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# Ferroptosis-protective membrane domains in quiescence

Amalia H Megarioti, Bianca M Esch, Alexandros Athanasopoulos, Dimitrios Koulouris, Manousos Makridakis, Vasiliki Lygirou, Martina Samiotaki, Jerome Zoidakis, Vicky Sophianopoulou, Bruno André, et al.

► To cite this version:

Amalia H Megarioti, Bianca M Esch, Alexandros Athanasopoulos, Dimitrios Koulouris, Manousos Makridakis, et al. Ferroptosis-protective membrane domains in quiescence. Cell Reports, 2023, 42 (12), pp.113561. 10.1016/j.celrep.2023.113561. hal-04607212

HAL Id: hal-04607212

https://hal.umontpellier.fr/hal-04607212v1

Submitted on 10 Jun 2024

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**Cell Reports**

# Ferroptosis-protective membrane domains in quiescence

## Graphical abstract

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

## Authors

Amalia H. Megarioti, Bianca M. Esch, Alexandros Athanasopoulos, ..., Bruno André, Florian Fröhlich, Christos Gournas

## Correspondence

florian.froehlich@uni-osnabrueck.de
(F.F.),
cgournas@spfb.brussels (C.G.)

## In brief

Quiescence is required for microorganismal survival under stress. Megarioti et al. show that eisosome membrane domains expand in quiescent cells and promote quiescence maintenance. FLPs, eisosome-resident ubiquinone reductases, generate extramitochondrial ubiquinol and protect PUFA-supplemented quiescent cells from accumulation of lipid peroxides and iron-driven ferroptotic cell death.

## Highlights

* Eisosome membrane domains expand in respiratory-active quiescent cells

* Eisosomes support quiescence maintenance

* Eisosomes stabilize flavodoxin-like proteins (FLPs) at the plasma membrane

* Eisosomes and FLPs protect quiescent cells from lipid peroxidation and ferroptosis

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

# Ferroptosis-protective membrane domains in quiescence

Amalia H. Megarioti,<fnref n="1" />,<fnref n="2" /> Bianca M. Esch,<fnref n="3" /> Alexandros Athanasopoulos,<fnref n="1" /> Dimitrios Koulouris,<fnref n="1" /> Manousos Makridakis,<fnref n="4" /> Vasiliki Lygirou,<fnref n="4" /> Martina Samiotaki,<fnref n="5" /> Jerome Zoidakis,<fnref n="2" />,<fnref n="4" /> Vicky Sophianopoulou,<fnref n="1" /> Bruno André,<fnref n="6" /> Florian Fröhlich,<fnref n="3" />,<fnref n="7" />,* and Christos Gournas<fnref n="1" />,<fnref n="8" />,<fnref n="9" />,*

## SUMMARY

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

## INTRODUCTION

Quiescence is a reversible, non-proliferating state, during which cells display very low metabolic activity.<fnref n="1" /> In complex eukaryotes, stem cells, neurons, and oocytes are examples of quiescent cells. Stem cell quiescence is critical for tissue maintenance, while exit from quiescence is essential for normal metazoan development, sexual reproduction, wound healing, and cancer progression.<fnref n="1" /> Quiescence is also critical in microorganisms, as >98% of microbes exist in this non-growing state,<fnref n="1" /> allowing survival for long periods<fnref n="1" /> until more favorable conditions permit growth recovery. In the yeast *Saccharomyces cerevisiae*, cells differentiate into quiescent and senescent populations following glucose depletion (GD).<fnref n="2" /> Quiescent yeasts remain poorly studied, despite recent important efforts to identify subcellular quiescence-specific predictors, including the formation of actin bodies, microtube bundles, telomere hyperclusters, proteasome storage granules, stress granules, etc.<fnref n="3" />⁻<fnref n="5" /> Yet, many of these subcellular changes correlate only with growth arrest or quiescence and are not essential for quiescence entry/maintenance.<fnref n="3" />

An important exception is the reorganization of the mitochondrial network morphology typically observed at the onset of oxidative phosphorylation.<fnref n="6" /> Respiration is essential for quiescence development by supporting successive waves of internal-pH-dependent subcellular reorganizations before transition of the cytoplasm to a glass state.<fnref n="7" /> The role of the plasma membrane in quiescence, however, remains unexplored.

The plasma membrane performs essential roles in selective nutrient exchange and environmental sensing and communication. The protein and lipid constituents of the plasma membrane are highly compartmentalized.<fnref n="8" /> In yeast, several plasma membrane domains have been identified.<fnref n="9" /> The most studied is the “membrane compartment occupied by [the arginine transporter] Can1” (MCC)¹⁰ (Figure 1A). MCCs correspond to static furrow-like invaginations of the plasma membrane,¹¹ associate with a subcortical protein scaffold termed an eisosome,¹² have a distinct lipidic composition,¹³⁻¹⁵ and display structural and functional analogies to mammalian caveolae.¹⁶ A main component of eisosomes is Pil1, a highly abundant, self-assembling BAR (Bin, Amphiphysin, Rvs)-domain protein shaping the invagination.¹⁷

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

<footnote n="1">¹Microbial Molecular Genetics Laboratory, Institute of Biosciences and Applications, National Center for Scientific Research "Demokritos," 15341 Agia Paraskevi, Greece</footnote>

<footnote n="2">²Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece</footnote>

<footnote n="3">³Bioanalytical Chemistry Section, Department of Biology/Chemistry, Osnabrück University, 49076 Osnabrück, Germany</footnote>

<footnote n="4">⁴Biotechnology Division, Systems Biology Center, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece</footnote>

<footnote n="5">⁵Institute for Bioinnovation, Biomedical Sciences Research Center “Alexander Fleming,” 16672 Vari, Greece</footnote>

<footnote n="6">⁶Molecular Physiology of the Cell Laboratory, Université Libre de Bruxelles (ULB), IBMM, 6041 Gosselies, Belgium</footnote>

<footnote n="7">⁷Center for Cellular Nanoanalytic Osnabrück (CellNanOs), Osnabrück University, 49076 Osnabrück, Germany</footnote>

<footnote n="8">⁸Present address: Yeast Amino Acid Signaling and Transport Group, Labiris Research Institute, 1070 Brussels, Belgium</footnote>

<footnote n="9">⁹Lead contact</footnote>

<footnote>*Correspondence: florian.froehlich@uni-osnabrueck.de (F.F.), cgournas@spfb.brussels (C.G.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2023.113561</footnote>
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Pil1 is the main organizer of eisosomes, whereas the role of its Lsp1 paralog remains unknown.¹² Nce102 is an additional organizer of eisosomes.¹⁰,¹⁸ In the absence of Pil1 or Nce102, MCC/eisosome plasma membrane patches disassemble, and eisosome-resident proteins either become homogeneous at the plasma membrane or relocalize to the cytoplasm.¹⁰,¹²,¹⁸ MCC-resident membrane proteins include tetraspan members of the Nce102/MARVEL and Sur7 families and several nutrient transporters⁹,¹⁹ (Figure 1A). Eisosomes host regulators of sphingolipid biosynthesis and membrane stress, participating in the spatial separation of components of signaling cascades, including TORC2.¹⁸,²⁰⁻²² Eisosomes also act as ubiquitylation-protective membrane domains for specific nutrient transporters.⁹,²³⁻²⁶ During short-term nutrient starvation, eisosomes recruit stress granule components and conceal transporters from endocytosis, allowing efficient recovery upon nutrient read-dition.²³,²⁷⁻²⁹ Yet, the role of eisosomes in quiescent cells and long-term starvation has not been investigated.

