBD-PC and NBD-PE liposomes or NBD-PC and NBD-PE scrambling assays. Traces represent the mean of 3 to 5 transport experiments from independent reconstitutions. (I) Quantification of residual fluorescence in liposomes versus Ist2 PLs upon dithionite quenching. The residual fluorescence measured in panel H at 500 s was plotted. Data are a mean ± SD of 3-5 biological replicates (independent reconstitutions).

1246
1247 **Figure 3. Mechanistic analysis of phospholipid scrambling by Ist2.** (A) The path followed by the lipid head groups during scrambling, predicted on the basis of lipid-protein contact during unbiased CG MD simulations. In the left panel, amino acids are colored based on the average duration of contacts with the PC head group; on the right panel, the color code indicates the average number of contacts during the MD simulation. At the center, the protein is rotated by 90 degrees and only 4 helices are shown, with residues showing the highest number of contacts highlighted. (B) Comparison of predicted models of Ist2 generated using AlphaFold 2 and trROSETTA. Note the variable proximity between TM4 (dark blue) and TM6 (pink) in the open and the close Ist2 model. (C) Membrane thickness, averaged over all simulations of the closed structural model (upper panel) and open structural model (lower panel). Thickness was calculated as the average distance between lipid head groups in each leaflet. The color scale indicates the average value of membrane in each position around the protein. Membrane thinning is visible around the open model, particularly in the position corresponding to the scrambling path.

1261
1262 **Figure 4. The influence of mutations in Ist2 N-terminal domain on the cellular lipidome**
1263 Localization of WT and truncated versions of Ist2
1264 tagged with GFP in the chromosomal *IST2* locus. The schematic illustrates Ist2 anchored to the
1265 endoplasmic reticulum (ER) via its N-terminal domain and tethered to the plasma membrane
1266 (PM) through its C-terminal domain. Corresponding images show GFP-Ist2, Ist2ΔC-GFP and
1267 GFP-ΔNIST2 localization. Scale bar = 5 µm. (B, C) Thin layer chromatography (TLC) analysis of
1268 lipid classes of the indicated yeast strains (B) and lipid classes distribution, separated into
1269 neutral lipids (C, non-polar) and phospholipids (C, polar) to facilitate viewing. For each group,
1270 lipid classes are expressed as percentage of the corresponding fraction (non-polar = 100% or
1271 polar = 100%). Quantification was performed separately on bottom and top halves of the TLC
1272 plate. (D) Schematic representation of PS pathways in yeast. PS is synthetized at the ER by the
1273 PS synthase Cho1 and can be transported to the PM by the LTPs Osh6/7. PS can be converted to PE by the PS decarboxylase Psd1 at the mitochondria or Psd2 in the endosome. (E, F) TLC analysis of lipid classes of the indicated yeast strains (E) and phospholipid class distribution (F). 1274 1275 Quantification of phospholipid classes from (E), expressed as percentage of the polar fraction (polar = 100%). (G,H) TLC of neutral lipid classes in yeast strains (G) and lipid class distribution 1276 1277 Equal phospholipid masses were applied for each sample in B, E and G. Strains labelled A, 1278 or not specified, were grown with agitation and strains labelled B were grown without it. The 1279 abbreviations used for standards (Std) refer to: SE: cholesterol ester; TG: triglyceride (18:1 or 1280 14:0); Cho: cholesterol; Erg: ergosterol; DG: diglycerides (1:3 or 1:2); FA: free fatty acid; MG: 1281 monoglyceride; Cer: ceramide; phytoSp: phytosphingosine; PG: phosphatidylglycerol; PE: 1282 phosphatidylethanolamine; CL: heart cardiolipin; PA: phosphatidic acid; PS: phosphatidylserine; 1283 PC: phosphatidylcholine; LPC: lysophosphatidylcholine. Graphs show the mean ± range of two 1284 independent replicates. (I) Genetic interaction between IST2 and CHO1. The expression of 1285 CHO1 is driven by the Gal promotor. Ten-fold serial dilutions were spotted onto galactose plates 1286 to allow the expression of CHO1 and glucose plates to shut down the expression of CHO1. 1287 Plates were incubated for 3 days at 22°C or 30°C. (J) Growth kinetics of ist2Δpsd1Δ cells 1288 expressing WT and mutated versions of Ist2, tagged with BFP at N-ter, grown in SC–His 1289 medium at 30°C without ethanolamine supplementation. Growth of these strains upon addition 1290 of ethanolamine is shown in Fig. S2D. The growth curves represent mean ± range of 1291 absorbance at 600 nm (OD<sub>600</sub>) of two independent biological replicates over time (hours). Each 1292 experiment is representative of at least three independent biological replicates. 1293 1294

1295 **Figure 5.** Effect of *IST2* WT or mutant over-expression on the COPII vesicular pathway at the ER. (A) Overview of the COPII vesicle formation at the ER. (B) Viability assay of the indicated yeast strains. Ten-fold serial dilutions of overnight cultures were spotted on YPD plates and incubated for 6 days at 25°C, 30°C, and 37°C. (C) Viability assay of *sec23-1* cells expressing Ist2ΔC at different levels from a plasmid. Ten-fold serial dilutions of overnight cultures were spotted on SC-URA plates and incubated for 6 days at 25°C and 30°C. The triangle indicates increasing expression of Ist2ΔC, from an empty plasmid (p0) to plasmids with ADH promoter for intermediate or GPD promoter for high expression. (D) Close-up view of the transmembrane helices TM3, TM4, and TM6 of Ist2 that are implicated in lipid scrambling. Residues L229, E240, T267, R359, Q363, and Y366, conserved with residues involved in lipid scrambling in TMEM16, are displayed as red sticks. These residues were mutated to alanine or to tryptophan in Ist2_6A and Ist2_6W mutants, respectively. (E) Growth kinetics of *sec23-1* cells overexpressing WT and mutated versions of Ist2ΔC, tagged with HaloTag, grown in SC-URA medium at 30°C. The growth curves represent the mean ± range of absorbance at 600 nm (OD<sub>600</sub>) of two independent biological replicates, measured in technical duplicates over time (minutes). Each experiment is representative of at least three independent biological replicates. (F) Localization of WT and 6A Ist2 mutant tagged with GFP in the endogenous *IST2* locus. Scale bar = 5 µm. (G) Viability assay of *sec23-1* cells overexpressing WT and mutant versions of Ist2ΔC from a centromeric plasmid with a GPD promotor. Ten-fold serial dilutions of overnight cultures were spotted on SC-URA plates and incubated for 6 days at 25°C and 30°C. p0 refers to an empty plasmid (pRS316). (H) Growth kinetics of *SEC23-AID*, *SEC24-AID*, *SEC31-AID* and WT cells with an empty plasmid (p0), pGPD-Ist2ΔC or pGPD-Ist2ΔC_6A grown in SC-URA medium at 30°C in the absence (-, empty symbols) or presence (+, full symbols) of indole-3-acetic acid (IAA). The arrows indicate the time IAA addition; its concentration was optimized separately for each strain. The growth curves represent the mean ± range of absorbance at 600 nm (OD<sub>600</sub>) of two independent biological replicates, measured in technical duplicates over time (minutes). Each experiment is representative of at least three independent biological replicates. (I) Localization of ER exit sites visualized by Sec16sfGFP in cells overexpressing WT and 6A
versions of Ist2ΔC. (J) Distribution of ER exit site sizes (µm²) in the same strains as in (I). The graph shows the relative abundance of ER exit sites with sizes between 0 and 2 µm², grouped into 0.1 µm² bins. The significance was assessed by Dunn's multiple comparisons test, P>0.05 (ns), P<0.0001 (****). (K) Maturation (trafficking) of the soluble vacuolar hydrolase, CPY, in *sec23-1* control cells (p0) or in *sec3-1 cells* overexpressing WT and 6A versions of Ist2ΔC, as revealed by pulse-chase experiments. Two independent clones for Ist2ΔC-6A are shown. Time of chase (in minutes) is indicated; 'p' marks precursor (ER) forms, "m" indicates mature (post-Golgi) forms. Arrows indicate the 55 min time-point, when differences between strains are most visible. Each experiment is representative of 2 - 3 independent experiments. See Fig. S3H for quantification of all experiments.

