# Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation

Yong Mi Choi, Dalila Ajjaji, Kaelin Fleming, Peter Borbat, Meredith Jenkins, Brandon Moeller, Shaveen Fernando, Surita Bhatia, Jack Freed, John Burke, et al.

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Yong Mi Choi, Dalila Ajjaji, Kaelin Fleming, Peter Borbat, Meredith Jenkins, et al. Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation. Nature Communications, 2023, 14 (1), pp.3204. 10.1038/s41467-023-38725-w. hal-04234703

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Submitted on 10 Oct 2023

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Structure and dynamics of human perilipin 3 membrane association

Yong Mi Choi¹ª, Dalila Ajjaji²ª, Kaelin D. Fleming³, Peter P. Borbat⁴,⁵, Meredith L. Jenkins³,
Brandon E Moeller³, Shaveen Fernando⁶, Surita Bhatia⁶, Jack H. Freed⁴,⁵, John E. Burke³,⁷*,
Abdou Rachid Thiam²*, Michael V. Airola¹*

¹Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook NY 11794, USA

²Laboratoire de Physique de l'École normale supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université Paris Cité, F-75005 Paris, France

³Department of Biochemistry and Microbiology, University of Victoria, Victoria BC V8N 1A1, Canada

⁴National Biomedical Resource for Advanced Electron Spin Resonance Technology (ACERT), Cornell University, Ithaca NY 14853, USA

⁵Department of Chemistry and Chemical Biology, Cornell University, Ithaca NY 14853, USA

⁶Department of Chemistry, Stony Brook University, Stony Brook NY 11794, USA

⁷Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

ªThese authors contributed equally

*Address correspondence to:

Michael V. Airola, michael.airola@stonybrook.edu

Abdou Rachid Thiam, thiam@ens.fr

John E. Burke, jeburke@uvic.ca

ABSTRACT
Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.
## 50 INTRODUCTION

Lipid droplets (LDs) act as energy reservoirs in cells. They contain a neutral lipid core of mainly triacylglycerols (TAGs) and cholesterol esters with a phospholipid monolayer that surrounds the neutral lipid core. This creates a membrane environment for LDs that is distinct from membrane bilayers and recruits several LD-associated proteins [1-3]. In addition to a major role in energy storage, LDs are also important cellular hubs that traffic proteins and lipids between organelles, regulate ER stress, and contribute to viral infections [4-6].

LDs are formed in the ER where neutral lipids are synthesized [7, 8]. Mechanistically, LD formation involves several steps [9] including accumulation of neutral lipids in the outer ER membrane leaflet, neutral lipid nucleation aided by the seipin complex and associated factors (e.g. LDAF1) [10], formation of nascent LDs that bud from the ER [11, 12] and LD growth and maturation [13, 14]. Several lines of evidence support the idea that the phospholipid composition of the ER membrane is locally edited to promote LD assembly or recruit specific proteins important for LD formation such as seipin and ORP proteins [7, 15-17]. Although there is still uncertainty regarding the phospholipid composition at the initial stages of LD formation, evidence from yeast suggest diacylglycerol (DAG), the direct precursor of TAG, is enriched in discrete ER subdomains where LD biogenesis is initiated [15, 18]. DAG can also promote TAG nucleation and impact the architecture of LDs on the ER [15, 19].

Perilipins (PLINs) are the major class of proteins that coat the surface of LDs [20-26]. There are five PLINs in humans that bind LDs at various stages of their initiation and maturation [21]. For example, PLIN1 is the major PLIN that binds to mature LDs in adipocytes, while PLIN2 and PLIN5 reside on mature LDs in liver and muscle cells, respectively [27]. In contrast, PLIN3 displays near ubiquitous expression and binds to early LDs as they bud from the ER but is later displaced by other PLINs as LDs grow and mature [10, 28]. PLIN3 is stable and not degraded in the absence of LDs. This allows PLIN3 to translocate from the cytoplasm to sites of early LD formation, where PLIN3 is well established to act as a marker for the biogenesis of early LDs across species [10, 28, 29].

PLINs share a conserved protein domain architecture composed of an N-terminal PAT domain, followed by variable stretches of 11-mer repeats and a C-terminal 4-helix bundle [30, 31]. The 11-mer repeats form amphipathic helixes that are sufficient to recruit PLINs to LDs [30-32], while the 4-helix bundle in some PLINs can also bind LDs [30, 33]. The binding of amphipathic
helices, such as those found in the 11-mer repeats, to membrane interfaces is greatly influenced by the level or presence of phospholipid packing defects [34, 35]. The degree of packing is determined by the type of lipid present (as indicated in the triangle in Fig. 1A). When it comes to the oil-water interface of LDs, the recruitment of amphipathic helices is greater when the level of phospholipid packing is lower [35, 36], i.e. when there are more packing defects. Although the PAT domain is the most conserved domain among PLINs, its function is not clear, as it has not been demonstrated to bind to membranes or LDs.

Here, we examine the mechanism of PLIN3 recruitment to membrane bilayers and LDs using a combination of *in vitro* and cell culture assays, and analyze the structural changes induced by membrane binding using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and pulsed-dipolar electron spin resonance spectroscopy (PD-ESR). We found that human PLIN3 is recruited to membrane bilayers enriched in the TAG precursors phosphatidic acid (PA) and DAG. By delineating the roles of the PAT domain and 11-mer repeats, we define an expanded PAT domain that is sufficient for PLIN3 to bind DAG enriched membranes, while the 11-mer repeats are sufficient to bind LD monolayers. We confirm that DAG enrichment can drive PLIN3 recruitment to ER membranes in cells, and this requires both the PAT domain and 11-mer repeats. Structurally, the PAT domain and 11-mer repeats form inducible alpha helices to drive membrane association and the PAT domain forms a tertiary structure upon membrane binding. Taken together, this study provides molecular insight into how PLIN3 is recruited to early LDs and reveals a novel function for the PAT domain of PLIN3 in DAG binding.

105
106
107
108 **RESULTS**

109 **The Triglyceride Precursors DAG and PA Recruit PLIN3 to Membrane Bilayers**

110 To systematically define how lipid composition affects PLIN3 recruitment to membrane bilayers,
111 we purified recombinant human PLIN3 from *Escherichia coli* and used liposome co-
112 sedimentation assays to monitor membrane binding. Liposomes were prepared using multiple
113 freeze/thaw cycles and characterized by dynamic light scattering (DLS) **(Fig. S1A)**. Liposomes
114 were made from phospholipids with different acyl-chain combinations comprised of either
115 palmitoyl-oleoyl (PO) or di-oleoyl (DO) phospholipids **(Fig. 1A)**. PO phospholipids have one
116 unsaturated acyl chain and are representative of the typical lipid composition in ER membranes.
117 DO phospholipids contain two unsaturated acyl chains, have increased membrane packing
118 defects [34, 37-39] and are enriched in ER membranes after oleate supplementation, which
119 stimulates LD formation [15, 40-42]. Initial liposome co-sedimentation experiments varied the
120 ratio of neutral phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as
121 PC and PE represent the major lipids on both the cytoplasmic face of the ER and surface of LDs,
122 PE increases both PLIN2 binding to liposomes [43] and PLIN3 insertion into mixed lipid
123 monolayers at phospholipid-oil interfaces [44] and the ratio of PC to PE has previously been
124 shown to regulate protein distribution on LDs [43, 45]. Under all PC-to-PE ratios tested, PLIN3
125 did not bind liposomes comprised solely of PC and PE **(Fig. 1B)**.

126
127 We next asked whether addition of other lipids that are synthesized in the ER could recruit
128 PLIN3 to membranes by generating PC/PE liposomes containing 20mol% of either phosphatidic
129 acid (PA), phosphatidylserine (PS), diacylglycerol (DAG), phosphatidylinositol (PI) or C18:1
130 ceramide. Regardless of lipid composition, PLIN3 did not bind to any PO-based liposomes **(Fig.
131 1C, 1D)**. However, PLIN3 was recruited to DO-based liposomes enriched in DAG or PA [37, 38,
132 46] **(Fig. 1C, 1D)**. Addition of DOPS and ceramide resulted in a minor increase in PLIN3 binding
133 in DO-based liposomes, while the addition of PI had no effect **(Fig. 1C, 1D)**.

134
135 PLIN3 recruitment to DO-based liposomes depended on the surface concentration of DAG, with
136 5mol% DAG able to induce ~10% binding, and 20mol% DAG inducing 35% binding **(Fig. 1E)**.
137 The addition of DOPA further increased PLIN3 binding in DAG-containing liposomes, which
138 suggested a synergistic effect of PA and DAG on PLIN3 membrane recruitment **(Fig. 1F)**.
139 Increasing the total liposome concentration caused the majority of PLIN3 to bind membranes,
140 but 5% of PLIN3 remained in the soluble fraction **(Fig. S2A)**. DLS characterization indicated
141 DAG enriched liposomes had two populations based on size **(Fig. S1C, S1D)**, which is likely
due to the fusogenic properties of DAG [46]. The addition of DOPA or PLIN3 protein decreased or eliminated the population of the larger (>400 nm) liposomes (Fig. S1C, S1D). We concluded that PLIN3 binds liposomes containing DAG and/or PA, which are notably the membrane-lipid precursors for triglyceride synthesis, and PLIN3 can also remodel membranes, consistent with previous studies [29].

PLIN3 Binds to Liposomes with Membrane Packing Defects

LDs have increased membrane packing defects in comparison to membrane bilayers [47]. Previously, PLIN4 has been shown to have increased binding to liposomes composed of methyl branched diphytanoyl (4ME) phospholipids that create shallow lipid-packing defects that can be accessed by hydrophobic insertion of peripheral membrane proteins, such as PLINs [31, 48]. The packing defects induced by 4ME phospholipids are greater than DO phospholipids because of the wider space between lipids [48]. In comparison to DO and PO liposomes, 4ME phospholipids significantly increased PLIN3 liposome association with ~70% binding observed with a mixture of 4ME-PC and 4ME-PE (Fig. 1G). Consistent with our previous observations, 4ME-PA further increased PLIN3 binding, while 4ME-PS and C18:1 ceramide decreased binding (Fig. 1G). The effect of DAG was unable to be assessed as addition of DAG to 4ME-PC/PE disrupted proper liposome formation. Taken together, this suggests PLIN3 membrane binding is also dependent on lipid packing defects, and PA can further enhance PLIN3 membrane binding.