Ferroptosis is an iron-dependent form of non-apoptotic cell death in mammalian cells caused by increased peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids.³⁰⁻³² The two main mechanisms protecting cells against ferroptosis are the reduction of lipid peroxides to lipid alcohols by the GPX4 glutathione peroxidase³³ and the inhibition of lipid-radical-mediated autoxidation by ubiquinol, the reduced form of ubiquinone, generated by the Fsp1 reductase.³⁴,³⁵ In fungi, ferroptotic conidial cell death has been reported,³⁶,³⁷ while in yeast, the glutathione peroxidases Gpx1/2/3 are involved in protection from peroxidation.³⁸,³⁹ Whether this peroxidation is iron dependent has not been reported. Moreover, ubiquinone serves lipid-peroxidation-protective roles in extramitochondrial membranes,⁴⁰,⁴¹ and ferroptosis inhibitors can rescue the sensitivity to peroxidation of a ubiquinone-deficient mutant,⁴² but the ubiquinone reductases involved remain uncharacterized.

In this work, we identify an essential role of eisosomes in protecting quiescent yeasts from ferroptosis. Eisosomes expand in quiescent cells and stabilize the ubiquinone reductases, named flavodoxin-like proteins (FLPs),⁴³ which are involved in the extramitochondrial regeneration of ubiquinol in yeast. FLPs act in par-

allel with the glutathione peroxidases in protecting quiescent cells from detrimental lipid peroxidation and ferroptosis. The induction of both eisosomes and FLPs is requisite for the fitness and the long-term survival of quiescent yeasts.

## RESULTS

### Eisosomes expand upon glucose depletion, and this requires the BAR-domain Lsp1 protein

Yeasts cultivated in buffered minimal medium typically show distinct phases of growth. Preadapted cells first grow exponentially for 30 h until they consume half of the glucose (G/2) (Figure 1B). For the next 12 h, they grow slower until GD. This phase is followed by a 6-day post-GD (PGD) slow growth, corresponding to utilization via oxidative phosphorylation of the ethanol produced during fermentation (Figure 1B). Since quiescent yeasts are formed following GD and during respiratory growth,⁶ the conditions above seem appropriate to study the formation of quiescent cells in minimal media, as confirmed below. Moreover, the growth deceleration observed at G/2 (Figure 1B) likely reflects cell preparation for quiescence entry, previously suggested to begin one division before GD.³,⁴⁴ To identify proteins involved in the transition to quiescence, we compared the proteome of cells from exponential phase (EXP) and upon GD and focused on pronounced changes in expression. This analysis identified 592 upregulated and 148 downregulated proteins (Figure 1C and Table S1). Gene Ontology (GO)-term analysis of the PGD downregulated dataset identified an enrichment of proteins involved in mRNA translation, rRNA maturation, and biosynthesis of amino acids and nucleobases (Figure S1B), consistent with these cells being slow growing and in the process of becoming quiescent. The PGD upregulated dataset showed enrichment in constituents of aerobic respiration, carbohydrate metabolism, glutathione metabolism, and cellular oxidant detoxification (Figure S1B), in line with the PGD shift from fermentation to oxidative phosphorylation.³,⁶ Accordingly, GO-term enrichment analysis for cellular components identified vacuoles, mitochondria, and lipid droplets (Figure 1D), which have known roles in quiescence/respiratory growth.⁶,⁷,⁴⁵⁻⁴⁷ In addition to these

### Figure 1. Eisosomes expand upon glucose depletion, and this requires the BAR-domain Lsp1 protein

(A) Graphical representation of the "membrane compartment occupied by Can1" (MCC)/eisosome. The main components of eisosomes are annotated.

(B) Line charts of cell density (green, cells/mL), glucose (blue), and ethanol (purple) concentrations (% w/v) at the different stages of cultures in minimal medium. Data from six (ethanol) or five (cell density) independent experiments are plotted over time as mean ± SD. Glucose concentration of the average cell density per time point was calculated from the scatterplot in Figure S1. Color coding of the different growth phases is utilized throughout the article.

(C) Volcano plot (Student's t difference vs. -log₁₀(p value)) comparison of the proteome of WT cells at GD vs. EXP phase (Table S1). Proteins with significant >1.9 (magenta) and < -1.9 (green) log₂(fold change) are indicated. All eisosomal proteins identified are annotated.

(D) Left: bar chart of the top GO-term-enriched cellular components in upregulated proteins of (C). The fold enrichment and p values are shown. Right: heatmap of the mean log₂(fold change) of all the proteins belonging to the indicated cellular components and identified in (C).

(E) Middle-section confocal microscopy images of a Pil1-GFP- and Lsp1-mCherry-expressing strain at different time points. Quantification: Pil1-GFP and Lsp1-mCherry single-cell mean fluorescence intensities (n = 35-97), normalized to the signal from EXP cells, are plotted in box-and-whiskers plots. \*\*\*p < 0.001; \*p < 0.05; ns, non-significant, p > 0.05, from one-way ANOVA.

(F) Western blot of total protein extracts from samples from (E) probed with anti-GFP, anti-mCherry, and anti-actin. Quantification: signal intensities, normalized to actin, from two independent experiments are plotted in line charts as mean fold change ± SD from EXP samples.

(G) Middle-section confocal microscopy of Lsp1-GFP-expressing WT and *pil1Δ* strains, at EXP and different time points PGD. Quantification: the single-cell MCC/total surface area ratios (n = 20-84) from surface-section images are plotted in box-and-whiskers plots. ND, not defined; \*\*\*p < 0.001; \*p < 0.05; ns, non-significant, p > 0.05, from one-way ANOVA.

(H) Surface-section Airyscan confocal microscopy of Pil1-mCherry-expressing WT and *lsp1Δ* strains at EXP and 7 days PGD. Quantification: the single-cell MCC/total surface area ratios and the Pil1-mCherry mean fluorescence intensities (n = 18-59), normalized to the signal from EXP cells, from surface-section images are plotted in box-and-whiskers plots. \*\*\*p < 0.001; ns, non-significant, p > 0.05, from one-way ANOVA. Scale bars: 1 µm. See also Figure S1.
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organelles, proteins of the eisosome cellular component showed the second highest enrichment (Figure 1D). Moreover, eisosomal proteins showed the highest mean fold increase in the whole PGD proteome, compared with all yeast cellular components (Figures 1C and 1D). Thus, we decided to characterize the role of eisosomes in quiescence.