**Figure 6.** The influence of *ist2* mutants on LDs and lipid transport. (A) Distribution and abundance of LDs stained with Bodipy in WT cells and cells with chromosomal mutations in *IST2*, as indicated. Scale bar = 5 µm. (B) Quantification of the number of LDs per cell in indicated strains (n≥60, N=3). The significance was assessed by Kruskal-Wallis and Dunn's post-hoc test, P>0.05 (ns), P<0.01 (**), P<0.0001 (****). (C) Illustration of the PLIN4-AH probe [2Q,2V], plotted using HelQuest (Gautier et al., 2008). The probe contains 4 repeats of the indicated sequence of 33 amino acids, fused to GFP (Copic et al., 2018). (D) Distribution of the AH probe in indicated yeast strains bearing chromosomal mutations in *IST2* (top panels), with total LD staining using AutoDot shown in bottom panels. Scale bar = 5 µm. (E) Quantification of percentage of cells with the AH probe localized to LDs. Data are representative of at least three independent biological replicates (n≥100). The significance was assessed by Kruskal-Wallis and Dunn's post-hoc test, P>0.05 (ns), P<0.01 (**), P<0.0001 (****). (F) Viability assay of the indicated yeast strains. Ten-fold serial dilutions of overnight cultures were spotted on YPD plates and incubated for 3 and 7 days at 20°C and 30°C. (G) Schematic representation of processes that are affected by phospholipid transport activity mediated by Ist2, either scrambling of phospholipids (yellow) or transfer of PS (green).

**Figure 7.** Characterization of the genetic interaction between *IST2* and *GET1* in regulating LD size. (A) Venn diagram showing genetic interactions of *IST2*, *GET1*, and *GET2* (SGD database). (B) Viability assay of the indicated yeast strains. Ten-fold serial dilutions of overnight cultures were spotted onto YPD agar medium and incubated for 3 days at 20°C, 30°C, and 37°C. (C) Distribution and abundance of LDs in indicated strains, visualized by chromosomally tagged Erg6-GFP. Scale bar = 5 µm. (D) Quantification of LD size in indicated strains (n≥150, N=3). LD diameters (µm) were measured from Erg6-GFP fluorescence. The significance was assessed by Kruskal-Wallis and Dunn's post-hoc test, and is represented as a letter, where conditions sharing the same letter are not significantly different. All conditions differ with P<0.0001 (****), except between *get1Δ* and *get1Δist2Δ* where P<0.001 (**). (E) Representative distribution of LD diameters per cell in indicated yeast strains (n =15 cells). The mean diameter is indicated by a black horizontal line and the error bars represent SD.
1370 References

1371

Abraham, M.J., Murtola, T., Schulz, R., Páll, S., Smith, J.C., Hess, B., Lindahl, E., 2015.

1372 GROMACS: High performance molecular simulations through multi-level parallelism from

1374 laptops to supercomputers. SoftwareX 1–2, 19–25. https://doi.org/10.1016/j.softx.2015.06.001

1375 Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore,

1376 L., Ballard, A.J., Bambrick, J., Bodenstein, S.W., Evans, D.A., Hung, C.-C., O'Neill, M., Reiman,

1377 D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., Bertolli, O., Bridgland,

1378 A., Cherepanov, A., Congreve, M., Cowen-Rivers, A.I., Cowie, A., Figurnov, M., Fuchs, F.B.,

1379 Gladman, H., Jain, R., Khan, Y.A., Low, C.M.R., Perlin, K., Potapenko, A., Savy, P., Singh, S.,

1380 Stecula, A., Thillaisundaram, A., Tong, C., Yakneen, S., Zhong, E.D., Zielinski, M., Žídek, A.,

1381 Bapst, V., Kohli, P., Jaderberg, M., Hassabis, D., Jumper, J.M., 2024. Accurate structure

1382 prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500.

1383 https://doi.org/10.1038/s41586-024-07487-w

1384 Arndt, M., Schweri, A., Dutzler, R., 2025. Structural basis for lipid transport at membrane contact

1385 sites by the IST2–OSH6 complex. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-025-

1386 01660-z

1387 Azouaoui, H., Montigny, C., Ash, M.R., Fijalkowski, F., Jacquot, A., Gronberg, C., Lopez-

1388 Marques, R.L., Palmgren, M.G., Garrigos, M., Maire, M. le, Decottignies, P., Gourdon, P.,

1389 Nissen, P., Champeil, P., Lenoir, G., 2014. A high-yield co-expression system for the purification

1390 of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by

1391 phosphatidylinositol-4-phosphate. PLoS ONE 9, e112176.

1392 Bao, X., Koorengevel, M.C., Groot Koerkamp, M.J.A., Homavar, A., Weijn, A., Crielaard, S.,

1393 Renne, M.F., Lorent, J.H., Geerts, W.J., Surma, M.A., Mari, M., Holstege, F.C.P., Klose, C., De

1394 Kroon, A.I.P.M., 2021. Shortening of membrane lipid acyl chains compensates for

1395 phosphatidylcholine deficiency in choline-auxotroph yeast. EMBO J. 40, e107966.

1396 https://doi.org/10.15252/embj.2021107966

1397 Bean, B.D.M., Whiteway, M., Martin, V.J.J., 2022. The MyLO CRISPR-Cas9 toolkit: a

1398 markerless yeast localization and overexpression CRISPR-Cas9 toolkit. G3

1399 GenesGenomesGenetics 12, jkac154. https://doi.org/10.1093/g3journal/jkac154

1400 Bell, R.M., Ballas, L.M., Coleman, R.A., 1981. Lipid topogenesis. J. Lipid Res. 22, 391–403.

1401 Bernetti, M., Bussi, G., 2020. Pressure control using stochastic cell rescaling. J. Chem. Phys.

1402 153, 114107. https://doi.org/10.1063/5.0020514

1403 Bethel, N.P., Grabe, M., 2016. Atomistic insight into lipid translocation by a TMEM16

1404 scramblase. Proc. Natl. Acad. Sci. U. S. A. 113, 14049–14054.