PLIN3 Binding to Artificial Lipid Droplets

As the major function of PLIN3 is to bind to emerging LDs as they bud from the ER, we next assessed the ability of recombinant PLIN3 to bind artificial lipid droplets (ALDs) in vitro using a flotation assay [10, 28, 29, 49]. ALDs were generated with a triolein neutral lipid core surrounded by a phospholipid monolayer of DOPC and DOPE in 1:2.5 molar ratio of phospholipids to TAG. ALDs with 80mol% DOPC and 20mol% DOPE showed 50% binding of PLIN3 (Fig. S2B). The addition of the anionic phospholipids PI4P decreased PLIN3 binding to ALDs. In contrast, PA, PS and PI slightly decreased binding, ceramide did not affect binding, and DAG slightly increased PLIN3 binding. However, none of these effects were statistically significant. The modest impact of these PA, PS, PI, DAG, and ceramide, which all can induce negative curvature or charge in membrane bilayers, may be due to the basal occurrence of significant phospholipid packing voids on the ALD surface as compared with bilayers [36, 47]; while the strongly charged PI4P would instead mask these and diminish binding [47].
176
177 **The PAT Domain and 11-mer Repeats are Disordered in the Absence of Membranes**
178 We next sought to examine how the structure of PLIN3 changes upon membrane binding, first
179 focusing on the structure of PLIN3 in the absence of membranes. For these experiments, we
180 used hydrogen deuterium exchange mass spectrometry (HDX-MS), which measures the
181 exchange of amide hydrogens with deuterated solvent. This method acts as a readout for
182 protein conformational dynamics with regions that form secondary structures undergoing slower
183 deuterium exchange than disordered regions, which lack intramolecular hydrogen bonds and
184 secondary structure [50, 51]. A brief pulse of deuterated solvent is useful for identifying regions
185 within a protein that lack structure compared to ordered regions [52, 53].

186
187 We determined the absolute exchange of PLIN3 after a 3sec pulse of deuterium incorporation at 0°C (equivalent to ~0.3sec at 20°C) using a fully deuterated control. After the deuterium pulse,
188 the first 200 residues of PLIN3 comprising the PAT domain and 11-mer repeats were fully 189 deuterated, indicating these regions are completely disordered in the absence of membranes
190 (Fig. 2A). The 4-helix bundle and α/β domain were largely ordered with comparatively low rates 191 of deuterium incorporation (Fig. 2A), which is consistent with prior structural studies of PLIN3 192 [54, 55].
193

194
195 **The PAT Domain and 11-mer Repeats are the Major Drivers of PLIN3 Membrane Association**
196 We next sought to determine any conformational changes that occur during membrane binding
197 using HDX-MS. Having established optimal conditions for PLIN3 membrane binding, we
198 measured the deuterium exchange rate over various time points (3, 30, 300, and 3000sec) in
199 the absence or presence of 4ME liposomes composed of 60mol% 4ME-PC, 20mol% 4ME-PE,
200 and 20mol% 4ME-PA.
201

202
203 In the presence of membranes, striking differences in deuterium exchange were observed
204 throughout the PLIN3 sequence (Fig. 2E, Table S1, source data). The most notable
205 differences were large decreases in deuterium exchange in the PAT domain and 11-mer repeat
206 regions. In comparison to previous results that demonstrated the 11-mer repeats are sufficient
207 for the lipid droplet association [30, 31], this suggests that the PAT domain and 11-mer repeats
208 are both major contributors to membrane binding. Consistently, we found that a purified PAT/11-
209 mer repeats fragment displayed similar membrane recruitment as full length PLIN3 to DO
210 liposomes enriched in DAG and PA, and 4ME liposomes (Fig. 2C, 2D).

211
212 The HDX-MS results suggested that membrane binding induces the formation of a secondary
213 structure in the PAT domain and 11-mer repeats. This is consistent with both AlphaFold and
214 RoseTTAFold models that predict the PAT domain and 11-mer repeats form amphipathic alpha
215 helices, with the PAT domain adopting a triangular globular structure and the 11-mer repeats
216 forming a series of extended alpha helices (Fig. 2B, 3B). Based on these results we concluded
217 that the PAT domain and 11-mer repeats of PLIN3 are intrinsically disordered in the absence of
218 membranes, with inducible amphipathic alpha helices being stabilized upon membrane binding.
219

220 Notably, many of the peptides with the PAT domain and 11-mer repeats displayed a bimodal
221 distribution of deuterium exchange upon membrane binding (Fig. S3A, S3B). This differs from a
222 canonical distribution where the degree of deuteriation is gaussian in individual peptides. The
223 observed bimodal distributions are consistent with either EX1 exchange [56], or there being two
224 distinct protein conformations in the sample. Depending on the membrane residency of PLIN3,
225 bimodal distributions can be consistent with observations of the free and membrane bound state.
226 Given our previous observation that the PAT and 11-mer repeats are intrinsically disordered in
227 the absence of membranes, the simplest interpretation is that the PAT domain and 11-mer
228 repeats cycle between two states: 1) a membrane bound alpha helical state on membranes that
229 is strongly protected from H-D exchange and 2) an intrinsically disordered solution state that
230 rapidly exchanges with deuterated solvent upon dissociation from membranes and leads to
231 rapid H-D exchange. Therefore, the half-life of bimodal exchange can act as a surrogate for
232 membrane residency time for disordered regions of the protein.
233

234 The protection from deuterium exchange and the non-gaussian bimodal distribution for most
235 peptides in the PAT domain persisted over a longer time course (~300sec) in comparison to
236 peptides within the 11-mer repeat regions (~30sec) (Fig. S4A). This trend of longer HDX
237 protection correlated with the degree of sequence conservation among PLINs (Fig. 3A). Taken
238 together this supports a role for the PAT domain in membrane binding and suggests a similar
239 role for the highly conserved PAT domain in other PLINs.
240

241 In contrast to the N-terminal PAT domain and 11-mer repeats, the C-terminal 4-helix bundle
242 displayed a large increase in deuterium exchange (Fig. 2E, S4A). The most apparent increase
243 in deuterium exchange was in the middle of the 4-helix bundle. We hypothesized this was due
244 to the 4-helix bundle unfolding upon membrane binding, with this possibly contributing to
245 membrane recruitment. To test this, we examined the ability of a purified PLIN3 4-helix bundle
246 fragment (residues 197-427) to bind membranes. Consistent with our hypotheses, the isolated
247 4-helix bundle bound to the 4ME-PC/PE/PA liposomes that were used in the HDX-MS
248 experiment (Fig. 2C, 2D). However, the 4-helix bundle did not bind to 4ME-liposomes lacking
249 PA, or to any DO-based liposomes even when PA and DAG were present (Fig. 2C, 2D). We
250 concluded that the 4-helix bundle of PLIN3 can unfold and bind membranes, but only under very
251 specific condition such as the presence of accumulated PA on the membrane having packing
252 defects induced by 4ME-PC/PE. This conclusion is supported by the PA accumulation at the
253 nascent LD formation stie where lipid packing defects occur [17].

254
255
256 To understand how lipid composition influences the structure of PLIN3, we again applied HDX-
257 MS, using variable liposome compositions of 4ME-PC/PE and DOPC/PE/DAG (Fig. 2F, 2G). In
258 line with of previous HDX-MS and liposome sedimentation experiments, we observed protection
259 from H-D exchange in the PAT domain and 11-mer repeats using 4ME-PC/PE and
260 DOPC/PE/DAG liposomes, with the magnitude and duration of H-D exchange protection in
261 these regions correlating with the observed fraction of PLIN3 bound in liposome sedimentation
262 assays (Fig. S4B, 1D and 1G).
263

264 In contrast, with the 4ME-PC/PE and DOPC/PE/DAG lipid compositions, the 4-helix bundle did
265 not display large increases in deuterium exchange indicating that 4-helix bundle is not involved
266 in membrane binding in the absence of PA. Taken together, these results confirm that the PAT
267 domain and 11-mer repeats are the major lipid interacting regions of PLIN3, and that the 4-helix
268 bundle forms a stable tertiary structure that in general does not contribute to membrane
269 association, unless PA is present in membrane areas with shallow lipid packing defects. This
270 condition is particularly fulfilled at nascent LD formation sites.
271

271
272 **An expanded PAT domain binds DAG enriched membranes**

273 Our results suggested a role for the PAT domain in PLIN3 membrane association. However,
274 several studies [30, 31] have previously established the 11-mer repeats of PLINs are sufficient
275 for membrane binding, and to our knowledge, the PAT domain alone has not been
276 demonstrated to bind membranes. Complicating matters, the precise boundaries of the PAT
domain have been difficult to establish. Previous studies have suggested the PAT domain
consists of the N-terminal 97 residues in PLIN3 [30], which are highly conserved with other
PLINs (Fig. 3A). However, high sequence conservation between PLINs continues beyond
residue 97 and ends at residue 115 in PLIN3 (Fig. 3A). This suggests the PAT domain may be
larger than previously expected. Consistent with the sequence conservation, both Alphafold [57]
and RoseTTA fold [58] predict a triangular alpha helical tertiary structure for the PAT domain
that encompasses residues 22-116 (Fig. 3B).

To investigate the PAT domain boundaries and membrane interaction capabilities, we purified
several C-terminal extended PAT domain constructs and the corresponding 11-mer repeats
counterparts (Fig. 3C) and assessed the ability of these fragments to bind DO liposomes
containing either 20mol% PA or 20mol% DAG. As an important note, we were unable to purify
the previously suggested PAT domain construct (residues 1-97), as this construct was not
stably expressed in Escherichia coli. This is consistent with the poor expression of the PAT
domain of PLIN1 in yeast [30].