We verified that the abundance of the core eisosomal proteins Pil1, Lsp1, and Nce102 gradually increased upon glucose exhaustion and until ethanol depletion (Figures 1E, 1F, and S1C). In fermenting cells, their expression and the density of eisosomes were low. An ~4-fold increase in expression was observed upon GD, reminiscent of cells shifted to acute carbon starvation.²⁷,²⁸ Yet, this increase was rather minor compared with the ~20-fold gradual increase observed during ethanol utilization, suggesting that respiration could be involved in eisosome expansion (analyzed below).

Next, we characterized eisosome assembly following GD. Similar to fermentation,¹⁰,¹²,¹⁸ Pil1 and Nce102 proved essential, as the proportion of Lsp1-GFP foci at the plasma membrane was drastically reduced in *pil1Δ* or *nce102Δ* cells (Figures 1G and S1D). Nce102 seemed to promote eisosome assembly by regulating Pil1 phosphorylation (Figure S1E), as previously reported for fermenting yeasts.¹⁸ Intriguingly, eisosome-like Lsp1 spots were detected at the plasma membrane of *pil1Δ* cells at PGD time points (Figure 1G). These Lsp1 spots colocalized with two MCC-resident proteins, namely Ynl194c¹⁰ (Figure S1F) and the Can1(7KR) variant resistant to ubiquitylation and endocytosis²³ (Figure S1G), suggesting that they correspond to intact eisosomes. Given that Lsp1 is neither necessary nor sufficient for eisosome formation during fermentation¹² (Figure 1G), but can form furrows during stress,⁴⁸ these results indicate that Lsp1 exerts its main physiological role in quiescence. Consistently, we could observe an important reduction of the MCC/total surface area in Lsp1-lacking quiescent cells, which was not due to reduced Pil1 levels (Figure 1H).

The above results show that eisosomes are among the most extensively expanding cellular components upon shift from fermentation to respiration and identify Lsp1 as an additional, quiescence-specific eisosome organizer.

## Eisosomes expand in respiratory-active quiescent yeasts

Following GD, cells displayed heterogeneity regarding eisosome expansion and assembly. The majority showed eisosome expansion, yet a small percentage displayed eisosome disassembly and/or defective eisosome expansion (Figure 2A). We thus examined whether this heterogeneity correlates with the previously reported formation of quiescent and senescent cells in this phase.² Density-gradient enrichment of quiescent cells² revealed that eisosome expansion occurred mainly in dense cells (Figures S2A and S2B). The less-dense, senescent-enriched fraction² was heterogeneous and enriched in cells with defective eisosome expansion/assembly, but nevertheless contained cells with normal eisosome expansion (Figures S2A and S2B). These observations are consistent with recent reports suggesting that cell density cannot accurately discriminate quiescent and senescent cells.⁶ Thus, we turned to the more refined marker of quiescence, namely the vesicular morphology of the mitochondrial network³ at the single-cell level, and examined at different time points PGD if it correlates with Pil1 induction and eisosome assembly (Figures 2A and 2B). Mitochondrial distribution changed from network-like in fermenting cells to vesicular (respiratory competent) in 90% of cells at the beginning of ethanol utilization, indicative of quiescence⁶ (Figures 2B and 2C). The PGD population further contained 7% apparently senescent cells displaying globular mitochondria and 3% non-fluorescent cells, reportedly dead.⁶ Both non-quietes categories increased over time. Notably, quiescent cells were mostly homogeneous and the vast majority displayed normal eisosome expansion and peripheral Pil1 distribution (Figures 2B, 2C, and S2C and Table S2). In

<caption>**Figure 2.** Eisosomes expand in respiratory-active quiescent yeasts</caption>

(A) Middle-section confocal microscopy images of a Pil1-mCherry-expressing strain representative of all cell categories identified regarding eisosome assembly and expansion.
(B) Left: schematic representation of the distribution of mitochondria according to Laporte et al.⁶ and the correlation between mitochondrial distribution and eisosome expansion/assembly at different time points PGD. Right: middle-section confocal microscopy images of a Pil1-mCherry and Ilv3-GFP-expressing strain representative of the predominant categories identified.
(C) Stacked bar chart displaying the classification of cells from different time points PGD according to mitochondrial distribution (as in B) and the eisosome induction and/or assembly (as in A). Data from four independent experiments are represented as the mean ± SD (detailed in Table S2, along with chi-squared values); representative quantifications of Pil1 intensity are provided in Figure S2C.
(D) Pie charts illustrating the probability of a cell with optimal eisosome assembly and with or without Pil1 induction to be quiescent, senescent, or dead (according to B). Detailed calculations are provided in Table S3.
(E) Middle-section confocal microscopy of a Pil1-mCherry and Ilv3-GFP-expressing strain at different time points PGD (control) or shifted at G/2 to glucose-free medium supplemented with 1.18% w/v ethanol (+EtOH) or not (–C). Quantification: Pil1-mCherry single-cell fluorescence intensities (n = 23–109), normalized to the signal from EXP cells, are plotted in box-and-whiskers plots. ***p < 0.001; ns, non-significant, p > 0.05, from one-way ANOVA.
(F) Western blot of total protein extracts from a Pil1-mCherry-expressing strain collected at the time points and conditions of (E) and probed with anti-mCherry and anti-actin. Quantification: signal intensities, normalized to actin, from two to four independent experiments are plotted in line charts as mean fold change ± SD from EXP samples. ***p < 0.001; *p < 0.05; ns, non-significant, p > 0.05, from two-way ANOVA.
(G) Left: schematic representation of the mitochondrial respiratory chain. The respiratory complexes and the corresponding mutants are indicated. Middle and right: middle-section confocal microscopy of Pil1-mCherry and Ilv3-GFP-expressing WT or *atp1Δ* strains at EXP and different time points PGD. Quantification: Pil1-mCherry single-cell fluorescence intensities (n = 36–118), normalized to the signal from EXP WT cells, are plotted in box-and-whiskers plots. ***p < 0.001; ns, non-significant, p > 0.05, from one-way ANOVA.
(H) Western blot of total protein extracts from Pil1-mCherry-expressing WT or *atp1Δ* strains collected at the time points and conditions of (G) and probed with anti-mCherry and anti-actin. Quantification: signal intensities, normalized to actin, from two to four independent experiments are plotted in line charts as mean fold change ± SD from WT EXP samples. ***p < 0.001; **p < 0.01; *p < 0.05; ns, non-significant, p > 0.05, from two-way ANOVA. Scale bars: 1 µm. See also Figure S2.
A

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contrast, senescent cells were heterogeneous regarding eisosome expansion/assembly, with the majority, however, showing defective induction and cytoplasmic redistribution of Pil1, while intermediate situations were also detected (Figures 2B, 2C, and S2C and Table S2). Finally, dead cells showed mostly eisosome disassembly. Inversely, eisosome expansion showed very high correlation with vesicular mitochondria (Figure 2D and Table S3). A cell with induced Pil1 had more than 96% probability of being quiescent until ethanol depletion. Moreover, cells showing both proper eisosome expansion and assembly had more than 89% probability of being quiescent at all time points. Although it was difficult to predict the fate of cells without Pil1 induction at early time points, these cells eventually tended to be mostly senescent or dead. These results indicate that the state of eisosomes can be a reliable marker of cell fate.