1405 https://doi.org/10.1073/pnas.1607574113

1406 Bigay, J., Antonny, B., 2013. Curvature, Lipid Packing, and Electrostatics of Membrane

1407 Organelles: Defining Cellular Territories in Determining Specificity. Dev. Cell 23, 886–895.

1408 https://doi.org/10.1016/j.devcel.2012.10.009

1409 Bretscher, M.S., Raff, M.C., 1975. Mammalian plasma membranes. Nature 258, 43–49.

1410 https://doi.org/10.1038/258043a0

1411 Brunner, J.D., Lim, N.K., Schenck, S., Duerst, A., Dutzler, R., 2014. X-ray structure of a calcium-

1412 activated TMEM16 lipid scramblase. Nature 516, 207–212. https://doi.org/10.1038/nature13984

1413 Bushell, S.R., Pike, A.C.W., Falzone, M.E., Rorsman, N.J.G., Ta, C.M., Corey, R.A., Newport,

1414 T.D., Christianson, J.C., Scofano, L.F., Shintre, C.A., Tessitore, A., Chu, A., Wang, Q., Shrestha,
1415 L., Mukhopadhyay, S.M.M., Love, J.D., Burgess-Brown, N.A., Sitsapesan, R., Stansfeld, P.J., Huiskonen, J.T., Tammaro, P., Accardi, A., Carpenter, E.P., 2019. The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K. Nat. Commun. 10, 1–16. https://doi.org/10.1038/s41467-019-11753-1

1419 Bussi, G., Donadio, D., Parrinello, M., 2007. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101. https://doi.org/10.1063/1.2408420

1421 Cao, S.-Y., Liu, Y.-S., Gao, X.-D., Kinoshita, T., Fujita, M., 2023. A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins. J. Biochem. (Tokyo) 174, 109–123. https://doi.org/10.1093/jb/mvad041

1424 Chauhan, N., Farine, L., Pandey, K., Menon, A.K., Bütikofer, P., 2016. Lipid topogenesis — 35years on. BBA - Mol. Cell Biol. Lipids 1861, 757–766. https://doi.org/10.1016/j.bbalip.2016.02.025

1427 Cheng, X., Jo, S., Lee, H.S., Klauda, J.B., Im, W., 2013. CHARMM-GUI micelle builder for pure/mixed micelle and protein/micelle complex systems. J. Chem. Inf. Model. 53, 2171–2180. https://doi.org/10.1021/ci4002684

1430 Chorlay, A., Monticelli, L., Ferreira, J.V., M'barek, K.B., Ajjaji, D., Wang, S., Johnson, E., Beck, R., Omrane, M., Beller, M., Carvalho, P., Thiam, A.R., 2019. Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER. Dev. Cell 1–26. https://doi.org/10.1016/j.devcel.2019.05.003

1434 Collado, J., Kalemanov, M., Campelo, F., Bourgoint, C., Thomas, F., Loewith, R., Martínez-Sánchez, A., Baumeister, W., Stefan, C.J., Fernández-Busnadiego, R., 2019. Tricalbin-Mediated Contact Sites Control ER Curvature to Maintain Plasma Membrane Integrity. Dev. Cell 51, 476–487.e7. https://doi.org/10.1016/j.devcel.2019.10.018

1438 Copic, A., Antoine-Bally, S., Giménez-Andrés, M., Garay, C.L.T., Antonny, B., Manni, M.M., Pagnotta, S., Guihot, J., Jackson, C.L., 2018. A giant amphipathic helix from a perilipin that is adapted for coating lipid droplets. Nat. Commun. 9, 1332. https://doi.org/10.1038/s41467-018-03717-8

1442 Copic, A., Latham, C.F., Horlbeck, M.A., D'Arcangelo, J.G., Miller, E.A., 2012. ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p. Science 335, 1359–1362. https://doi.org/10.1126/science.1215909

1445 Costanzo, M., VanderSluis, B., Koch, E.N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S.D., Pelechano, V., Styles, E.B., Billmann, M., Leeuwen, J. van, Dyk, N. van, Lin, Z.-Y., Kuzmin, E., Nelson, J., Piotrowski, J.S., Srikumar, T., Bahr, S., Chen, Y., Deshpande, R., Kurat, C.F., Li, S.C., Li, Z., Usaj, M.M., Okada, H., Pascoe, N., Luis, B.-J.S., Sharifpoor, S., Shuteriqi, E., Simpkins, S.W., Snider, J., Suresh, H.G., Tan, Y., Zhu, H., Malod-Dognin, N., Janjic, V., Przulj, N., Troyanskaya, O.G., Stagljar, I., Xia, T., Ohya, Y., Gingras, A.-C., Raught, B., Boutros, M., Steinmetz, L.M., Moore, C.L., Rosebrock, A.P., Caudy, A.A., Myers, C.L., Andrews, B., Boone, C., 2016. A global genetic interaction network maps a wiring diagram of cellular function. Science 353. https://doi.org/10.1126/science.aaf1420

1454 D'Ambrosio, J.M., Albanese, V., Copic, A., 2019. Following Anterograde Transport of Phosphatidylserine in Yeast in Real Time. Methods Mol. Biol. Clifton NJ 1949, 35–46. https://doi.org/10.1007/978-1-4939-9136-5_4

1457 D'Ambrosio, J.M., Albanese, V., Lipp, N.-F., Fleuriot, L., Debayle, D., Drin, G., Čopič, A., 2020. Osh6 requires Ist2 for localization to ER-PM contacts and efficient phosphatidylserine transport in budding yeast. J. Cell Sci. 133, jcs243733. https://doi.org/10.1242/jcs.243733
1460 Derganc, J., Antonny, B., Copic, A., 2013. Membrane bending: the power of protein imbalance.

1461 Trends Biochem. Sci. 38, 576–584. https://doi.org/10.1016/j.tibs.2013.08.006

1462 Dias Araújo, A.R., Buvry, O., Antonny, B., Debayle, D., 2025. Separation of polar and neutral

1463 lipids from mammalian cell lines by high-performance thin-layer chromatography. J. Chromatogr.

1464 A 1741, 465610. https://doi.org/10.1016/j.chroma.2024.465610

1465 Dieudonné, T., Herrera, S.A., Laursen, M.J., Lejeune, M., Stock, C., Slimani, K., Jaxel, C.,

1466 Lyons, J.A., Montigny, C., Pomorski, T.G., Nissen, P., Lenoir, G., 2022. Autoinhibition and

1467 regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic

1468 cholestatic disorders. eLife 11, e75272. https://doi.org/10.7554/eLife.75272

1469 Drin, G., 2023. Creating and sensing asymmetric lipid distributions throughout the cell. Emerg.

1470 Top. Life Sci. 7, 7–19. https://doi.org/10.1042/ETLS20220028

1471 Du, Z., Su, H., Wang, W., Ye, L., Wei, H., Peng, Z., Anishchenko, I., Baker, D., Yang, J., 2021.

1472 The trRosetta server for fast and accurate protein structure prediction. Nat. Protoc. 16, 5634–

1473 5651. https://doi.org/10.1038/s41596-021-00628-9

1474 Eddy, S.R., 2011. Accelerated Profile HMM Searches. PLoS Comput. Biol. 7, e1002195.

1475 https://doi.org/10.1371/journal.pcbi.1002195

1476 Encinar Del Dedo, J., Fernández-Golbano, I.M., Pastor, L., Meler, P., Ferrer-Orta, C., Rebollo,

1477 E., Geli, M.I., 2021. Coupled sterol synthesis and transport machineries at ER-endocytic contact

1478 sites. J. Cell Biol. 220, e202010016. https://doi.org/10.1083/jcb.202010016

1479 Entian, K.D., Schuster, T., Hegemann, J.H., Becher, D., Feldmann, H., Güldener, U., Götz, R.,

1480 Hansen, M., Hollenberg, C.P., Jansen, G., Kramer, W., Klein, S., Kotter, P., Kricke, J.,

1481 Launhardt, H., Mannhaupt, G., Maierl, A., Meyer, P., Mewes, W., Munder, T., Niedenthal, R.K.,

1482 Rad, M.R., R\textbackslash backslash"ohmer, A., R\textbackslash backslash"omer, A., Hinnen, A., 1999. Functional

1483 analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol.