In general, fragments encompassing the PAT domain bound strongly to liposomes in the
presence of DAG, with modest liposome binding in the presence of PA (Fig. 3D). In contrast,
the 11-mer repeats bound to liposomes containing either PA or DAG (Fig. 3D). Taken together,
the data suggests that the PAT region does form a domain that spans residues 22-116 in PLIN3.
Notably, this expanded PAT domain is capable of membrane binding and displays a preference
for binding DAG enriched membranes, while the 11-mer repeats of PLIN3 do not.

299
300 Conformational Rearrangements of the PAT Domain upon Membrane Binding

We first sought to confirm the effects of liposome binding on secondary structure of PLIN3 by
circular dichroism (CD) (Fig. S5). Liposomes were prepared with 4ME-PC/PE/PA, which
recruited all the PLIN3 constructs (full length, PAT, 11-mer repeats, PAT/11-mer repeats, 4-helix
bundle) in the previous liposome co-sedimentation experiments. For full length PLIN3, the
presence of liposomes increased overall helicity as observed by an increased negative peak
around 222nm in the CD spectra. In comparison, the CD spectra of the 4-helix bundle was
largely unaffected by the presence of liposomes and was consistent with a stable alpha helical
structure. In contrast, the PAT/11-mer repeats adopted a mostly random coil structure in
solution with a negative peak around 200nm, and a shift to alpha helices in the presence of
liposomes as indicated by a large negative peak at 222nm. Liposomes induced similar changes
311 in the CD spectra for both the PAT domain and 11-mer repeats alone. Taken together, this
312 confirms that the increase in helicity observed in full length PLIN3 by membranes was due to
313 the PAT/11-mer repeats undergoing a disorder/alpha helical transition. This is in line with our
314 HDX-MS results and prior studies of PLIN2 and PLIN3 fragments [55, 59].

315
316 Next, to investigate if membranes induced conformational changes in the PAT domain, we
317 applied the pulsed-dipolar spectroscopy (PDS) technique of double electron-electron resonance
318 (DEER) [60, 61] to full length PLIN3 in solution and bound to liposomes. PDS is a collection of
319 several, based on recording electron spin-echo (ESE), pulse ESR techniques [62-66] routinely
320 applied to characterize protein conformations by providing accurate constraints in the distance
321 range of ~10-90Å. The amplitude of detected ESE depends on dipolar coupling between the
322 spins of unpaired electrons in nitroxide spin labels covalently bound to engineered cysteine
323 residues. Stepping out pulse separation in the sequence produces the time domain ESE
324 envelope, from which the distances could be reconstructed. [62-65].

325
326 Two sets of residues (25 and 96; 37 and 114) were selected for spin labeling, which were
327 respectively 71 and 77 residues apart in the primary sequence but predicted to be in close
328 proximity by AlphaFold and RoseTTAFold (Fig. 4A-4C). Comparison of the DEER decoherence
329 (decay) times indicated that the PAT domain is less structured and/or intrinsically disordered in
330 solution versus when bound to liposomes, which is consistent with our HDX-MS and CD
331 analysis (Fig. 4B, 4D). One can estimate, based on ~20% extent of decay in solution compared
332 to liposomes that the decoherence time is of the order of 20µs, which corresponds to nearly a
333 100Å separation expected for a random polypeptide chain with stiffness [67]. In contrast, with
334 liposomes the decoherence times are fast enough to decay to background well within the
335 evolution time of 1.2 µs used in the liposome DEER measurements. The decays do not show
336 oscillations, as is frequently observed for spin pairs with narrow distance distributions [68-71].
337 As a control to rule out lateral aggregation [69], we attached a single spin-label to residue 37C
338 and did not observe a dipolar evolution (DEER shape) that could indicate a pair with a shorter
339 than ~80Å separation based on only slightly concave shape of the signal (Fig. 4E). The DEER
340 data thus suggest that conspicuous dipolar signals in the doubly labeled proteins result from
341 intramolecular interactions, rather than being caused by intermolecular interactions on
342 membranes.

343
Distance distributions could be obtained in the presence of liposomes. The results showed semi-broad distance spreads of ~20-40Å for the 25C/96C pair and ~20-35Å for the 37C/114C pair (Fig. 4F, 4G). These distances are comparable but do not exactly match the simulated distances using the AlphaFold and RoseTTAFold structure predictions that have only modest confidence levels (Fig. 4A) [57, 58]. For comparison, an extended alpha helix would result in distances of ~100Å for these two sites. We also checked the spectral shape by recording field-swept echo with pulse separation of 250ns and did not notice any conspicuous broadening that could indicate a shorter range of distances (<15Å). We do however see from continuous wave (CW) ESR of 37C/114C (Fig. S6) that there might be a sizeable fraction of spins in the 15-20Å range whose contribution to the distance distribution will be significantly attenuated, since DEER has low sensitivity to distances in this range. The conformations with this distance range correlate well with AlphaFold predictions. The spread of the P(r) to longer distances could originate from the mobility of the C-terminal helix where residue 114C is located (Fig. 4C). Taken together, we concluded that the PAT domain does form a folded domain when bound to membranes and this domain is likely mobile with a structure similar but not identical to the AlphaFold and RoseTTAFold predictions.

DAG recruits PLIN3 to membrane bilayers and droplets in vitro

To verify a role for the PAT domain in DAG binding, we conducted an independent set of experiments to test if purified PLIN3 with GFP fused to the N-terminus bound DAG enriched membranes using droplet embedded vesicles (DEVs) [7, 35, 45, 72]. DEVs have emerged as a powerful in vitro system to model emerging LDs that bud from membrane bilayers. A major advantage of DEVs is the ability to not only monitor membrane association, but to also examine the protein distribution and preference for droplet monolayers versus membrane bilayers.

DEVs were generated by adding the neutral lipids TAG or DAG to giant unilamellar vesicles (GUVs) of DOPC that were marked with fluorescent phospholipids (Fig. 5A). As observed previously [45, 73], full length PLIN3 was strongly recruited to the monolayer surface of triolein droplets with almost no bilayer signal (Fig. 5B, 5D). Addition of diolein (DO-DAG) generated small droplet buds, and full length PLIN3 was recruited equally to the droplet and bilayer surface (Fig. 5C, 5D). Residues 1-204 of PLIN3 that contained both the PAT domain and 11-mer repeats behaved nearly identical to full length PLIN3 in both TAG and DAG containing DEVs (Fig. 5B, 5C and 5E).
Next, we tested fragments of the PAT domain and 11-mer repeats using both triolein and diolein DEVs. The 11-mer repeats preferentially bound the droplet surface of triolein DEVs with the magnitude of signal dependent on the length of the 11-mer repeats (Fig. 5B, 5G and 5I). In contrast, PAT domain fragments did not bind to the droplet or bilayer surface of triolein DEVs (Fig. 5B, 5F and 5H). The PAT domain was recruited to diolein DEVs, and the largest fragment (residues 1-116) bound to both the droplet and bilayer surface with similar magnitudes to full length PLIN3 (Fig. 5C, 5F and 5H). The 11-mer repeats also bound to both the droplet and bilayer surface of diolein DEVs, but to a lesser extent (Fig. 5C, 5G and 5I). These results are consistent with our previous hypothesis that the PAT domain is larger than previously expected and that this functional PAT domain binds DAG enriched membranes, while the 11-mer repeats can also bind DAG enriched membranes but display a preference for TAG containing droplets over membrane bilayers.

**DAG accumulation is sufficient to recruit PLIN3 to the ER in cells**

DAG has previously been proposed to recruit PLIN3 to the ER in cells by blocking its hydrolysis or acylation or by the exogenous addition of DAG [74]. This is consistent with the current model for LD formation, where DAG accumulates at the site of TAG nucleation on the ER membrane [18, 19, 75], the local high concentration of neutral lipids promotes LD nucleation through seipin [15], and cytoplasmic PLIN3 marks these sites [10, 49]. We sought to test whether DAG accumulation was sufficient for PLIN3 recruitment using an independent system and also assess what fragments of PLIN3 were necessary and sufficient for ER recruitment.

To best visualize PLIN3 recruitment to ER membranes, we generated intracellular giant ER vesicles (GERVs) by submitting cells to hypotonic medium [72, 76, 77] (Fig. 6A). After exchange to hypotonic medium, cells were pretreated with DMSO or the DGAT inhibitors (DGATi), followed by oleic acid to induce TAG or DAG synthesis [77] (Fig. 6A). Confocal microscopy was used to visualize cells prior and after exchange to hypotonic medium, and after oleic acid treatment. Imaging was done in the following minutes after the treatments.

As expected, the majority of subcellular localization of GFP-tagged PLIN3 was cytoplasmic in both normal and hypotonic media without addition of oleic acid (Fig. 6B, upper panels), and co-localized with LDs (Fig. 6D). Under conditions of DGAT inhibition, PLIN3 co-localized to the outer periphery of GERVs (Fig. 6B, yellow arrow, 6E and 6F), which suggests DO-DAG accumulation is sufficient to recruit PLIN3 to ER membrane bilayers.
412
413 In line with our previous results, a construct containing both the PAT domain and 11-mer
414 repeats showed a similar subcellular localization as full length PLIN3 under all conditions (Fig.
415 6C-6F). This suggests the PAT domain and 11-mer repeats are sufficient for PLIN3 recruitment
416 to both DAG enriched membrane bilayers and TAG containing LDs, which might also contain
417 DAG whose concentration could increase the protein binding level. Interestingly, constructs
418 containing only the PAT domain (residues 1-116) or only the 11-mer repeats (residues 114-204),
419 which bound to DEVs in a DAG-dependent manner, remained cytoplasmic under all conditions
420 (Fig. 6D, 6E, S7). Taken together, we concluded that PLIN3 is capable of binding both DAG
421 enriched ER membranes and early LDs, and that the PAT domain and 11-mer repeats are both
422 necessary and synergize to perform these functions in cells.