In support of eisosomes expanding specifically in respiratory-active cells, shifting cells from G/2 to fresh, carbon-free medium provoked only a minor induction in Pil1 expression (Figures 2E and 2F), as previously reported.²⁷ This indicates that glucose starvation is insufficient to promote optimal eisosome expansion. Consistently, the formation of stress granules, visible upon acute glucose starvation,²⁷ did not increase PGD, while cells displaying stress granules were mostly senescent, based on Pil1 expression/localization (Figure S2D). Antithetically, full eisosome expansion PGD required both GD and the supplementary presence of ethanol (Figures 2E and 2F) or another non-fermentable carbon source (Figure S2E). The requirement of respiration for eisosome expansion was further confirmed by the defective PGD Pil1 induction in mutants lacking essential components of respiratory chain complexes II, III, and IV and rho0 cells (Figures 2G, 2H, S2F, and S2G).

In total, the above results strongly indicate that eisosomes are specifically induced in respiratory-active quiescent cells, while

optimal eisosome expansion and assembly correlate strongly with quiescence maintenance.

## Eisosomes promote respiratory growth and are required for long-term quiescence maintenance

Next, we focused on identifying the physiological role of eisosomes in quiescent cells, using the double *pil1Δ/sp1Δ* (*mccΔ*) mutant, which totally lacks eisosomes at PGD time points (Figure S1G). The *mccΔ* strain grew identical to the wild type (WT) during fermentation. However, this mutant displayed a more pronounced slowdown at G/2, and it showed limited growth thereafter (Figure 3A), a phenotype intermediate between the WT and the *atp1Δ* respiratory-deficient strain. This defective PGD growth could be largely complemented by ectopic expression of either *PIL1* or *LSP1* (Figure S3A), confirming that the phenotype is due to the absence of eisosomes. The above suggests that *mccΔ* cells might suffer from defective PGD respiratory metabolism. Indeed, cultures of the *mccΔ* strain showed reduced consumption of ethanol, similar to a respiration-deficient mutant (Figure 3B). This impaired respiratory growth was not due to defective induction of autophagy (Figure S3B), which is essential for adaptation to respiration.⁴⁹ Rather, *mccΔ* cells displayed defective mitochondrial functionality following GD and, more specifically, problematic mitochondrial membrane potential (MMP). More specifically, *mccΔ* cells showed defective targeting of Pre-Cox4-mCherry to mitochondria, a process known to require intact MMP,⁵⁰ comparable to WT cells treated with the MMP uncoupler CCCP (Figure 3C). This phenotype was also observed in cells with vesicular mitochondrial morphology. This indicates that, in the absence of eisosomes, mitochondria are not fully functional PGD, even in cells displaying a “respiratory-competent” phenotype.

As mutants with defective respiration are known to age and die much faster,⁶ we next sought to characterize the role of

<caption>**Figure 3.** Eisosomes promote respiratory growth and are required for long-term quiescence maintenance by stabilizing flavodoxin-like proteins</caption>

(A) Line chart of WT, *pil1Δ/sp1Δ* (*mccΔ*), *atp1Δ*, and *pst2Δ*γcp4Δ*frs1Δ* (*flpΔ*) cultures. Cell density measurements at different time points in EXP and PGD, from five independent experiments, are plotted as mean ± SD. ***p < 0.001; **p < 0.01, from two-way ANOVA.

(B) Percentage (w/v) ethanol measurements in supernatants of WT, *pil1Δ/sp1Δ* (*mccΔ*), and *atp1Δ* cultures, from four to six independent experiments at different time points PGD, are plotted as mean ± SD. ***p < 0.001; **p < 0.01; *p < 0.05, from two-way ANOVA.

(C) Left: schematic representation of the mitochondrial membrane potential (MMP) microscopy-based assay. WT, *pil1Δ/sp1Δ* (*mccΔ*), *pst2Δ*γcp4Δ*frs1Δ* (*flpΔ*), and *atp1Δ* strains were transformed with pMitoLoc plasmid⁵⁰ encoding Pre-SU9-GFP and Pre-Cox4-mCherry. Pre-SU9-GFP is targeted constitutively to the mitochondrial lumen, while targeting of Pre-Cox4-mCherry requires intact MMP.⁵⁰ Middle: middle-section merged confocal microscopy images of the indicated strains treated or not for 6 h with 30 µM CCCP and mutant strains, at EXP or at GD. Right: quantification: the single-cell Pearson correlation coefficient (PCC) of the two fluorescence signals (n = 38–232) is plotted in box-and-whiskers plots. ***p < 0.001; **p < 0.01; ns, non-significant, p > 0.05, from one-way ANOVA.

(D) Growth recovery of WT, *pil1Δ/sp1Δ* (*mccΔ*), and *pst2Δ*γcp4Δ*frs1Δ* (*flpΔ*) strains from different time points PGD, as percentage of colony-forming units (CFUs) from 3 to 19 independent experiments, are plotted in box-and-whiskers plots. ***p < 0.001; *p < 0.05 from unpaired t tests.

(E) Eisosomes are required for maintenance of quiescence. Middle-section confocal microscopy images of Ilv3-GFP-expressing WT and *pil1Δ/sp1Δ* (*mccΔ*) strains at GD and 7 days PGD (detailed in Figure S3D). Quantification: the percentages of WT and *pil1Δ/sp1Δ* (*mccΔ*) cells displaying cortical (quiescent, Q) or globular (senescent, S) mitochondria or lack of Ilv3-GFP fluorescence (dead, D) (as in Figures 2B and 2C) at different time points PGD are plotted in stacked bar charts. Data from two to four independent experiments are represented as mean ± SD. **p < 0.01; *p < 0.05, from two-way ANOVA.

(F) Volcano plot (Student's t difference vs. -log₁₀(p value)) comparison of the proteome of *pil1Δ/sp1Δ* (*mccΔ*) vs. WT cells at 3 days PGD (Table S4). Proteins with significant >0.50 (magenta) and < -0.50 (green) log₂(fold change) are indicated. Eisosomal proteins, proteins related to ROS, and proteins differing most are highlighted.