1484 Gen. Genet. MGG 262, 683–702. https://doi.org/10.1007/pl00013817

1485 Fabbre, A., Syska, C., Gobbi Sebinelli, H., Cardinal, A., Magdeleine, M., Al-Qatabi, N.,

1486 D'Ambrosio, J.M., Montigny, C., Lenoir, G., Čopič, A., Drin, G., 2025. Ist2 promotes lipid transfer

1487 by Osh6 via its membrane tethering and lipid scramblase activities.

1488 https://doi.org/10.1101/2025.05.06.652365

1489 Fairn, G.D., Schieber, N.L., Ariotti, N., Murphy, S., Kuerschner, L., Webb, R.I., Grinstein, S.,

1490 Parton, R.G., 2011. High-resolution mapping reveals topologically distinct cellular pools of

1491 phosphatidylserine. J. Cell Biol. 194, 257–275. https://doi.org/10.1083/jcb.201012028

1492 Falzone, M.E., Feng, Z., Alvarenga, O.E., Pan, Y., Lee, B., Cheng, X., Fortea, E., Scheuring, S.,

1493 Accardi, A., 2022. TMEM16 scramblases thin the membrane to enable lipid scrambling. Nat.

1494 Commun. 13, 2604. https://doi.org/10.1038/s41467-022-30300-z

1495 Falzone, M.E., Rheinberger, J., Lee, B.-C., Peyear, T., Sasset, L., Raczkowski, A.M., Eng, E.T.,

1496 Lorenzo, A.D., Andersen, O.S., Nimigean, C.M., Accardi, A., 2019. Structural basis of Ca(2+)-

1497 dependent activation and lipid transport by a TMEM16 scramblase. eLife 8.

1498 https://doi.org/10.7554/elife.43229

1499 Farkas, Á., Urlaub, H., Bohnsack, K.E., Schwappach, B., 2022. Regulated targeting of the

1500 monotopic hairpin membrane protein Erg1 requires the GET pathway. J. Cell Biol. 221,

1501 e202201036. https://doi.org/10.1083/jcb.202201036

1502 Feng, Z., Alvarenga, O.E., Accardi, A., 2024. Structural basis of closed groove scrambling by a

1503 TMEM16 protein. Nat. Struct. Mol. Biol. 31, 1468–1481. https://doi.org/10.1038/s41594-024-

1504 01284-9
1505 Filsbeck, J.M. von, Čopič, A., Delfosse, V., Vanni, S., Jackson, C.L., Bourguet, W., Drin, G., 2015.

1506 Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-

1507 phosphate. Science 349, 432–436. https://doi.org/10.1126/science.aab1346

1508 Frewein, M.P.K., Piller, P., Semeraro, E.F., Czakkel, O., Gerelli, Y., Porcar, L., Pabst, G., 2023.

1509 Distributing aminophospholipids asymmetrically across leaflets causes anomalous membrane

1510 stiffening 1–11. https://doi.org/10.1016/j.bpj.2023.04.025

1511 Gabler, F., Nam, S., Till, S., Mirdita, M., Steinegger, M., Söding, J., Lupas, A.N., Alva, V., 2020.

1512 Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Curr. Protoc. Bioinforma. 72,

1513 e108. https://doi.org/10.1002/cpbi.108

1514 Gameiro, E., Juárez-Núñez, K.A., Fung, J.J., Shankar, S., Luke, B., Khmelinskii, A., 2025.

1515 Genome-wide conditional degron libraries for functional genomics. J. Cell Biol. 224,

1516 e202409007. https://doi.org/10.1083/jcb.202409007

1517 Gapsys, V., De Groot, B.L., Briones, R., 2013. Computational analysis of local membrane

1518 properties. J. Comput. Aided Mol. Des. 27, 845–858. https://doi.org/10.1007/s10822-013-9684-0

1519 Gautier, R., Douguet, D., Antonny, B., Drin, G., 2008. HELIQUEST: a web server to screen

1520 sequences with specific \textbackslash backslash text \textbackslash text \textbackslash text \textbackslash sc \textbackslash t \textbackslash i \textbackslash t \textbackslash ilde \textbackslash helical properties. Bioinformatics 24, 2101–

1521 2102. https://doi.org/10.1093/bioinformatics/btn392

1522 Ghanbarpour, A., Valverde, D.P., Melia, T.J., Reinisch, K.M., 2021. A model for a partnership of

1523 lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis. Proc.

1524 Natl. Acad. Sci. U. S. A. 118, e2101562118. https://doi.org/10.1073/pnas.2101562118

1525 Gomez-Navarro, N., Melero, A., Li, X.-H., Boulanger, J., Kukulski, W., Miller, E.A., 2020. Cargo

1526 crowding contributes to sorting stringency in COPII vesicles. J. Cell Biol. 219, e201806038.

1527 https://doi.org/10.1083/jcb.201806038

1528 Goren, M.A., Morizumi, T., Menon, I., Joseph, J.S., Dittman, J.S., Cherezov, V., Stevens, R.C.,

1529 Ernst, O.P., Menon, A.K., 2014. Constitutive phospholipid scramblase activity of a G protein-

1530 coupled receptor. Nat. Commun. 5, 5115. https://doi.org/10.1038/ncomms6115

1531 Gowers, R., Linke, M., Barnoud, J., Reddy, T., Melo, M., Seyler, S., Domański, J., Dotson, D.,

1532 Buchoux, S., Kenney, I., Beckstein, O., 2016. MDAnalysis: A Python Package for the Rapid

1533 Analysis of Molecular Dynamics Simulations. Presented at the Python in Science Conference,

1534 Austin, Texas, pp. 98–105. https://doi.org/10.25080/Majora-629e541a-00e

1535 Hama, Y., Morishita, H., Mizushima, N., 2022. Regulation of ER-derived membrane dynamics by

1536 the DedA domain-containing proteins VMP1 and TMEM41B. EMBO Rep. 23, e53894.

1537 https://doi.org/10.15252/embr.202153894

1538 Hoffmann, P.C., Bharat, T.A.M., Wozny, M.R., Boulanger, J., Miller, E.A., Kukulski, W., 2019.

1539 Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged

1540 by Rod-Shaped Structures. Dev. Cell 51, 488-502.e8.