423

424
425 **DISCUSSION**
426 Here we find that PLIN3 not only binds LDs, but also membrane bilayers enriched in DAG and
427 PA. These results are in line with other studies that revealed PLIN3 binding to DAG [74, 78, 79].
428 In our hands, DAG binding was observed with multiple in vitro systems (e.g. liposomes, DEVs)
429 and DAG was also sufficient for PLIN3 recruitment to the ER in cells. Cellular recruitment to
430 membranes by DAG is consistent with a seminal study that used inhibitors to block DAG
431 hydrolysis or acylation and exogenous DAG to promote ER recruitment of PLIN3 [74].
432 Independent experiments in yeast have also demonstrated that membrane-anchored PLIN3 is
433 sufficient to bind to DAG enriched subdomains in the ER [78, 79]. Here we show that oleate
434 addition in combination with DGAT inhibition is sufficient for the subcellular redistribution of
435 PLIN3 to the ER, which mimics normal the LD biogenesis pathway.
436

437 There are several mechanistic implications of PLIN3 recruitment to DAG enriched membranes.
438 First, this implies that PLIN3 is recruited to sites of LD formation, not only through TAG
439 generation, but also at the initial stages when DAG begins to accumulate. This raises a likely
440 possibility that PLIN3 may play an active role in the early stages of LD formation by stabilizing
441 DAG enriched regions on ER [78], and define sites of LD formation, before or in concomitant
442 with seipin. Once PLIN3 binds to accumulated DAG, it might coat the curved surface of a
443 growing DAG/TAG lens and regulate LD budding, in conjunction with seipin.
444

445 PA is another TAG precursor that is present at sites where LD originate [17] and can bind to
446 seipin [16]. In addition to DAG, it was found to have a significant impact on the recruitment of
447 PLIN3 to membranes in vitro. Thus, it may increase the translocation of PLIN3 to early LD
448 formation sites. These two TAG precursors appear to provide specificity for the association of
449 PLIN3 with membranes. From a curvature standpoint, DAG has a more negative curvature
450 compared to PA and PE, whereas PE has a more negative curvature than PA [80]. This
451 suggests that membrane curvature cannot solely account for the major role of PA in PLIN3
452 membrane binding specificity. However, from a surface charge perspective, PA and PE together
453 may act synergistically by increasing charge of PA on the membrane [46, 81, 82]. In this
454 scenario, PLIN3 recruitment to LD nucleation sites could be enhanced by specific recognition of
455 PA, potentially through the 4-helix bundle. Therefore, PLIN3 membrane association may not
456 only be determined by membrane packing defects, but could also involve selective physical
457 interactions between PLIN3 and DAG or PA. This idea is also supported by a previous study
that found that the LD binding properties of PLINs are sensitive to the polar residue composition of their amphipathic helices [83].

In this study, we attempted to clarify the function and boundaries of the PAT domain. Our HDX-MS results using full length PLIN3 clearly implicate both the PAT domain and 11-mer repeats in membrane binding. In addition, we were able to define a functional PAT domain that encompasses all of the conserved residues within PLINs and is longer than previously suggested. This expanded PLIN3 PAT domain is sufficient to bind DAG enriched membranes, but not LDs. In contrast, the 11-mer repeats display some affinity for DAG enriched membranes and are necessary to bind LD monolayers. Our overall conclusion is that the PAT domain and 11-mer repeats serve synergistic functions, as the individual regions are necessary for both LD and DAG recruitment in cells.

The PAT domain is predicted to adopt a triangular tertiary structure by both AlphaFold and RoseTTAFold. The DEER distance measurements and CW ESR data are not identical to these predictions, but do indicate that when bound to membranes the PAT domain adopts a folded domain. This conclusion is supported by our HDX-MS results that found the peptides within the PAT domain display longer protection times from H-D exchange, which could be due to either a tertiary structure more resistant to unfolding or a longer membrane residency time. We note that the membrane bound PAT domain structure is likely dynamic and additional distance measurements at distinct sites are needed to verify the accuracy of the predicted triangular structures.

To our knowledge, our finding that the PAT domain of PLIN3 is sufficient to bind DAG represents the first functional role for the PAT domain in any PLIN. This suggests that the highly conserved PAT domain in other PLINs may serve similar or related functions. For example, could the PAT domain be a general sensor for DAG in membrane bilayers? Or could the PAT domain of other PLINs potentially bind to other neutral lipids (e.g. CEs, TAGs, retinol esters)? Given the previously observed differing neutral lipid preference of PLINs [20] and the involvement of the PAT domain in lipid/membrane binding, this now raises the questions if this is due to the PAT domain preference, the 11-mer repeat preference, or the synergistic action of the combined PAT/11-mer repeat units.
Lastly, our results are consistent with previous studies that found the PLIN3 4-helix bundle does not bind LDs, but other PLINs 4-helix bundles can bind LDs [33]. While the domain boundaries of the 4-helix bundles seem reasonable given structure of mouse PLIN3 [54], the variable membrane binding affinities we observed for the PAT domain and 11-mer repeats does raise the question if stability of 4-helix bundle, and consequently the ability to unfold and bind membranes or LDs in other PLINs may depend on the domain boundaries of specific constructs.
Thus, we suggest it is reasonable to revisit the ability of the 4-helix bundles from other PLINs using constructs that can be stably purified in vitro to rule out potential artifacts from the use of unstable 4-helix bundles.

500
501
## 502 MATERIALS AND METHODS

### 503 Protein expression and purification

The genes encoding PLIN3 constructs were codon-optimized for expression in *Escherichia coli* and cloned into pTHT, which is a derivative of pET28 that contains a TEV cleavable N-terminal 6x His tag. For GFP tagged PLIN3 constructs, monomeric superfolder GFP (msfGFP) was inserted between the 6x His tag and the N-terminus of PLIN3. PLIN3 plasmids were transformed into BL21(DE3) RIPL cells and protein expression was induced with 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 3h. Cells was harvested by centrifugation at 3,320 x g for 20mins and stored at -80°C until use. Cell pellets were resuspended with buffer A containing 500mM NaCl, 20 mM Tris-HCl pH 7.5, 5% glycerol, 5mM 2-mercaptoethanol (BME) and lysed by sonication. Cell lysates were centrifuged at 81,770 x g for 1h at 4°C and the resulting supernatant was incubated with 5mL of Ni-NTA resin for 2h at 4°C prior to loading onto a gravity column. The Ni-NTA resin was washed with buffer B containing 500mM NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol, 5mM BME and 60mM imidazole. Human PLIN3 proteins were eluted with buffer C containing 500mM NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol, 5mM BME and 300mM imidazole. Eluted protein was applied to a HiLoad 26/600 Superdex 200 pg column (GE life sciences) equilibrated with 150mM NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol, and 5mM BME. The average yield of full length PLIN3, PAT/11-mer repeats and 4-helix bundle throughout expression and purification was between 5-10mg from a 1L culture of *Escherichia coli*. The yield of PAT or 11-mer repeats constructs or GFP tagged PLIN3 fragments was ~1mg/L. Protein was analyzed by SDS-PAGE, concentrated using a centrifugal tube (10K MWCO, Pall Corporation), aliquoted and stored at -80°C.

### 525 Lipids

The following lipids were purchased from Avanti Polar Lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, catalog #850375), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, catalog #850725), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA, catalog #840875), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, catalog #840035), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, catalog #850457), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, catalog #850757), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA, catalog #840857), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, catalog #840034), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME-PC, catalog #850356), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME-PE, catalog #850402), 1,2-diphytanoyl-
535 sn-glycero-3-phosphate (4ME-PA, catalog #850406), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (4ME-PS, catalog #850408), 1-palmitoyl-2-oleoyl-sn-glycerol (DAG, catalog #800815), L-α-phosphatidylinositol (Soy PI, catalog #84044), L-α-phosphatidylinositol-4-phosphate (Brain, Porcine (PI(4)P, catalog #840045), N-stearoyl-D-erythro-sphingosine (C18:1 ceramide, catalog #860518). 1,2,3-Trioleoyl Glycerol was purchased from Cayman Chemicals (TAG, catalog #26950).

541
542 Liposome co-sedimentation assay
543 To prepare liposomes, lipids were dried under a nitrogen gas stream and dissolved with buffer
544 containing 50mM NaCl and 50mM HEPES pH 8.0 with a total lipid concentration of 2mM, unless
545 otherwise noted. Liposomes were generated by repeated cycles of flash freezing in liquid
546 nitrogen, thawing in 37°C water bath, and vortexing (ref Traikia et al). Liposomes were
547 characterized using dynamic light scattering and showed uniform distribution for most lipid
548 compositions. Lamellarity analysis of liposomes was not carried out, but liposomes likely
549 contained a mixture of multi-lamellar vesicles and unilamellar vesicles. For liposome co-
550 sedimentation assays, 20 µL of 12µM purified protein was incubated with 40µL of 2 mM
551 liposomes for 40mins at 23°C. Protein fractions bound to liposomes were isolated by
552 centrifugation at 100,000 x g for 70mins at 4°C and analyzed by SDS-PAGE.

553
554 Circular Dichroism
555 Circular dichroism (CD) spectra of purified PLIN3 full length and its constructs were measured
556 on Spectropolarimeter (Jasco, J-715). 0.12~0.24mg/ml of protein was incubated with 1~2mM
557 liposomes containing 4ME-PC/PE/PA for 40mins prior to measurement. Liposomes/protein
558 mixture was prepared in the buffer containing 20mM Tris pH 7.5 and 150mM NaCl. CD spectra
559 was measured between 190nm and 260nm in increments of 1nm, with a bandwidth of 50nm
560 and an averaging time of 1min at 25°C. 10 iterations of spectra were averaged and was
561 reported into a mDeg, which was converted to molar ellipticity (m°*M/(10*L*C) of which unit is
562 expressed in degree cm² dmol⁻¹. Molar ellipticity of each protein fragments in the absence or
563 presence were plotted using GraphPad prism software. Comparisons of spectra in the presence
564 and the absence of liposome were assessed using unpaired t test with p<0.05 of statistical
565 significance.