(G) Middle-section confocal microscopy images of strains expressing Pil1-mCherry and GFP-tagged versions of Ycp4, Rfs1, or Pst2 at GD. Quantification: the single-cell Pearson correlation coefficient (PCC) values from surface sections of the two fluorescence signals (n = 67–98) are plotted in box-and-whiskers plots.

(H) Western blots of total protein extracts from WT and *mccΔ* strains expressing GFP-tagged versions of Ycp4, Rfs1, or Pst2 at EXP and different time points PGD probed with anti-GFP and anti-actin. Quantification: signal intensities, normalized to actin, from two or three independent experiments are plotted in line charts as mean fold change ± SD from WT EXP samples. ***p < 0.001; **p < 0.01; *p < 0.05, from two-way ANOVA.

(I) Middle-section confocal microscopy from the strains and time points of (H). Quantification: the single-cell membrane-to-total fluorescence intensities (n = 40–117) are plotted in box-and-whiskers plots. ***p < 0.001; ns, p > 0.05, from one-way ANOVA. Scale bars: 1 µm. See also Figure S3.
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eisosomes in quiescent cell survival. In agreement with previous reports,²,⁶ growth recovery of the WT was close to 80% the first week PGD and remained high for at least 1 month. In contrast, the *mccΔ* and other strains lacking MCC organizers displayed only 50% survival after the first week, and this dropped to 15% within 1 month (Figures 3D and S3C). The reduced long-term survival was further confirmed at the single-cell level using the morphology of the mitochondrial network (Figures 3E and S3D). *mccΔ* cells seemed fully capable of forming vesicular mitochondria following GD, indicating no defects in initial quiescent cell formation. However, the percentage of quiescent cells containing vesicular mitochondria declined over time much faster than that of the WT. The mutant population acquired significantly increased percentages of both senescent cells containing globular mitochondria and non-fluorescent dead cells (Figures 3E and S3D). Overall, these results show that eisosomes support respiratory growth after GD and are required for long-term quiescence maintenance and growth recovery.

## Eisosomes promote quiescence maintenance by stabilizing the flavodoxin-like proteins

For characterizing how eisosomes affect quiescence maintenance, we compared the proteome of WT and *mccΔ* cells (Figure 3F and Table S4) during the ethanol utilization phase (Figure 1B). This analysis identified 59 downregulated and 90 upregulated proteins in the mutant (Table S4). Fifty-eight percent of the downregulated proteins overlap with the PGD-upregulated proteins (Figure S3E), suggesting partially defective adaptation to quiescence. Indeed, proteins involved in important quiescence-related biological processes³ (Figure 3G), such as respiration-related metabolism of carbohydrates and fatty acids, were enriched in the downregulated proteins of the *mccΔ* strain. As expected, the top category identified was eisosome assembly. Intriguingly, the dataset of proteins downregulated in the *mccΔ* was enriched in proteins involved in reactive oxygen species (ROS) metabolism and detoxification. The downregulation of these processes after the PGD shift to respiration, when increased mitochondrial ROS production occurs,⁷ could contribute to the defective quiescence preservation of the *mccΔ* (Figure S3F). Our attention was drawn by Rfs1, an eisosome-resident peripheral membrane protein with a potential antioxidant role.¹⁰,⁴³ Rfs1 is one of the three (along with Pst2 and Ycp4) poorly characterized FLPs of yeast.¹⁰ All three FLPs were upregulated PGD (Figure 1C), suggesting an important quiescence-specific role. As during fermentation,¹⁰ FLPs showed mainly eisosomal localization PGD (Figure 3G), while they were many-fold induced in quiescent cells (Figure 3H). Ycp4 displayed cytoplasmic and eisosomal localization, while Pst2 and Rfs1 were found at the plasma membrane and showed enrichment in eisosomes (Figures 3G and 3I). Most importantly, in *mccΔ* cells, all three FLPs were significantly less abundant (Figure 3H); Pst2 and Rfs1 became homogeneous at the plasma membrane, while Rfs1 and especially Ycp4 additionally showed defective recruitment to the plasma membrane (Figure 3I). Although the mechanism of plasma membrane/eisosomal recruitment of FLPs is unknown, Ycp4 is palmitoylated,⁵¹ and this modification could assist its tethering to membranes. The above results show that the total levels of FLPs at the plasma

membrane of the *mccΔ* strain are much reduced. To determine whether the phenotypes of the *mccΔ* strain could be attributed to the lower levels of FLPs, we examined the phenotype of the FLP-lacking (*flpΔ*) strain. The *flpΔ* not only phenocopied the *mccΔ* for defective PGD growth, long-term survival, and MMP, but also showed stronger defects (Figures 3A, 3C, and 3D). These defects were not due to impaired eisosome assembly (Figure S3G). Consistently, the PGD proteome of the *flpΔ* strain showed, similar to that of the *mccΔ*, defective induction of proteins involved in mitochondrial respiration and the metabolism of carbohydrates and fatty acid acids (Figures S3H, S3I, and Table S4). We conclude that stabilization of the FLPs at the plasma membrane is an important function of eisosomes for promoting long-term quiescence maintenance.

## Eisosomes and flavodoxin-like proteins protect quiescent cells from lipid peroxidation and ferroptosis

Next, we characterized how the lack of FLPs affects quiescent cells. Pst2 shows reductase activity against several quinones, including ubiquinone.⁵² The reduced form of ubiquinone, ubiquinol, is part of the respiratory chain but also a lipophilic antioxidant protecting yeasts from lipid peroxidation, with roles in extramitochondrial membranes.⁴⁰,⁴¹ We hypothesized that FLPs could act analogous to the plasma-membrane-localized ubiquinol-generating Fsp1 reductase, which protects human cells from ferroptosis.³⁴,³⁵ Consistently, *mccΔ* cells and especially *flpΔ* cells were more sensitive than the WT to supplementation with PUFAs (linolenic acid [LNA] and linoleic acid [LA]), which are more prone to peroxidation,⁵³ in respiratory media (Figures 4A and S4A). Among the FLPs, deletion of *PST2* caused the most pronounced effect, while additional deletion of *RFS1* and *YCP4* further enhanced LNA sensitivity (Figure S4B). The sensitivity of the *mccΔ* to LNA could be complemented by ectopic expression of *PIL1* and, to a lesser extent, *LSP1* (Figure S4C). Hyper-sensitivity was more evident to PUFAs than to monounsaturated (MUFAs) or saturated fatty acids (Figure S4A), pointing to accumulation of peroxidized phospholipids at the plasma membrane of the mutants. To directly measure peroxidized phospholipids, we performed targeted lipidomics of PGD cells from cultures supplemented or not with either MUFAs (oleic acid, OA) or PUFAs (LA or LNA). We determined the peroxidation of the plasma-membrane-enriched phospholipid phosphatidylethanolamine (PE) as a readout, since PE can incorporate exogenous fatty acids into its backbone,⁵⁴ while peroxidation of PE is considered the main driver of ferroptosis in human cells.⁵⁵ Indeed, externally supplied MUFAs and PUFAs were incorporated into the PE of all strains to similar levels (Figure S4D). Most importantly, 2 days PGD, we could identify a several-fold increase in peroxidized PUFA-containing PE, specifically in *mccΔ* and *flpΔ* cells grown in the presence of PUFAs (Figures 4B and 4C). These results strongly indicate an important role of FLPs and ubiquinol in counteracting lipid peroxidation, mainly in respiratory conditions and quiescent cells.