1541 https://doi.org/10.1016/j.devcel.2019.09.019

1542 Holthuis, J.C.M., Menon, A.K., 2014. Lipid landscapes and pipelines in membrane homeostasis.

1543 Nature 510, 48–57. https://doi.org/10.1038/nature13474

1544 Huang, D., Xu, B., Liu, L., Wu, L., Zhu, Y., Ghanbarpour, A., Wang, Y., Chen, F.-J., Lyu, J., Hu,

1545 Y., Kang, Y., Zhou, W., Wang, X., Ding, W., Li, X., Jiang, Z., Chen, J., Zhang, Xu, Zhou, H., Li,

1546 J.Z., Guo, C., Zheng, W., Zhang, Xiuqin, Li, P., Melia, T., Reinisch, K., Chen, X.-W., 2021.

1547 TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis.

1548 Cell Metab. 33, 1655-1670.e8. https://doi.org/10.1016/j.cmet.2021.05.006
1549 Humphrey, W., Dalke, A., Schulten, K., 1996. VMD: Visual molecular dynamics. J. Mol. Graph.

1550 14, 33–38. https://doi.org/10.1016/0263-7855(96)00018-5

1551 Hutchings, J., Stancheva, V.G., Brown, N.R., Cheung, A.C.M., Miller, E.A., Zanetti, G., 2021.

1552 Structure of the complete, membrane-assembled COPII coat reveals a complex interaction

1553 network. Nat. Commun. 12, 2034. https://doi.org/10.1038/s41467-021-22110-6

1554 Jahn, H., Bartoš, L., Dearden, G.I., Dittman, J.S., Holthuis, J.C.M., Vácha, R., Menon, A.K.,

1555 2023. Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

1556 Nat. Commun. 14, 8115. https://doi.org/10.1038/s41467-023-43570-y

1557 Jan, L.Y., Jan, Y.N., 2024. Wide-ranging cellular functions of ion channels and lipid scramblases

1558 in the structurally related TMC, TMEM16 and TMEM63 families. Nat. Struct. Mol. Biol.

1559 https://doi.org/10.1038/s41594-024-01444-x

1560 Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart,

1561 A., Doenges, G., Schwob, E., Schiebel, E., Knop, M., 2004. A versatile toolbox for PCR-based

1562 tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution

1563 cassettes. Yeast Chichester Engl. 21, 947–962. https://doi.org/10.1002/yea.1142

1564 Jha, A., Chung, W.Y., Vachel, L., Maleth, J., Lake, S., Zhang, G., Ahuja, M., Muallem, S., 2019.

1565 Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca

1566 2+signaling complexes at the ER/PM compartment. EMBO J. 38, 14483–15.

1567 https://doi.org/10.15252/embj.2018101452

1568 Jiang, T., Yu, K., Hartzell, H.C., Tajkhorshid, E., 2017. Lipids and ions traverse the membrane

1569 by the same physical pathway in the nhTMEM16 scramblase. eLife 6, 174a.

1570 https://doi.org/10.7554/elife.28671

1571 Joshi, A.S., Fei, N., Greenberg, M.L., 2016. Get1p and Get2p are required for maintenance of

1572 mitochondrial morphology and normal cardiolipin levels. FEMS Yeast Res. fow019.

1573 https://doi.org/10.1093/femsyr/fow019

1574 Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool,

1575 K., Bates, R., Zídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S.A.A., Ballard, A.J.,

1576 Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., Back, T., Petersen, S., Reiman,

1577 D., Clancy, E., Zielinski, M., Steinegger, M., Pacholska, M., Berghammer, T., Bodenstein, S.,

1578 Silver, D., Vinyals, O., Senior, A.W., Kavukcuoglu, K., Kohli, P., Hassabis, D., 2021. Highly

1579 accurate protein structure prediction with AlphaFold. Nature 596, 583–589.

1580 https://doi.org/10.1038/s41586-021-03819-2

1581 Kalienkova, V., Mosina, V.C., Bryner, L., Oostergetel, G.T., Dutzler, R., Paulino, C., 2019.

1582 Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. eLife 8,

1583 213. https://doi.org/10.7554/elife.44364

1584 Kalienkova, V., Mosina, V.C., Paulino, C., 2021. The Groovy TMEM16 Family: Molecular

1585 Mechanisms of Lipid Scrambling and Ion Conduction. J. Mol. Biol. 433, 166941.

1586 https://doi.org/10.1016/j.jmb.2021.166941

1587 Kannan, M., Lahiri, S., Liu, L.-K., Choudhary, V., Prinz, W.A., 2017. Phosphatidylserine

1588 synthesis at membrane contact sites promotes its transport out of the ER. J. Lipid Res. 58, 553–

1589 562. https://doi.org/10.1194/jlr.m072959

1590 Klose, C., Surma, M.A., Gerl, M.J., Meyenhofer, F., Shevchenko, A., Simons, K., 2012. Flexibility

1591 of a Eukaryotic Lipidome – Insights from Yeast Lipidomics. PLoS ONE 7, e35063-11.

1592 https://doi.org/10.1371/journal.pone.0035063
1593 Kralt, A., Carretta, M., Mari, M., Reggiori, F., Steen, A., Poolman, B., Veenhoff, L.M., 2014.

1594 Intrinsically Disordered Linker and Plasma Membrane-Binding Motif Sort Ist2 and Ssy1 to

1595 Junctions. Traffic Cph. Den. 16, 135–147. https://doi.org/10.1111/tra.12243

1596 Kroon, P., Grunewald, F., Barnoud, J., Van Tilburg, M., Souza, P., Wassenaar, T., Marrink, S.,

1597 2024. Martinize2 and Vermouth: Unified Framework for Topology Generation.

1598 https://doi.org/10.7554/eLife.90627.2

1599 Kunzelmann, K., Cabrita, I., Wanitchakool, P., Ousingsawat, J., Sirianant, L., Benedetto, R.,

1600 Schreiber, R., 2016. Modulating Ca2+ signals: a common theme for TMEM16, Ist2, and TMC.

1601 Pflüg. Arch. - Eur. J. Physiol. 468, 475–490. https://doi.org/10.1007/s00424-015-1767-4

1602 Lee, B.-C., Khelashvili, G., Falzone, M., Menon, A.K., Weinstein, H., Accardi, A., 2018. Gating

1603 mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase. Nat.

1604 Commun. 9, 3251. https://doi.org/10.1038/s41467-018-05724-1

1605 Lee, J., Cheng, X., Swails, J.M., Yeom, M.S., Eastman, P.K., Lemkul, J.A., Wei, S., Buckner, J.,

1606 Jeong, J.C., Qi, Y., Jo, S., Pande, V.S., Case, D.A., Brooks, C.L., MacKerell, A.D., Klauda, J.B.,

1607 Im, W., 2016. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and

1608 CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory

1609 Comput. 12, 405–413. https://doi.org/10.1021/acs.jctc.5b00935

1610 Lenoir, G., D'Ambrosio, J.M., Dieudonné, T., Čopič, A., 2021. Transport Pathways That

1611 Contribute to the Cellular Distribution of Phosphatidylserine. Front. Cell Dev. Biol. 9, 737907.

1612 https://doi.org/10.3389/fcell.2021.737907

1613 Li, D., Rocha-Roa, C., Schilling, M.A., Reinisch, K.M., Vanni, S., 2024. Lipid scrambling is a

1614 general feature of protein insertases. Proc. Natl. Acad. Sci. U. S. A. 121, e2319476121.