566
567 Artificial lipid droplet flotation assay
568 Artificial lipid droplets (ALDs) were prepared by mixing phospholipids and triacylglycerols 569 (TAGs). Di-oleoyl phospholipids were dried under a nitrogen gas stream. Dried lipids were 570 resuspended with lipid droplet flotation assay buffer containing 50mM NaCl and 50mM HEPES 571 pH 8.0 and TAGs in the molar ratio of 2:5. ALDs were formed by repeating the cycles of vortex 572 for 10sec and rest for 10sec. ALDs were further vortexed before use for the assay. 100µL of 573 ALDs were mixed with 5µL of human PLIN3 and 105µL of lipid flotation assay buffer to give a 574 final concentration of 5µM protein and 1.5mM phospholipids. The average diameters of different 575 ALDs were measured by dynamic light scattering (DLS). The concentration of ALDs were 576 determined by optical density at 600nm. The mixture of human PLIN3 and ALDs was incubated 577 for 1h at 23°C. To generate a sucrose gradient, 140µL of a 75% sucrose solution was added to 578 210µL of the protein-ALD mixture to give a final concentration of 30% sucrose. The resulting 579 mixture (320µL) was transferred to an ultra-centrifuge tube. 260µL of a 25% sucrose solution 580 was applied on top of 30% sucrose/protein/ALDs mixture. Lastly, 60µL of lipid flotation assay 581 buffer was laid on the top. ALDs were floated by centrifugation at 76,000 x g for 3h at 20°C with 582 sucrose gradient of 30% (bottom), 25% (middle), and 0% (top). Three fractions of 100µL from 583 the top, 260µL from the middle and 280µL from the bottom were collected and analyzed by 584 SDS-PAGE. 585

586 **Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)**
587

Sample preparation

588 HDX reactions for PLIN3 deuterium pulse were conducted in a final reaction volume of 10µL 589 with a final concentration 2.12µM PLIN3. Exchange was carried out in triplicate for a single time 590 point (3 sec at 0 °C). Hydrogen deuterium exchange was initiated by the addition of 9µL of D2O 591 buffer solution (20mM HEPES pH 7.5, 100mM NaCl) to 1µL of protein, to give a final 592 concentration of 84.9% D2O. Exchange was terminated by the addition of 60µL acidic quench 593 buffer at a final concentration 0.6M guanidine-HCl and 0.9% formic acid. Samples were 594 immediately frozen in liquid nitrogen at -80°C. Fully deuterated samples were generated by first 595 denaturing the protein in 3M guanidine for 1h at 20°C. Following denaturing, 9µL of D2O buffer 596 was added to the 1µL of denatured protein and allowed to incubate for 10mins at 20°C before 597 quenching with 0.6M guanidine-HCl and 0.9% formic acid. Samples were immediately frozen in 598 liquid nitrogen at -80°C, and all timepoints were created and run in triplicate. 599
600 HDX reactions comparing PLIN3 in the presence of PA liposomes (60mol% 4ME-PC, 20mol% 4ME-PE, 20mol% 4ME-PA) were conducted in a final reaction volume of 10µL with a final protein concentration of 3µM and final liposome concentration of 1mM. Protein and liposomes were preincubated together for 2mins at 20°C before the addition of 7µL D2O buffer solution (50mM HEPES pH 8.0, 50mM NaCl) for a final concentration of 63% D2O. Exchange was carried out for 3sec, 30sec, 300sec and 3000sec, and exchange was terminated by the addition of 60µL acidic quench buffer at a final concentration 0.6M guanidine-HCl and 0.9% formic acid. Samples were immediately frozen in liquid nitrogen at -80°C, and all timepoints were created and run in triplicate.

609
610 HDX reactions comparing PLIN3 in the presence of two different liposomes (60mol% DOPC, 20mol% DOPE, 20mol% DAG liposomes and 80mol% 4ME-PC, 20mol% 4ME-PE liposomes) were conducted in a final reaction volume of 10µL with a final protein concentration of 3µM and final liposome concentration of 500µM. Protein and liposomes were preincubated together for 2mins at 20°C before the addition of 8µL D2O buffer solution (50mM HEPES pH 8.0, 50mM NaCl) for a final concentration of 72% D2O. Exchange was carried out for 3sec, 30sec, 300sec and 3000sec, and exchange was terminated by the addition of 60µL acidic quench buffer at a final concentration 0.6M guanidine-HCl and 0.9% formic acid. Samples were immediately frozen in liquid nitrogen at -80°C, and all timepoints were created and run in triplicate.

619
620 Protein digestion and MS/MS data collection
621 Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2°C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The protein was run over either one (at 10°C) or two (at 10°C and 2°C) immobilized pepsin columns (Trajan; ProDx protease column, 2.1mm x 30mm PDX.PP01-F32) at 200µL/min for 3mins. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7mm column (2.1 x 5mm); Waters 186003975). The trap was subsequently eluted in line with an ACQUITY 1.7µm particle, 100 × 1mm2 C18 UPLC column (Waters), using a gradient of 3-35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11mins immediately followed by a gradient of 35-80% over 5mins. Mass spectrometry experiments acquired over a mass range from 150 to 2200m/z using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5kV.
633
634 *Peptide identification*
635 Peptides were identified from the non-deuterated samples of PLIN3 using data-dependent
636 acquisition following tandem MS/MS experiments (0.5sec precursor scan from 150-2000m/z;
637 twelve 0.25sec fragment scans from 150-2000m/z). MS/MS datasets were analyzed using
638 PEAKS7 (PEAKS), and peptide identification was carried out by using a false discovery based
639 approach, with a threshold set to 1% using a database of known contaminants found in Sf9 and
640 E. coli cells [84]. The search parameters were set with a precursor tolerance of 20ppm,
641 fragment mass error 0.02 Da, charge states from 1-8, leading to a selection criterion of peptides
642 that had -10logP scores of 22.8 for the pulse experiment and 20.9 for the liposome experiment.
643

644 *Mass Analysis of Peptide Centroids and Measurement of Deuterium Incorporation*

645 HD-Examiner Software (Sierra Analytics) was used to automatically calculate the level of
646 deuterium incorporation into each peptide. All peptides were manually inspected for correct
647 charge state, correct retention time, appropriate selection of isotopic distribution, etc.
648 Deuteration levels were calculated using the centroid of the experimental isotope clusters.
649 Results are presented as relative levels of deuterium incorporation, with the only correction
650 being applied correcting for the deuterium oxide percentage of the buffer utilized in the
651 exchange (63% and 72%). For the experiment with a fully deuterated sample, corrections for
652 back exchange were made by dividing the pulse %D value by the fully deuterated %D value and
653 multiplying by 100. The raw %D incorporation for the fully deuterated sample is included in the
654 source data, with the average back exchange being 33%, and ranging from 10-60%.
655 Differences in exchange in a peptide were considered significant if they met all three of the
656 following criteria: ≥5% change in exchange and ≥0.4 Da difference in exchange. The raw HDX
657 data are shown in two different formats. Samples were only compared within a single
658 experiment and were never compared to experiments completed at a different time with a
659 different final D2O level. The data analysis statistics for all HDX-MS experiments are in
660 Supplemental Table 1 according to the guidelines of [85]. The mass spectrometry proteomics
661 data have been deposited to the ProteomeXchange Consortium via the PRIDE partner
662 repository with the dataset identifier PXD025717 [86].

663
664 **Dynamic light scattering**
The size of liposomes was measured by dynamic light scattering (Brookhaven Instruments NanoBrook Omni), with or without full length PLIN3. Liposomes were prepared in the buffer containing 50mM HEPES pH 8.0 and 50mM NaCl by repeating the freeze/thaw cycles to prevent multilamellar vesicle formation. Each protein construct was incubated with liposomes in 1:200 of molar ratio at 25°C for 40mins. DLS was conducted using a scattering angle of 90°, and the liposome/protein mixture was equilibrated for 5mins after loading into the instrument to get a uniform temperature and minimize any loading effects prior to measurement. The number average size distribution (%) was considered as a relative concentration of particles with a certain size. For analysis, measurements with large outlier peaks, which were suspected to be due to the dust particles or aggregated vesicles, were discarded, and three runs that did not contain outlier peaks were used for the data analysis. The mode of the distribution (e.g., the size having the highest peak in the number average size distribution) was chosen from each of these three measurements and averaged to obtain the reported average diameter. A representative size distribution is shown for each sample.

## Site-directed spin-labeling

Recombinant PLIN3 cysteine-substituted proteins were purified using Ni-NTA resin in a buffer 681 500mM NaCl, 20mM Tris pH 7.5, 5% glycerol and eluted in a buffer of 500mM NaCl, 20mM Tris 682 pH 7.5, 5% Glycerol, 300mM imidazole. Elution was spin-labeled with 1µg/mL (S-(1-oxyl-683 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate), MTSL, (Santa 684 Cruz Biotech) dissolved in acetonitrile at 4°C overnight. The spin-labeled proteins were further 685 purified by HiLoad 26/600 Superdex 200 pg column (GE life sciences) in a buffer of 150mM 686 NaCl, 20mM Tris pH 7.5 to remove unreacted spin labels. 687

## Sample preparations for DEER spectroscopy

Protein samples in solutions were prepared at ca. 40-60µM concentrations in 20mM Tris at pH 690 7.5, 150mM NaCl, and 40% glycerol. Liposome samples containing 10µM protein and 3.33 mM 691 lipid were prepared by mixing 30µM protein stocks with 5mM liposome solutions using 1 to 2 692 aliquots followed by 30mins incubation at room temperature. The liposomes were freshly 693 prepared by rehydration of lyophilized 80/20 mol% 4ME-PC/PE followed by seven freeze/thaw 694 cycles. Freshly-prepared 20µL of spin-labeled samples were loaded into 1.8mm inner diameter 695 Pyrex sample tubes (Wilmad-LabGlass), frozen by plunging in liquid nitrogen, and stored in 696 liquid nitrogen for PDS measurements. 697

698
699 **DEER data collection and analysis**

700 PDS DEER measurements were performed at 60K with a home-built Ku band pulse ESR
701 spectrometer operating around 17.3GHz [64, 87]. A four-pulse DEER sequence [88] was
702 employed using for spin-echo detection π/2- and π-pulses with respective widths of 16 and 32ns,
703 the π-pulse for pumping was 16ns. The detection frequency matched the peak at the low-field
704 spectral edge, while pumping was performed at a lower by 70MHz frequency, corresponding to
705 the central maximum. A 32-step phase cycle [89] was applied to suppress unwanted
706 contributions to the signal. Nuclear electron spin-echo envelope modulation (ESEEM) caused
707 by surrounding protons was suppressed using the data from four measurements. In this method
708 the initial pulse separations and the start of detection were advanced by 9.5ns in subsequent
709 measurements, i.e. by the quarter period of the 26.2MHz nuclear Zeeman frequency for protons
710 at 0.615T corresponding to the working frequency. The four signal traces were summed to
711 achieve deep suppression of nuclear ESEEM, [90].