Consistent with the above view, *flpΔ* and *mccΔ* cells showed only limited sensitivity to LNA in glucose medium. Moreover, they were less sensitive to LNA than the *coq3Δ* strain (Figure 4A), which totally lacks ubiquinone and is hypersensitive to lipid peroxidation.⁴¹ This mild phenotype could be explained by a
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contribution of the Gpx1/2/3 glutathione peroxidases reported to counteract lipid peroxidation during fermentation.³⁹ Indeed, a strain carrying deletions of *GPX1/2/3* (*gpxΔ*) showed hypersensitivity to LNA in glucose but was less sensitive to LNA than the *flpΔ* in respiratory conditions (Figure 4D). These results suggest that Gpx1/2/3-glutathione is the principal lipid-ROS scavenging mechanism during fermentation, while during quiescence FLP-ubiquinol have a more prominent role. In support of FLPs acting in parallel with the Gpx1/2/3 to protect quiescent cells from peroxidation, a strain carrying deletion of all six genes (*gpxΔflpΔ*) displayed synthetic hypersensitivity in respiratory conditions (Figure 4D). We further confirmed genetically that FLPs were essential for the extramitochondrial role of ubiquinone in counteracting lipid peroxidation, by deleting *CQD1*, which is required for mitochondrial import of ubiquinone⁴⁰ (Figure 4D). Lack of *Cqd1* results in excess ubiquinone export to extramitochondrial membranes and rescues the hypersensitivity of the *gpxΔ* strain to LNA.⁴⁰ Importantly, deletion of *FLPs* was epistatic over this *Cqd1*-dependent rescue of the LNA hypersensitivity of the *gpxΔ* (Figure 4D), strongly indicating that FLPs are involved in the regeneration of ubiquinol in extramitochondrial membranes. Consistently, we could detect a decrease in the ratio of ubiquinol/ubiquinone in total cell extracts, specifically for PUFA-supplemented *mccΔ* and *flpΔ* cells, shortly after GD (Figure 4E). Yet, this decrease recovered later on, consistent with the presence of additional ubiquinone reductases in mitochondria that are upregulated PGD (Table S1).⁵⁶ The above shows that FLPs are the main extramitochondrial ubiquinone reductases in PUFA-supplemented yeasts upon GD.

The results above highlight the importance of FLPs in counter-
acting lipid peroxidation in PUFA-containing cultures, consistent
with PUFAs being more prone to peroxidation.⁵³ Although yeast
does not biosynthesize PUFAs, our results (Figure 3) reveal an
essential role for eisosomes and FLPs in quiescent cells without

PUFA supplementation, suggesting that the phenotypes could
be caused by peroxidation of MUFAs. Indeed, we could detect
a 2.5-fold increase in peroxidized PE-incorporated MUFA phos-
pholipids in the *flpΔ* mutant in non-supplemented cultures upon
GD (Figure 4F). Interestingly, this difference was not evident at
later time points. In an effort to counteract this initial accumula-
tion of MUFA peroxides, we treated the cultures with the lipid
peroxidation scavenger *α*-tocopherol.⁴¹ Importantly, *α*-tocoph-
erol treatment could significantly rescue the defects in PGD
growth and long-term growth recovery of mutants lacking
FLPs or MCCs (Figures 4G, 4H, and S4E and Table S5). Signifi-
cant *α*-tocopherol rescue was also observed in ubiquinone-lack-
ing *coq3Δ* mutants but not in WT or *atp1Δ* respiration-deficient
cells, indicating that the rescue concerns peroxidation-protect-
tive roles of ubiquinone and not respiratory-chain-related roles.

The above findings indicate that an initial accumulation of
MUFA peroxides is responsible for the reduced long-term sur-
vival of the *flpΔ* and *mccΔ* strains. They also suggest that quies-
cent yeasts without PUFA supplementation do not succumb to
ferroptosis. However, we could identify nearly 100% cell death
in *gpxΔflpΔ* cells within 2 days of supplementation with the
PUFA LA at 10 mM (Figure 4I). This PUFA-induced cell death
was totally reversed by co-treatment with *α*-tocopherol, strongly
pointing to peroxidation of lipids as the cause. Co-treatment with
the Fe²⁺ chelator bathophenanthroline disulfonic acid disodium
salt hydrate (BPS) could similarly largely rescue the PUFA-
induced cell death and, to a lesser extent, the growth recovery
of the *gpxΔflpΔ* (Figures 4I and S4F). This suggests that iron is
essential for peroxidation of PUFA-containing phospholipids in
quiescent yeasts. Nearly identical results were obtained for
*gpxΔ* cells under the same conditions (Figure 4I), confirming
that Gpx1/2/3 have the major peroxidation-protective role during
fermentation. Importantly, *mccΔ* and *flpΔ* populations showed
10% and 17% PUFA-induced cell death, respectively, under

<caption>**Figure 4. Eisosomes and FLPs protect quiescent cells from lipid peroxidation and ferroptosis**</caption>

(A) Drop tests of WT, *pil1Δsp1Δ* (*mccΔ*), *pst2Δycp4Δrfs1Δ* (*flpΔ*), and *coq3Δ* strains in minimal medium with glucose or lactic acid as the carbon source, supplemented or not with 0.1 mM linolenic acid (LNA).

(B) Heatmap of the percentage abundance of each oxidized (<O>) and peroxidized (<2O>) PE species normalized to total PE in PUFA (LA, LNA)- and MUFA (OA)-supplemented and unsupplemented WT, *mccΔ*, and *flpΔ* cells. The coloring threshold was set to 0.1% abundance.

(C) The percentage abundance of the total oxidized and peroxidized PE detected in PUFA (LA, LNA)- and MUFA (OA)-supplemented and unsupplemented WT, *mccΔ*, and *flpΔ* cells is plotted as box and whiskers. \*\*\*p < 0.01; \*p < 0.05; ns, p > 0.05, from unpaired t tests.