1615 https://doi.org/10.1073/pnas.2319476121

1616 Li, Y.E., Wang, Y., Du, X., Zhang, T., Mak, H.Y., Hancock, S.E., McEwen, H., Pandzic, E.,

1617 Whan, R.M., Aw, Y.C., Lukmantara, I.E., Yuan, Y., Dong, X., Don, A., Turner, N., Qi, S., Yang,

1618 H., 2021. TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and

1619 phosphatidylserine. J Cell Biol 220. https://doi.org/10.1083/jcb.202103105

1620 Liu, D., Yuan, H., Chen, S., Ferro-Novick, S., Novick, P., 2024. Different ER-plasma membrane

1621 tethers play opposing roles in autophagy of the cortical ER. Proc. Natl. Acad. Sci. U. S. A. 121,

1622 e2321991121. https://doi.org/10.1073/pnas.2321991121

1623 Long, T., Li, D., Vale, G. calo, Jiang, Y., Schmiege, P., Yang, Z.J., McDonald, J.G., Li, X., 2024.

1624 Molecular insights into human phosphatidylserine synthase 1 reveal its inhibition promotes LDL

1625 uptake. Cell 187, 5665–5678.e18. https://doi.org/10.1016/j.cell.2024.08.004

1626 Lorent, J.H., Levental, K.R., Ganesan, L., Rivera-Longsworth, G., Sezgin, E., Doktorova, M.,

1627 Lyman, E., Levental, I., 2020. Plasma membranes are asymmetric in lipid unsaturation, packing

1628 and protein shape. Nat. Chem. Biol. 16, 644–652. https://doi.org/10.1038/s41589-020-0529-6

1629 Maeda, K., Anand, K., Chiapparino, A., Kumar, A., Poletto, M., Kaksonen, M., Gavin, A.-C.,

1630 2013. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

1631 Nature 501, 257–261. https://doi.org/10.1038/nature12430

1632 Malvezzi, M., Chalat, M., Janjusevic, R., Picollo, A., Terashima, H., Menon, A.K., Accardi, A.,

1633 2013. Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat.

1634 Commun. 4, 2367–9. https://doi.org/10.1038/ncomms3367

1635 Manford, A.G., Stefan, C.J., Yuan, H.L., MacGurn, J.A., Emr, S.D., 2012. ER-to-Plasma

1636 Membrane Tethering Proteins Regulate Cell Signaling and ER Morphology. Dev. Cell 23, 1129–

1637 1140. https://doi.org/10.1016/j.devcel.2012.11.004
1638 Matoba, K., Kotani, T., Tsutsumi, A., Tsuji, T., Mori, T., Noshiro, D., Sugita, Y., Nomura, N., Iwata, S., Ohsumi, Y., Fujimoto, T., Nakatogawa, H., Kikkawa, M., Noda, N.N., 2020. Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion. Nat. Struct. Mol. Biol. 27, 1185–1193. https://doi.org/10.1038/s41594-020-00518-w

1642 Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S.Y., Hamamoto, S., Schekman, R., Yeung, T., 1998. COPII-Coated Vesicle Formation Reconstituted with Purified Coat Proteins and Chemically Defined Liposomes. Cell 93, 263–275. https://doi.org/10.1016/S0092-8674(00)81577-9

1646 Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A., Schwudke, D., 2008. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 49, 1137–1146. https://doi.org/10.1194/jlr.D700041-JLR200

1649 Melero, A., Chiaruttini, N., Karashima, T., Riezman, I., Funato, K., Barlowe, C., Riezman, H., Roux, A., 2018. Lysophospholipids Facilitate COPII Vesicle Formation. Curr. Biol. 28, 1950-1958.e6. https://doi.org/10.1016/j.cub.2018.04.076

1652 Monticelli, L., Kandasamy, S.K., Periole, X., Larson, R.G., Tieleman, D.P., Marrink, S.-J., 2008. The MARTINI Coarse-Grained Force Field: Extension to Proteins. J. Chem. Theory Comput. 4, 819–834. https://doi.org/10.1021/ct700324x

1655 Moretti, F., Bergman, P., Dodgson, S., Marcellin, D., Claerr, I., Goodwin, J.M., DeJesus, R., Kang, Z., Antczak, C., Begue, D., Bonenfant, D., Graff, A., Genoud, C., Reece-Hoyes, J.S., Russ, C., Yang, Z., Hoffman, G.R., Mueller, M., Murphy, L.O., Xavier, R.J., Nyfeler, B., 2018. TMEM 41B is a novel regulator of autophagy and lipid mobilization. EMBO Rep. 19, e45889. https://doi.org/10.15252/embr.201845889

1660 Morita, K., Hama, Y., Izume, T., Tamura, N., Ueno, T., Yamashita, Y., Sakamaki, Y., Mimura, K., Morishita, H., Shihoya, W., Nureki, O., Mano, H., Mizushima, N., 2018. Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation. J. Cell Biol. 217, 3817–3828. https://doi.org/10.1083/jcb.201804132

1664 Morra, G., Razavi, A.M., Menon, A.K., Khelashvili, G., 2022. Cholesterol occupies the lipid translocation pathway to block phospholipid scrambling by a G protein-coupled receptor. Structure 30, 1208-1217.e2. https://doi.org/10.1016/j.str.2022.05.010

1667 Nieto, V., Crowley, J., Santos, D., Monticelli, L., 2023. Birth of an organelle: molecular mechanism of lipid droplet biogenesis. https://doi.org/10.1101/2023.07.28.550987

1669 Olsson, M.H.M., Søndergaard, C.R., Rostkowski, M., Jensen, J.H., 2011. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p K_a Predictions. J. Chem. Theory Comput. 7, 525–537. https://doi.org/10.1021/ct100578z

1672 Pedersen, M.E., Gad, S.I., Østergaard, J., Jensen, H., 2019. Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach. Anal. Chem. 91, 4975–4979. https://doi.org/10.1021/acs.analchem.9b00537

1675 Peter, A.T.J., Schie, S.N.S. van, Cheung, N.J., Michel, A.H., Peter, M., Kornmann, B., 2022. Rewiring phospholipid biosynthesis reveals resilience to membrane perturbations and uncovers regulators of lipid homeostasis. EMBO J. 41, e109998. https://doi.org/10.15252/embj.2021109998

1679 Petkovic, M., Oses-Prieto, J., Burlingame, A., Jan, L.Y., Jan, Y.-N., 2020. TMEM16K is an interorganelle regulator of endosomal sorting. Nat. Commun. 1–16. https://doi.org/10.1038/s41467-020-17016-8
1682 Pettersen, E.F., Goddard, T.D., Huang, C.C., Meng, E.C., Couch, G.S., Croll, T.I., Morris, J.H.,
1683 Ferrin, T.E., 2021. UCSF ChimeraX: Structure visualization for researchers, educators, and
1684 developers. Protein Sci. Publ. Protein Soc. 30, 70–82. https://doi.org/10.1002/pro.3943