712
713 The solution samples had phase memory times, T<sub>m</sub>’s, of about 2.5µs, so the data could be
714 recorded up to 3µs evolution time (t<sub>m</sub>). Such evolution times did not provide sufficient decay of
715 the dipolar coherence to the background level, providing however clear indication of
716 unstructured nature of the protein in solution. Any residual secondary structure could be probed
717 by using multiple labeling sites, however other techniques did not encourage this undertaking.
718 For solutions the DEER data were acquired in less than 12h. Faster phase relaxation times
719 (~1µs) and low spin concentrations in liposome samples required signal averaging for ~24h to
720 facilitate good reconstruction by Tikhonov regularization. [91, 92]

721
722 For liposomes, time domain DEER data, V(t), were recorded and preprocessed using standard
723 approaches [62, 65, 88, 93] before their reconstruction into distance distributions, P(r)'s. The
724 first step is to remove signal decay caused by intermolecular dipole-dipole couplings, followed
725 by subtracting the residual background from the spins whose partner was not flipped by the
726 pump or missing. This was done, as usual, by fitting the latter points (about a half of the record)
727 of ln[V(t)] to a low-order polynomial, usually a straight line for solutions (and often for liposomes),
728 extrapolating it to zero time, and subtracting out from ln[V(t)]; so that the antilog yields u(t) = d(t)
729 + 1. Here d(t) is the dipolar evolution representing part. Once u(t) is normalized as u(t)/u(0), it
730 serves as a typical form of DEER data presentation, while v(t)=(u(t)-1)/u(0), gives background-
731 free "dipolar" data to be converted to a distance distribution between spins in pairs. We used L-
732 curve Tikhonov regularization [91] for distance reconstruction. The Tikhonov regularization utility
733 allowed selection of either the signal or its derivatives in the Tikhonov functional and selection of
734 the regularization parameter. The latter increases P(r) smoothness at the expense of
735 introducing some broadening.

736
737 Dipolar signal amplitude ("modulation depth") is given by v(0). This would be accurate to the
738 extent the background or V(∞), the asymptotic value of V(t), is known. This depth, tabulated by
739 calculations for typical pulse sequence setup and verified in numerous experiments, is a
740 measure of a fraction of spins in pairs or oligomers [69, 94, 95]. It is thus useful in estimate of
741 spin-labeling efficiency and protein concentration.

742
743 **Cell culture**
744 Cos7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with
745 10% heat inactivated fetal bovine serum (Life Technologies), 4.5gL⁻¹ D-glucose, 0.1gL⁻¹ sodium
746 pyruvate (Life Technologies) and 1% penicillin-streptomycin (Life Technologies). The cells were
747 cultured at 37°C under a 5% CO₂ atmosphere.

748
749 **Transfection**
750 When indicated, Cos7 cells (60–70% confluence) plated into a 35mm cell-culture Mattek dishes
751 (with a glass coverslip at the bottom), (MatTek Corp. Ashland, MA). were transfected with 3µg of
752 plasmid DNA/ml using Polyethylenimine HCl MAX (Polysciences) following the manufacturer's
753 instructions. For co-expressions, RFP-KDEL or GFP-Plin3 constructs in equal concentrations
754 (1.5–2µg for each one) were transfected to cells 24h prior observation.

755
756 **Giant unilamellar vesicles, GUVs**
757 GUVs were prepared by electroformation following the protocol described in [96]. Phospholipids,
758 dioleoyl phosphatidylcholine (PC) (70%) and dioleoyl phosphatidylethanolamine (PE) (29%),
759 Rhodamine-DOPE 1% (w/w)), were purchased from Avanti Polar Lipids. The lipid mixture, in
760 chloroform at 0.5mM, was dried on an indium tin oxide (ITO)-coated glass plate. The lipid film
761 was desiccated for 1h. The chamber was sealed with another ITO- coated glass plate. The
762 lipids were then rehydrated with a sucrose solution (275mOsm). Electroformation was done
763 using 100Hz AC voltage at 1.0 to 1.4Vpp and maintained for at least 1h.

764
765 Droplet-embedded vesicles (DEVs) preparation
766 Artificial LDs (aLDs) were prepared in HKM buffer: 50mM HEPES, 120mM Kacetate, and 1mM
767 MgCl₂ (in Milli-Q water) at pH 7.4 and 275±15mOsm. To do so, 5µL of the lipid oil solution
768 (triolein or diacylglycerol purchased from Sigma) was added to 45mL of HKM buffer and the
769 mixture was sonicated as described in [97]. The resulting emulsion was then mixed with GUVs
770 for five minutes under rotator to generate DEVs [97]. DEVs were then placed on a glass
771 coverslip pretreated with 10%(w/w) BSA and washed three times with buffer.
772

773 Protein binding to DEVs
774 For the protein binding experiments, 50µL HKM were deposited on the BSA-treated glass, 30µL
775 of the DEV solution added and 1.5µL purified PLIN3 or PLIN3 fragments added (in the case of
776 107-204 and 1-106 fragments, 3µL were added). The final protein concentration in the solution
777 was between 1µM to 4µM depending on the protein. For quantifications, 10-15 DEVs were
778 considered and the signal on the droplet and bilayer was determined by Fiji, via drawing 5-10
779 points-thick line profiles.
780

780
781 Neutral lipid synthesis induction
782 Wherever relevant, cells were exposed for 1h to 350µM oleic acid (OA) coupled to bovine serum
783 albumin (BSA) (0.2% weight/volume) to induce neutral lipids' synthesis. LipidTox DeepRed
784 (Thermo Fischer), was used to visualize lipid droplets or membranes enriched in neutral lipids.
785

786 Giant Intra-Cellular ER vesicle experiments
787 For GERV experiments, Cos7 cells were first transfected for 24h with the indicated eGFP
788 plasmids and RFP-KDEL. The culture media of the cells was next replaced by a hypotonic
789 culture media (DMEM:H2O, 1:20). The cells were then incubated at 37°C, 5% CO2 for 5mins, to
790 induce GERVs, and then they were visualized. Neutral lipids' synthesis was triggered by the
791 addition of OA and Z-stacks imaging of different entire cells was performed before and 15mins
792 after OA administration, as described in [77]. For the DGAT1 (Sigma, PF-04620110) and
793 DGAT2 (Sigma, PF-06424439) inhibitors used in Cos7, the dilution applied was 1/1000 for a
794 final concentration of 3µg/mL. In the GERV protocol, the inhibitors were added to the cell
795 medium before cell transfection or during the hypotonic solution addition. The signal of PLIN3 or
796 fragments thereof was quantified for tens of GERVs by depicting the mean signal on the GERVs
797 to which was subtracted the bulk cytosolic signal; this resulted signal was normalized to the
798 cytosolic signal.
799
800 **Structure prediction by AlphaFold and RoseTTAFold**

801 Structure prediction of PLIN3 was carried out by AlphaFold and RoseTTAFold. Two different

802 confidence values were generated (pLDDT from AlphaFold and rmsd from RoseTTAFold).

803 These values were converted using PHENIX to get relative confidence level from two softwares,

804 AlphaFold and RoseTTAFold [98].

805

806

807

808 **REFERENCES**

809

810 1. Thiam, A.R., R.V. Farese, Jr., and T.C. Walther, *The biophysics and cell biology of lipid droplets*. Nat Rev Mol Cell Biol, 2013. **14**(12): p. 775-86.

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1043

1044

1045
1046 Acknowledgements

1047 This work was supported by in part by the NIGMS (R35GM128666, MVA), the Sloan Research
1048 Foundation (MVA), the Natural Science and Engineering Research Council of Canada
1049 (Discovery Grant 2020-04241, JEB) with salary support from the Michael Smith Foundation for
1050 Health Research (Scholar award 17868, JEB), the ANR (18-CE11-0012-01-MOBIL, CE11-0032-
1051 02-LIPRODYN and 21-CE13-0014-LIPDROPER, ART), and the NSF (CBET 1903189, SB and
1052 DGE 1922639, SR). ESR study, conducted at ACERT, was supported by NIH/NIGMS grants
1053 1R24GM146107 and P41GM103521.
1054

1055
1056
1057 Author contributions
1058
1059 YMC generated constructs for E. coli and mammalian cell expression, purified all proteins,
1060 conducted liposome sedimentation, artificial lipid droplet binding assays, CD, DLS, prepared
1061 figures, and wrote the initial draft. DA conducted all DEV and cell culture experiments. KDF,
1062 MLJ, and BEM setup, analyzed, and/or prepared figures and text from the HDX-MS data. PPB
1063 designed, acquired, and analyzed all PDS-ESR data. JEB, ART, JHF and MVA supervised the
1064 work and provided funding support.

1065
1066 Competing Interest Statement

1067 JEB reports personal fees from Scorpion Therapeutics and Olema Oncology; and research
1068 grants from Novartis.

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1071
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1076
<figure><img src="image_2.png" /></figure>

<caption>**Figure 1.** Triglyceride precursors DAG and PA recruit PLIN3 to membrane bilayers</caption>

A) Structures of representative phospholipids used in this study.