(D) Drop tests of WT, *gpx1Δgpx2Δgpx3Δ* (*gpxΔ*), *pst2Δycp4Δrfs1Δ* (*flpΔ*), *gpxΔflpΔ*, *coq3Δ*, *cqd1ΔgpxΔ*, and *cqd1ΔgpxΔflpΔ* strains in minimal medium with glucose or lactic acid as the carbon source, supplemented or not with 0.1 mM LNA. Scheme: graphical representation of the interpretation of the drop tests. *Cqd1* is involved in the import of ubiquinone (CoQ6) in mitochondria, and *cqd1Δ* rescues the PUFA hypersensitivity of *gpxΔ* cells by excess ubiquinone export from the mitochondrial membrane (MM) to extramitochondrial membranes (EMM).¹⁰ Additional lack of FLPs is epistatic over *Cqd1*-dependent rescue of the PUFA hypersensitivity of *gpxΔ* cells, indicating that FLPs are required for extramitochondrial ubiquinol (CoQ6H2)-mediated protection from lipid peroxidation.

(E) The ratio of ubiquinol (CoQ6H2) to ubiquinone (CoQ6) in total cell extracts detected in unsupplemented or PUFA (6 mM LA)-supplemented WT, *mccΔ*, and *flpΔ* cells 1 and 3 days PGD is plotted as box and whiskers. \*\*\*p < 0.001; \*p < 0.05; ns, p > 0.05, from unpaired t tests.

(F) The percentage abundance of the total oxidized and peroxidized PE detected in unsupplemented WT and *flpΔ* quiescent cells upon glucose depletion (GD) and 2, 4, and 6 days PGD is plotted as box and whiskers. \*p < 0.05; ns, p > 0.05, from unpaired t tests.

(G) Line charts of WT, *pil1Δsp1Δ* (*mccΔ*), *pst2Δycp4Δrfs1Δ* (*flpΔ*), *atp1Δ*, and *coq3Δ* yeast cultures in minimal medium, split and supplemented or not with 0.5 mM *α*-tocopherol (+*α*-TOH) at OD₆₀₀ 3. Cell density measurements at different time points in EXP and PGD from five independent experiments are plotted as mean ± SD. Statistical analysis from two-way ANOVA is provided in Table S5.

(H) Growth recovery of WT, *pil1Δsp1Δ* (*mccΔ*), *pst2Δycp4Δrfs1Δ* (*flpΔ*), *gpxΔ*, and *gpxΔflpΔ* yeast cultures in minimal medium supplemented or not with 1 mM *α*-tocopherol (+*α*-TOH) at OD₆₀₀ 3, as percentage of CFU from 3 to 10 independent experiments, is plotted in box-and-whiskers plots. \*\*\*p < 0.001; \*\*p < 0.01; \*p < 0.05, from unpaired t tests.

(I and J) Percentage of dead, methylene blue-stained cells at 2 days PGD of WT, *gpxΔ*, and *gpxΔflpΔ* strains and at 2 and 4 days PGD for WT, *mccΔ*, and *flpΔ* cells treated or not with 10 mM linoleic acid (LA) and 1 mM *α*-tocopherol (*α*-TOH) or 100 µM iron chelator (BPS) at OD₆₀₀ 3. Data from two to eight independent experiments are plotted in box-and-whiskers plots. \*\*\*p < 0.001; \*\*p < 0.01; \*p < 0.05, from unpaired t tests. See also Figure S4.
<figure><img src="image_51.png" /><figcaption>Figure 5. Eisosomes and FLPs protect quiescent yeasts from ferroptosis in parallel with glutathione peroxidases</figcaption></figure>

Schematic summary of the results. Eisosomes promote quiescence maintenance by stabilizing FLPs. FLPs counteract ferroptosis and cell death by re-generating ubiquinol (CoQ₆H₂), thereby inhibiting the lipid-radical (PLOO') autoxidation of MUFAs and PUFAs,³⁴,³⁵ similar to α-tocopherol (α-TOH). In parallel, the Gpx1/2/3 glutathione (GSH) peroxidases reduce peroxidized MUFAs and PUFAs (PLOOH) to lipid alcohols (PLOH).³³,³⁹ The iron chelator (BPS) rescues from ferroptosis in PUFA-supplemented cultures.

the same conditions. For the *flpΔ*, this percentage further increased to 21% within two additional days in quiescence (Figure 4J). This cell death could similarly be reversed by treatment with either α-tocopherol or BPS.

We conclude that the eisosome-resident, ubiquinol-generating FLPs act in parallel with Gpx1/2/3 to prevent quiescent yeast cell death induced by peroxidation of PUFA and MUFA phospholipids (Figure 5). In the presence of exogenously supplied PUFAs, lipid peroxides accumulate in an iron-dependent way and can lead to cell death of a strain lacking FLPs and/or Gpx1/2/3, pointing to the existence of ferroptosis in PUFA-supplemented quiescent yeasts.

DISCUSSION

In this work, we identify MCCs/eisosomes as a highly expanding plasma membrane compartment in quiescence. We further show that eisosomes are essential for quiescence maintenance and the long-term survival of quiescent cells. The ubiquinol-generating,⁵² eisosome-resident FLPs, in particular, protect quiescent yeasts from phospholipid peroxidation. The FLP Pst2 affects survival in the stationary phase,⁵⁷ but the mechanism remained unknown. Our results are consistent with the high abundance of eisosomes in quiescent conidiospores and ascospores of *Aspergillus nidulans*,⁵⁸,⁵⁹ the peroxidation-protective roles and the increased levels of ubiquinol in stationary phase,⁴⁰,⁴¹,⁶⁰ and the known antioxidant roles of FLPs in *Candida albicans* against exogenous quinones and PUFAs.⁴³,⁶¹ The absence of eisosomes causes similar but less severe phenotypes than the lack of FLPs (Figures 3A, 3C, and 3D), consistent with the reduced membrane abundance/recruitment of FLPs in

the *mccΔ* (Figures 3H and 3I). Yet, it cannot be excluded that eisosomes have additional, FLP-independent roles in protection from lipid peroxidation. For example, the abundance of Gpx3/Hyr1, which reduces hydroperoxyphospholipids³⁹ and contributes to protection from ferroptosis (Figure 4), is reduced in quiescent *mccΔ* cells (Figure 3F), but the underlying mechanism remains elusive. Other properties of eisosomes, like membrane curvature, the particular lipidic composition,¹³⁻¹⁵,⁶² and TORC2 signaling,²⁰,²¹ could additionally affect protection from peroxidation. Interestingly, our proteomics data suggest previously unnoticed interactions between eisosomes and fatty acid oxidation (Figures S3F and S3I), a process potentially involved in sensitivity to lipid peroxidation.⁶³