1685 Pinot, M., Vanni, S., Pagnotta, S., Lacas-Gervais, S., Payet, L.A., Ferreira, T., Gautier, R., Goud,
1686 B., Antonny, B., Barelli, H., 2014. Polyunsaturated phospholipids facilitate membrane
deformation and fission by endocytic proteins. Science 345, 693–697.
1687
1688 https://doi.org/10.1126/science.1255288

1689 Ploier, B., Menon, A.K., 2016. A Fluorescence-based Assay of Phospholipid Scramblase
1690 Activity. J. Vis. Exp. 54635. https://doi.org/10.3791/54635-v

1691 Pomorski, T., Menon, A.K., 2006. Lipid flippases and their biological functions. Cell. Mol. Life
1692 Sci. 63, 2908–2921. https://doi.org/10.1007/s00018-006-6167-7

1693 Pompon, D., Louerat, B., Bronine, A., Urban, P., 1996. [6] Yeast expression of animal and plant
1694 P450s in optimized redox environments, in: Methods in Enzymology. Elsevier, pp. 51–64.
1695 https://doi.org/10.1016/S0076-6879(96)72008-6

1696 Reinhard, J., Starke, L., Klose, C., Haberkant, P., Hammarén, H., Stein, F., Klein, O., Berhorst,
1697 C., Stumpf, H., Sáenz, J.P., Hub, J., Schuldiner, M., Ernst, R., 2024. MemPrep, a new
1698 technology for isolating organellar membranes provides fingerprints of lipid bilayer stress. EMBO
1699 J. 43, 1653–1685. https://doi.org/10.1038/s44318-024-00063-y

1700 Renne, M.F., Kroon, A.I.P.M. de, 2017. The role of phospholipid molecular species in
determining the physical properties of yeast membranes. FEBS Lett. 592, 1330–1345.
1701
1702 https://doi.org/10.1002/1873-3468.12944

1703 Rouser, G., Fleischer, S., Yamamoto, A., 1970. Two dimensional thin layer chromatographic
separation of polar lipids and determination of phospholipids by phosphorus analysis of spots.
1704
1705 Lipids 5, 494–496. https://doi.org/10.1007/BF02531316

1706 Sakuragi, T., Nagata, S., 2023. Regulation of phospholipid distribution in the lipid bilayer by
flippases and scramblases. Nat. Rev. Mol. Cell Biol. 24, 576–596.
1707
1708 https://doi.org/10.1038/s41580-023-00604-z

1709 Schekman, R., Novick, P., 2004. 23 Genes, 23 years later. Cell 116, S13–S15.
1710 https://doi.org/10.1016/S0092-8674(03)00972-3

1711 Schuldiner, M., Metz, J., Schmid, V., Denic, V., Rakwalska, M., Schmitt, H.D., Schwappach, B.,
1712 Weissman, J.S., 2008. The GET complex mediates insertion of tail-anchored proteins into the
1713 ER membrane 134, 634–645. https://doi.org/10.1016/j.cell.2008.06.025

1714 Sebinelli, H.G., Syska, C., Copic, A., Lenoir, G., 2024. Established and emerging players in
phospholipid scrambling: A structural perspective. Biochimie 1–12.
1715
1716 https://doi.org/10.1016/j.biochi.2024.09.008

1717 Sejdiu, B.I., Tieleman, D.P., 2021. ProLint: a web-based framework for the automated data
analysis and visualization of lipid–protein interactions. Nucleic Acids Res. 49, W544–W550.
1718
1719 https://doi.org/10.1093/nar/gkab409

1720 Shiomi, A., Nagao, K., Yokota, N., Tsuchiya, M., Kato, U., Juni, N., Hara, Y., Mori, M.X., Mori, Y.,
1721 Ui-Tei, K., Murate, M., Kobayashi, T., Nishino, Y., Miyazawa, A., Yamamoto, A., Suzuki, R.,
1722 Kaufmann, S., Tanaka, M., Tatsumi, K., Nakabe, K., Shintaku, H., Yesylevsky, S., Bogdanov,
1723 M., Umeda, M., 2021. Extreme deformability of insect cell membranes is governed by
phospholipid scrambling. Cell Rep. 35, 109219. https://doi.org/10.1016/j.celrep.2021.109219
1724
1725 Smith, M., Gay, L., Babst, M., 2024. ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion. J Cell Biol 223. https://doi.org/10.1083/jcb.202308137

1727 Smith, P., Lorenz, C.D., 2021. LiPyphilic: A Python Toolkit for the Analysis of Lipid Membrane Simulations. J. Chem. Theory Comput. 17, 5907–5919. https://doi.org/10.1021/acs.jctc.1c00447

1729 Sohn, M., Ivanova, P., Brown, H.A., Toth, D.J., Varnai, P., Kim, Y.J., Balla, T., 2016. Lenz-Majewski mutations in PTDSS1affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions. Proc. Natl. Acad. Sci. U. S. A. 113, 4314–4319.
1732 https://doi.org/10.1073/pnas.1525719113

1733 Soreanu, I., Hendler, A., Dahan, D., Dovrat, D., Aharoni, A., 2018. Marker-free genetic manipulations in yeast using CRISPR/CAS9 system. Curr. Genet. 64, 1129–1139.
1735 https://doi.org/10.1007/s00294-018-0831-y

1736 Souza, P.C.T., Alessandri, R., Barnoud, J., Thallmair, S., Faustino, I., Grünewald, F., 1973 Patmanidis, I., Abdizadeh, H., Bruininks, B.M.H., Wassenaar, T.A., Kroon, P.C., Melcr, J., Nieto, V., Corradi, V., Khan, H.M., Domański, J., Javanainen, M., Martinez-Seara, H., Reuter, N., Best, R.B., Vattulainen, I., Monticelli, L., Periole, X., Tieleman, D.P., De Vries, A.H., Marrink, S.J., 1974 2021. Martini 3: a general purpose force field for coarse-grained molecular dynamics. Nat. Methods 18, 382–388. https://doi.org/10.1038/s41592-021-01098-3

1742 Souza, P.C.T., Borges-Araújo, L., Brasnett, C., Moreira, R.A., Grünewald, F., Park, P., Wang, L., 1743 Razmazma, H., Borges-Araújo, A.C., Cofas-Vargas, L.F., Monticelli, L., Mera-Adasme, R., Melo, M.N., Wu, S., Marrink, S.J., Poma, A.B., Thallmair, S., 2024. GōMartini 3: From large 1745 conformational changes in proteins to environmental bias corrections.
1746 https://doi.org/10.1101/2024.04.15.589479

1747 Stephens, C.A., Van Hilten, N., Zheng, L., Grabe, M., 2025. Simulation-based survey of 1748 TMEM16 family reveals that robust lipid scrambling requires an open groove.
1749 https://doi.org/10.7554/eLife.105111.1

1750 Stevens, T., Esmon, B., Schekman, R., 1982. Early stages in the yeast secretory pathway are 1751 required for transport of carboxypeptidase Y to the vacuole. Cell 30, 439–448.
1752 https://doi.org/10.1016/0092-8674(82)90241-0

1753 Storey, M.K., Clay, K.L., Kutateladze, T., Murphy, R.C., Overduin, M., Voelker, D.R., 2001. 1754 Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is 1755 independent of its ability to form hexagonal phase structures. J. Biol. Chem. 276, 48539–48548.
1756 https://doi.org/10.1074/jbc.m109043200