B) SDS-PAGE analysis of human PLIN3 liposome sedimentation assay on the either palmitoyl-

oleoyl (PO)-liposomes or di-oleoyl (DO)-liposomes in different ratio of PC to PE. Lane S

(supernatant) and P (pellet) represent unbound and bound human PLIN3 to liposomes.

C) SDS-PAGE analysis of human PLIN3 recruitment to liposomes enriched with 20 mol% of the

additional lipids PS, PA, DAG, PI and ceramide.

D) Quantification of PLIN3 recruitment to liposomes in different lipid compositions (n=2).

Statistical analysis was performed using two-way ANOVA (****, p<0.0001).

E) SDS-PAGE analysis of human PLIN3 recruitment to DO-liposomes using different amounts

of DAG. DOPE concentration was held constant at 20mol% and DOPC was adjusted between

60 and 80mol% to maintain total phospholipid concentration. Bar graph shows addition of DAG

increases PLIN3 binding.

F) SDS-PAGE analysis of human PLIN3 recruitment to DO-liposomes enriched with different

amount of DOPA. The amounts of DOPE and DAG were kept constant at 20mol%. DOPC

concentration was adjusted between 40 and 60mol% to maintain total phospholipid

concentration. Bar graph shows addition of DOPA further increases PLIN3 binding.

G) SDS-PAGE analysis of human PLIN3 recruitment to 4ME liposomes enriched with 20 mol%

of the additional lipids 4ME-PA, 4ME-PS, ceramide. 4ME-PC and 4ME-PE concentrations

ranged between 60 ~80mol% and 20mol%, respectively. Statistical analysis for quantification of

liposome binding was performed using ordinary one-way ANOVA with Tukey's multiple

comparison test (n=2, ***, p<0.0001)
<figure><img src="image_3.png" /><figcaption>**Figure 2. HDX-MS analysis of PLIN3 membrane binding**</figcaption></figure>

A) Absolute percentage of deuterium incorporation after 3sec deuterium exposure of PLIN3 at 1°C in the absence of liposomes. Each point represents a single peptide, with them being graphed on the x-axis according to their central residue. A domain architecture of human PLIN3 was drawn to match the scale of x-axis of HDX-MS. N-terminal PAT domain (mustard) and 11-mer repeats (cyan) have no detectable secondary structure in the absence of liposomes.

B) A predicted 3D structure of human PLIN3 (UniProt: O60664) by AlphaFold (Left). The surface of PAT domain and 11-mer repeats were shown according to the hydrophobicity (Right).

C) SDS-PAGE and D) quantitative analysis of PAT/11-mer repeats and 4-helix bundle liposome binding. DO and 4ME-liposomes were prepared in the same way as Figure 1C and 1D.

Statistical analysis for quantification of liposome binding was performed using two-way ANOVA with Tukey's multiple comparison test (n=2).

E-G) Quantitative analysis of deuterium exchange differences of human PLIN3 in the presence of liposomes. The sum of the difference in the # of incorporated deuterons is shown for the absence and the presence of liposomes over all timepoints. The N-terminus composed of
1119 PAT/11-mer repeats was significantly protected from deuterium exchange (defined as > 5%
1120 change in exchange, > 0.4 Da mass difference in exchange, a p-value < 0.01 using a two-tailed
1121 Student's t-test). Each point represents an individual peptide, with those colored in red having a
1122 significant difference, with error bars showing standard deviation (n=3). Liposomes were
1123 generated with (E) 60mol% 4ME-PC, 20mol% 4ME-PE and 20mol% 4ME-PA, (F) 80mol% 4ME-
1124 PC, 20mol% 4ME-PE or (G) 60mol% DOPC, 20mol% DOPE and 20mol% DAG. A map of
1125 deuterium exchange rate according to all peptides throughout the entire PLIN3 was generated
1126 based on the AlphaFold predicted PLIN3 structure and color coded according to the legend. The
1127 full set of peptides is shown in the source data.

1128
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<figure><img src="image_4.png" /></figure>

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

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

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

<figure><img src="image_8.png" /><figcaption>Figure 3. An expanded PAT domain binds DAG enriched membranes</figcaption></figure>

A) Multiple sequence alignment of the N-terminus of human PLIN 1 (UniProt: O60240), PLIN2 UniProt: Q99541), PLIN3 (UniProt: O60664), and PLIN5 (UniProt: Q00G26). The previously suggested PAT domain (residues 1-97) is indicated with black arrow.

B) Putative PAT domain triangular structures predicted by both AlphaFold and RoseTTAFold.

C) Schematic of PAT domain constructs and the counterpart 11-mer repeats constructs.

D) Quantitative analysis of liposome recruitment for various PAT domain and 11-mer repeats constructs. Statistical analysis for quantification of liposome binding was performed using two-way ANOVA with Tukey's multiple comparison test (n=2).

1166

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<figure><img src="image_9.png" /><figcaption>Figure 4. Membrane-induced conformational rearrangements are consistent with a PAT domain tertiary structure</figcaption></figure>

A) Cartoon representation of the PAT domain structure predicted by AlphaFold2 and RoseTTAFold with relative confidence levels shown. The positions of the spin-labeled residues are indicated with arrows.

B) The hydrophobic surface of predicted PAT domain are depicted. The hydrophobic face of the helices is facing towards the reader.

C) The PAE (Predicted Aligned Error) value of AlphaFold2 for full length PLIN3 was plotted by ChimeraX and shown as an interactive 2D plot (bottom, right panel).

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1190 D) Time domain signals from DEER spectroscopy of double-labeled 25C/96C (left) and
1191 37C/114C (right) in solution (sol) and on liposomes (lip).
1192 E) Time domain signals from DEER spectroscopy of double-labeled 37C/114C and single-
1193 labeled 37C proteins on liposomes.
1194 F, G) Distance distributions from DEER spectroscopy of (E) 25C/96C and (F) 37C/114C
determined experimentally (exp) and from MtsslWizard simulations using the AlphaFold2 (AF2,
1195 sim) and RoseTTAFold (Rosetta, sim) predicted structures.
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<figure><img src="image_10.png" /><figcaption>Figure 5. DAG recruits PLIN3 to membrane bilayers and LD droplets in vitro</figcaption></figure>

A) A diagram of generation of DEVs by mixing oils and giant unilamellar vesicles (GUVs).

B, C) Quantification of the recruitment of GFP tagged PLIN3 constructs to droplet embedded vesicles (DEVs) containing (B) triolein or (C) Diolein. Statistical analysis was done with a Mann-Whitney non-parametric test. P<0.01 for all proteins. Three different experiments were performed and 10 to 15 DEVs quantified for each experiment.

D-I) Fluorescent microscopic images of N-terminal GFP tagged full length and PLIN3 constructs (green) comprised of PAT domain and 11-mer repeats to (DEVs) (magenta) that are generated with fluorescent labeled phospholipids and oils. F) and H) Recruitment of PAT domain constructs. Almost no recruitment of PAT domain constructs in the presence of Triolein. G) and I) Recruitment of 11-mer repeats constructs.

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

Noraml ER with
no treatment

+ Hypotonic
medium

<figure><img src="image_12.png" /></figure>

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

Giant ER Vesicles (GERVs)

- DGATi

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

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

Giant ER Vesicles (GERVs)
with LD biogenesis

+ OA

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

+ DGATi

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

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

DAG accumulation on
Giant ER Vesicles (GERVs)

<caption>B</caption>

Full length PLIN3

- DGATi

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

+ DGATi

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

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

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

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

Protein

LD

ER

Merge

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

Protein

LD

ER

Merge

<caption>C</caption>

Plin3 1-204

- DGATi

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

+ DGATI

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

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

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

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

Protein

LD

ER

Merge

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

Protein

LD

ER

Merge

<figure><img src="image_31.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_33.png" /></figure>
<figure><img src="image_34.png" /></figure>
<figure><img src="image_35.png" /><figcaption>A</figcaption></figure>

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

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

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

<figure><img src="image_39.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_41.png" /></figure>

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1291 Supplementary Figure 1.

1292 A) DLS analysis of liposomes with different lipid composition. For each liposome, three
measurements were plotted.

1293 B-F) DLS analysis of different liposomes with or without full length PLIN3. For each liposome, the number average size distribution (%) was recorded and plotted (n=3) in the presence and absence of full length PLIN3.

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<figure><img src="image_42.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_46.png" /></figure>

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**Supplementary Figure 2.**

A) SDS-PAGE and quantitative analysis of human PLIN3 recruitment by increasing the total amount of liposomes. The molar ratio of DOPC/DOPE/DOPA/DAG in 50:20:10:20 was kept same. Lane S represents unbound human PLIN3 from supernatant. Lane P represents pelleted human PLIN3 that bound to liposomes. Bar graph shows total increase in liposome amount results almost 100% recruitment of PLIN3 to liposome.

B) SDS-PAGE and quantitative analysis of human PLIN3 recruitment to ALDs generated with DO-phospholipids and TAGs (C16) by adding additional with 20 mol% of lipids such as PS, PA, DAG, PI, PI4P and ceramide. Top, middle and bottom fractions after sucrose-gradient centrifuge were indicated as T, M and B, respectively. Statistical analysis was performed using ordinary one-way ANOVA with Tukey's multiple comparison test (n=2, *, p=0.0258, **, p=0.004).

C) SDS-PAGE and quantitative analysis of human PLIN3 recruitment to liposomes in buffer containing 100mM NaCl and 20mM HEPES pH 7.0. Three different liposomes that were applied in HDX-MS were generated. Lane S represents unbound human PLIN3 from supernatant. Lane P represents pelleted human PLIN3 that bound to liposomes.
A

<figure><img src="image_47.png" /></figure>

B

<figure><img src="image_48.png" /></figure>

C

<figure><img src="image_49.png" /></figure>

D

<figure><img src="image_50.png" /><figcaption>Supplementary Figure 3.</figcaption></figure>

Representative bimodal distribution mass spectra from the peptides at PAT domain, 11-mer repeats, and 4-helix bundle of PLIN3 after deuterium exchange.
<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /><figcaption>Supplementary Figure 4.</figcaption></figure>

A) Deuterium exchange uptake plots for selected peptides in the PAT domain, 11-mer repeats and 4-helix bundle were plotted across all timepoints as 3s, 30s, 300s and 3000s. Data points in the absence and presence of liposomes were colored in black and red, respectively. Liposomes were generated with 60 mol% 4ME-PC, 20 mol% 4ME-PE and 20 mol% 4ME-PA). All peptides are shown in the source data.