1757 Suzuki, J., Umeda, M., Sims, P.J., Nagata, S., 2010. Calcium-dependent phospholipid 1758 scrambling by TMEM16F. Nature 468, 834–838. https://doi.org/10.1038/nature09583

1759 Tiberti, M.L., Antonny, B., Gautier, R., 2020. The transbilayer distribution of polyunsaturated 1760 phospholipids determines their facilitating effect on membrane deformation. Soft Matter 16, 1761 1722–1730. https://doi.org/10.1039/c9sm02107h

1762 Timcenko, M., Lyons, J.A., Januliene, D., Ulstrup, J.J., Dieudonne, T., Montigny, C., Ash, M.R., 1763 Karlsen, J.L., Boesen, T., Kühlbrandt, W., Lenoir, G., Moeller, A., Nissen, P., 2019. Structure 1764 and autoregulation of a P4-ATPase lipid flippase. Nature 571, 366–370.
1765 https://doi.org/10.1038/s41586-019-1344-7

1766 Toulmay, A., Whittle, F.B., Yang, J., Bai, X., Diarra, J., Banerjee, S., Levine, T.P., Golden, A., 1767 Prinz, W.A., 2022. Vps13-like proteins provide phosphatidylethanolamine for GPI anchor 1768 synthesis in the ER. J Cell Biol 221. https://doi.org/10.1083/jcb.202111095
1769 Tsuji, T., Cheng, J., Tatematsu, T., Ebata, A., Kamikawa, H., Fujita, A., Gyobu, S., Segawa, K.,

1770 Arai, H., Taguchi, T., Nagata, S., Fujimoto, T., 2019. Predominant localization of
1771 phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent

1772 redistribution. Proc. Natl. Acad. Sci. 116, 13368–13373.

1773 https://doi.org/10.1073/pnas.1822025116

1774 Van Vliet, A.R., Chiduza, G.N., Maslen, S.L., Pye, V.E., Joshi, D., De Tito, S., Jefferies, H.B.J.,
1775 Christodoulou, E., Roustan, C., Punch, E., Hervás, J.H., O'Reilly, N., Skehel, J.M., Cherepanov,

1776 P., Tooze, S.A., 2022. ATG9A and ATG2A form a heteromeric complex essential for
1777 autophagosome formation. Mol. Cell 82, 4324-4339.e8.

1778 https://doi.org/10.1016/j.molcel.2022.10.017

1779 Vance, D.E., Choy, P.C., Farren, S.B., Lim, P.H., Schneider, W.J., 1977. Asymmetry of
1780 phospholipid biosynthesis. Nature 270, 268–269. https://doi.org/10.1038/270268a0

1781 Wassenaar, T.A., Ingólfsson, H.I., Böckmann, R.A., Tieleman, D.P., Marrink, S.J., 2015.

1782 Computational Lipidomics with *insane*: A Versatile Tool for Generating Custom Membranes for
1783 Molecular Simulations. J. Chem. Theory Comput. 11, 2144–2155.

1784 https://doi.org/10.1021/acs.jctc.5b00209

1785 Webb, B., Sali, A., 2016. Comparative Protein Structure Modeling Using MODELLER. Curr.

1786 Protoc. Bioinforma. 54. https://doi.org/10.1002/cpbi.3

1787 Wolf, W., Kilic, A., Schrul, B., Lorenz, H., Schwappach, B., Seedorf, M., 2012. Yeast Ist2

1788 Recruits the Endoplasmic Reticulum to the Plasma Membrane and Creates a Ribosome-Free
1789 Membrane Microcompartment. PLoS ONE 7, e39703-13.

1790 https://doi.org/10.1371/journal.pone.0039703

1791 Wong, A.K.O., Young, B.P., Loewen, C.J.R., 2021. Ist2 recruits the lipid transporters Osh6/7 to
1792 ER-PM contacts to maintain phospholipid metabolism. J. Cell Biol. 220.

1793 https://doi.org/10.1083/jcb.201910161

1794 Wu, L., Wang, J., Wang, Y., Yang, J., Yao, Y., Huang, D., Hu, Y., Xu, X., Wang, R., Du, W., Shi,
1795 Y., Li, Q., Liu, L., Zhu, Y., Wang, X., Guo, Q., Xu, L., Li, P., Chen, X.-W., 2024. CLCC1 Governs

1796 Bilayer Equilibration at the Endoplasmic Reticulum to Maintain Cellular and Systemic Lipid

1797 Homeostasis. https://doi.org/10.1101/2024.06.07.596575

1798 Yang, H., Kim, A., David, T., Palmer, D., Jin, T., Tien, J., Huang, F., Cheng, T., Coughlin, S.R.,

1799 Jan, Y.N., Jan, L.Y., 2012. TMEM16F Forms a Ca2+-Activated Cation Channel Required for
1800 Lipid Scrambling in Platelets during Blood Coagulation. Cell 151, 111–122.

1801 https://doi.org/10.1016/j.cell.2012.07.036

1802 Yeung, T., Gilbert, G.E., Shi, J., Silvius, J., Kapus, A., Grinstein, S., 2008. Membrane

1803 phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213.

1804 https://doi.org/10.1126/science.1152066

1805
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<caption>Figure 2</caption>

A

<figure><img src="image_3.png" /></figure>

Residence time: 1.85 min
R<sub>h</sub>: 6.11 ± 0.23 nm

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>

E

<figure><img src="image_8.png" /></figure>

F

<figure><img src="image_9.png" /></figure>

G

<figure><img src="image_10.png" /></figure>

H

<figure><img src="image_11.png" /></figure>

I

<figure><img src="image_12.png" /></figure>
<caption>Figure 3</caption>

A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /></figure>
A

PM

ER

GFP-Ist2

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

Ist2ΔC-GFP

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

GFP-ΔNist2

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

B

SE

TG

Cho

FA

Cer

PG

PE

CL

PI

PS

PC

LPC

origin

<figure><img src="image_22.png" /></figure>

E

SE

ChoF

TG

DG1.3

Cho

FA

MG

Cer

phytoSp

LPC

origin

<figure><img src="image_23.png" /></figure>

G

ChoF

SE

TG 18:1

TG 14:0

DG 1.3

TG 1.2

Cho

FA

MG

LPC

origin

MG

LPC

origin

origin

<figure><img src="image_24.png" /></figure>

F

<figure><img src="image_25.png" /></figure>

H

<figure><img src="image_26.png" /></figure>

I

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

D

<figure><img src="image_30.png" /></figure>

J

<figure><img src="image_31.png" /></figure>
<figure><img src="image_32.png" /><figcaption>Figure 5</figcaption></figure>
A

<figure><img src="image_33.png" /></figure>

B

<figure><img src="image_34.png" /></figure>

C

<figure><img src="image_35.png" /></figure>

D

<figure><img src="image_36.png" /></figure>

E

<figure><img src="image_37.png" /></figure>

F

<figure><img src="image_38.png" /></figure>

G

<figure><img src="image_39.png" /></figure>
<figure><img src="image_40.png" /><figcaption>Figure 7</figcaption></figure>