B) Deuterium exchange uptake plots for selected peptides in the presence of two different liposomes are plotted across all timepoints as 3s, 30s, 300s and 3000s colored according to the legend. All peptides are shown in the source data.
<figure><img src="image_53.png" /><figcaption>**Supplementary Figure 5.** CD analysis of PLIN3 full length and various fragments with or without 4ME-PC/PE/PA liposomes</figcaption></figure>

<figure><img src="image_54.png" /></figure>
<table>
   <tr>
    <td>1361</td>
    <td><b>Supplementary Figure 6.</b> CW ESR spectrum of 37C/144C in lipid recorded at 9,434 GHz</td>
   </tr>
   <tr>
    <td>1362</td>
    <td>frequency. The Parameter Δ, defined [99] as the ratio of d1/d, is 0.45, which for the MTSL spin</td>
   </tr>
   <tr>
    <td>1363</td>
    <td>label indicates the distance in range of 1.5-2.0 nm [99, 100], i.e. somewhat shorter than</td>
   </tr>
   <tr>
    <td>1364</td>
    <td>reported by DEER which has reduced sensitivity to distances below 2.0 nm.</td>
   </tr>
   <tr>
    <td>1365</td>
    <td></td>
   </tr>
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    <td>1366</td>
    <td></td>
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   </tr>
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   </tr>
  </table>
<figure><img src="image_55.png" /><figcaption>**Supplementary Figure 7.**</figcaption></figure>

Subcellular localization of GFP tagged 114-204 and 1-116 constructs of PLIN3 was visualized in green in Cos7 cells under fluorescent microscope ZEISS LSM800 Airyscan. ER was visualized with ER specific marker RFP-KDEL in magenta. Lipid droplets were labeled with LipidTox Deepred to stain neutral lipids in cyan. After treated with hypotonic medium, cells were supplemented with oleate in the presence or absence of DGAT1/2 inhibitors.

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<table><thead><tr><th>Data set</th><th>PLIN3 order disorder Expt-1</th><th>PLIN3 Apo Expt-2</th><th>PLIN3 + 60% 4ME-PC, 20% 4ME-PE, 20% 4ME-PA liposomes Expt-2</th><th>PLIN3 Apo Expt-3</th><th>PLIN3 + 60% DOPC, 20% DOPE, 20% DAG liposomes Expt-3</th><th>PLIN3 + 80% 4ME-PC, 20% 4ME-PE liposomes Expt-3</th></tr></thead><tbody><tr><td>HDX reaction details</td><td>%D2O=84.9 % pH(read)=7.5 Temp=20°C</td><td>%D2O=63% pH(read)=8.0 Temp=20°C</td><td>%D2O=63% pH(read)=8.0 Temp=20°C</td><td>%D2O=72% pH(read)=8.0 Temp=20°C</td><td>%D2O=72% pH(read)=8.0 Temp=20°C</td><td>%D2O=72% pH(read)=8.0 Temp=20°C</td></tr><tr><td>HDX time course (seconds)</td><td>0.3s, fully deuterated</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td></tr><tr><td>HDX controls</td><td>FD</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td></tr><tr><td>Back-exchange</td><td>Corrected by fully deuterated sample</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td></tr><tr><td>Number of peptides</td><td>144</td><td>90</td><td>90</td><td>90</td><td>90</td><td>90</td></tr><tr><td>Sequence coverage</td><td>99.8%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td></tr><tr><td>Average peptide /redundancy</td><td>Length= 17.3 Redundancy = 5.4</td><td>Length= 13.0 Redundancy = 2.6</td><td>Length= 13.0 Redundancy = 2.6</td><td>Length= 13.0 Redundancy = 2.6</td><td>Length= 13.0 Redundancy = 2.6</td><td>Length= 13.0 Redundancy = 2.6</td></tr><tr><td>Replicates</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Repeatability</td><td>Average StDev=0.4%</td><td>Average StDev=0.6%</td><td>Average StDev=0.6%</td><td>Average StDev=0.8%</td><td>Average StDev=1.0%</td><td>Average StDev=0.9%</td></tr><tr><td>Significant differences in HDX</td><td>N/A</td><td>>5% and >0.4 Da and unpaired t-test ≤0.01</td><td>>5% and >0.4 Da and unpaired t-test ≤0.01</td><td>>5% and >0.4 Da and unpaired t-test ≤0.01</td><td>>5% and >0.4 Da and unpaired t-test ≤0.01</td><td>>5% and >0.4 Da and unpaired t-test ≤0.01</td></tr><tr><td>Data set</td><td>PLIN3 order disorder Expt-1</td><td>PLIN3 Apo Expt-2</td><td>PLIN3 + 60% 4ME-PC, 20% 4ME-PE, 20% 4ME-PA liposomes Expt-2</td><td>PLIN3 Apo Expt-3</td><td>PLIN3 + 60% DOPC, 20% DOPE, 20% DAG liposomes Expt-3</td><td>PLIN3 + 80% 4ME-PC, 20% 4ME-PE liposomes Expt-3</td></tr><tr><td>HDX</td><td>%D2O=84.9</td><td>%D2O=63%</td><td>%D2O=63%</td><td>%D2O=72%</td><td>%D2O=72%</td><td>%D2O=72%</td></tr></tbody></table>
<table><tr><td>reaction details</td><td>%<br>pH(read)=7.5<br>Temp=20°C</td><td>pH(read)=8.0<br>Temp=20°C</td><td>pH(read)=8.0<br>Temp=20°C</td><td>pH(read)=8.0<br>Temp=20°C</td><td>pH(read)=8.0<br>Temp=20°C</td><td>pH(read)=8.0<br>Temp=20°C</td></tr><tr><td>HDX time course (seconds)</td><td>0.3s, fully deuterated</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td></tr><tr><td>HDX controls</td><td>FD</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td></tr><tr><td>Back-exchange</td><td>Corrected by fully deuterated sample</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td><td>No correction, deuterium levels are relative</td></tr><tr><td>Number of peptides</td><td>144</td><td>90</td><td>90</td><td>90</td><td>90</td><td>90</td></tr><tr><td>Sequence coverage</td><td>99.8%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td></tr><tr><td>Average peptide /redundancy</td><td>Length=17.3<br>Redundancy=5.4</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td></tr><tr><td>Replicates</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Repeatability</td><td>Average<br>StDev=0.4%</td><td>Average<br>StDev=0.6%</td><td>Average<br>StDev=0.6%</td><td>Average<br>StDev=0.8%</td><td>Average<br>StDev=1.0%</td><td>Average<br>StDev=0.9%</td></tr><tr><td>Significant differences in HDX</td><td>N/A</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td></tr><tr><td>Data set</td><td>PLIN3 order disorder<br>Expt-1</td><td>PLIN3 Apo Expt-2</td><td>PLIN3 + 60% 4ME-PC,<br>20% 4ME-PE, 20% 4ME-PA<br>liposomes Expt-2</td><td>PLIN3 Apo Expt-3</td><td>PLIN3 + 60% DOPC, 20% DOPE, 20% DAG<br>liposomes Expt-3</td><td>PLIN3 + 60% 4ME-PC,<br>20% 4ME-PE<br>liposomes Expt-3</td></tr><tr><td>HDX reaction details</td><td>%D2O=84.9%<br>pH(read)=7.5<br>Temp=20°C</td><td>%D2O=63%<br>pH(read)=8.0<br>Temp=20°C</td><td>%D2O=63%<br>pH(read)=8.0<br>Temp=20°C</td><td>%D2O=72%<br>pH(read)=8.0<br>Temp=20°C</td><td>%D2O=72%<br>pH(read)=8.0<br>Temp=20°C</td><td>%D2O=72%<br>pH(read)=8.0<br>Temp=20°C</td></tr><tr><td>HDX time course (seconds)</td><td>0.3s, fully deuterated</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td><td>3s, 30s, 300s, 3000s</td></tr><tr><td>HDX controls</td><td>FD</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td></tr><tr><td>Back-exchange</td><td>Corrected by fully</td><td>No correction,</td><td>No correction,</td><td>No correction,</td><td>No correction,</td><td>No correction,</td></tr></table>
<table><thead><tr><th></th><th>deuterated<br>sample</th><th>deuterium<br>levels are<br>relative</th><th>deuterium<br>levels are<br>relative</th><th>deuterium<br>levels are<br>relative</th><th>deuterium<br>levels are<br>relative</th><th>deuterium<br>levels are<br>relative</th></tr></thead><tbody><tr><td>Number of<br>peptides</td><td>144</td><td>90</td><td>90</td><td>90</td><td>90</td><td>90</td></tr><tr><td>Sequence<br>coverage</td><td>99.8%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td><td>91.7%</td></tr><tr><td>Average<br>peptide<br>/redundancy</td><td>Length=17.3<br>Redundancy=5.4</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td><td>Length=13.0<br>Redundancy=2.6</td></tr><tr><td>Replicates</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td><td>3</td></tr><tr><td>Repeatability</td><td>Average<br>StDev=0.4%</td><td>Average<br>StDev=0.6%</td><td>Average<br>StDev=0.6%</td><td>Average<br>StDev=0.8%</td><td>Average<br>StDev=1.0%</td><td>Average<br>StDev=0.9%</td></tr><tr><td>Significant<br>differences<br>in HDX</td><td>N/A</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td><td>>5% and<br>>0.4 Da and<br>unpaired t-test ≤0.01</td></tr></tbody></table>