In the presence of PUFAs, we detected increased levels of peroxidized PUFA-containing PE, which is considered a main driver of ferroptosis in human cells.⁵⁵ PUFA-mediated phospholipid peroxidation seems iron dependent and can lead to cell death of a strain lacking both Gpx1/2/3 and FLPs (Figures 4I, 4J, and S4F). These two mechanisms, namely the one of glutathione and glutathione peroxidases and the one of ubiquinol and FLPs, act in parallel to protect yeasts from peroxidation under different physiological conditions (Figure 4D). The analogy to Gpx4 and Fsp1 acting in parallel in human cells³⁴,³⁵ strongly indicates that ferroptosis-like mechanisms are conserved in fungi, and they are important for quiescent fungal cell survival. As quiescent yeasts are critical for environmental spreading,⁶⁴ ferroptosis-protective mechanisms could be essential in LA-containing natural yeast habitats, like grapes. Ferroptosis-protective mechanisms could additionally have important roles in other PUFA-biosynthesizing/incorporating fungi, including pathogenic fungi. Interestingly, we have identified homologs of human Fsp1 in several PUFA-biosynthesizing filamentous fungi, including two homologs in *A. nidulans*, which are under investigation (V.S. and C.G., unpublished data). Quiescent yeasts are implicated in the development of persistence, a resistance to antimicrobial compounds targeting only growing cells.⁶⁵ Given that eisosomes are fungal-specific structures and abundantly expressed in dormant cells/spores of other fungal species as well,⁹,⁵⁸,⁵⁹,⁶⁶ eisosomal proteins could be promising targets for the development of novel, growth-independent fungal treatment strategies.

An important aspect of our work is the link of MUFA peroxidation with survival defects. MUFA peroxidation is considered less efficient than PUFA peroxidation, while MUFA supplementation promotes a ferroptosis-resistant state.⁵³,⁶³ Surprisingly, our results indicate that MUFA peroxidation can occur in the absence of PUFAs and can lead to growth defects that are rescued by addition of α-tocopherol (Figures 4B, 4C, 4F, 4G, and 4H). Yet, increased MUFA peroxidation occurs only shortly after GD, while MUFA peroxides do not accumulate in quiescent cells and do not cause rapid cell death (Figures 4B, 4C, 4F, and 4H). These results suggest that other mechanisms should counteract the accumulation of MUFA-lipid peroxides, yet these are not sufficient to rescue the defective long-term survival of *mccΔ* and *flpΔ* quiescent cells. Determining whether MUFA peroxidation is iron dependent is not feasible under current experimental conditions, since iron chelation would affect respiration and consequently quiescence. Interestingly, other transition metals have previously been connected to lipid peroxidation in yeast.³⁸
An important remaining question is why FLPs are localized in MCCs. Although the exact lipidic composition of MCCs is still lacking, evidence suggests that MCCs are enriched in sterols and phospholipids, including unsaturated phospholipids,¹³,¹⁵,¹⁹,⁶² and depleted in the saturated-fatty-acid-containing sphingolipids.¹⁴,⁶⁷,⁶⁸ Such a lipidic composition would make MCCs more sensitive to peroxidation than the rest of the plasma membrane, thus requiring increased levels of active, ubiquinol-producing FLPs. This view is also consistent with the membrane-tension-responsive roles of eisosomes,²⁰,²¹,⁶² as accumulation of lipid peroxides increases membrane tension and activates mechanosensitive channels as executors of mammalian ferroptotic cell death.⁶⁹ Importantly, a mechanosensitive channel in S. pombe has recently been reported to localize to eisosomes in an inactive state,⁷⁰ strongly favoring eisosomes as low-membrane-tension domains. Together with our work, these data propose that in fungi, too, activation of mechanosensitive channels might be the executors of ferroptosis.

Lipid peroxidation in human cells has been previously connected to defective mitochondrial function, but the causal relationship remained controversial and context dependent.⁷¹,⁷² In our conditions, α-tocopherol supplementation rescued the defects in PGD growth and growth recovery (Figures 4G and 4H), indicating that counteracting lipid peroxidation could rescue impaired mitochondrial function. It remains to be shown whether this defective mitochondrial function is due to accumulation of phospholipid peroxides in the organelle, as PUFA phospholipids accumulate there and are beneficial for respiratory growth,⁷³ or an adaptation mechanism to reduce the production of ROS.

Finally, our work uncovers a previously unnoticed relationship between ferroptosis-protective plasma membrane compartments and quiescence. Despite eisosomes not being conserved in humans, caveolae, which show structural and functional analogies,¹⁶ have recently been reported to be involved in ferroptosis.⁷⁴⁻⁷⁶ Another interesting analogy is the metabolism of certain cancer stem cells that, similar to quiescent yeasts, rely on oxidative phosphorylation rather than glycolysis.⁷⁷ As our results suggest that ubiquinol-dependent protection from ferroptosis is more important in quiescence than in fermentation (Figures 4D, 4I, and 4J), it would be interesting to exploit inhibition of ubiquinol-dependent ferroptosis-protective mechanisms as an approach to target quiescent cancer cells, which are resistant to conventional treatments.⁷⁷,⁷⁸

## Limitations of the study

In this study we used PE as a general readout for lipid peroxidation as it is known to be enriched at the plasma membrane and as a main driver for ferroptosis.⁵⁵ Our approach does not measure all peroxidized lipids and does not determine the subcellular localization of the lipids measured. Similarly, our method for the measurement of ubiquinol and ubiquinone does not determine which parts are in mitochondria or extramitochondrial membranes. Fractionation of membranes would have been challenging, as the hypersensitivity of ubiquinol to oxidation by air necessitates a one-step extraction protocol for total cellular lipids. Regarding long-term survival of *mccΔ* and *flpΔ* cells not supplemented with PUFAs, our data show that they do not die due to ferroptosis, and the mechanism involved remains to be determined.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

* Materials availability

* Data and code availability

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* METHOD DETAILS

* Strains, plasmids, and growth conditions

* Confocal microscopy, quantifications, and statistics

* Determination of the relationship between eisosome induction and the morphology of mitochondria

* Epifluorescence microscopy

* Western blotting

* Estimation of cell survival

* Proteomics

* Fatty acid supplementation and targeted lipidomics for Phosphatidylethanolamine

* Determination of ubiquinol/ubiquinone ratios

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2023.113561.

## ACKNOWLEDGMENTS

We thank T. Zakopoulou and V. Perpiniadis for technical assistance; Dr. S. Sharma, L. Santos Sousa, and Dr. G. Kapetanakis for fruitful discussions; S. Limar, S. Walter, and S. Schuback for assistance with the lipidomics experiments; and Dr. P. Syntichaki for providing materials. This research was co-financed by Greece and the European Union (European Social Fund-ESF) through the operational program "Human Resources Development, Education and Lifelong Learning 2014-2020" in the context of the project "Study of the role of eisosomal proteins in the quiescent state of fungal cells" (MIS 5047827) awarded to C.G. and V.S. and by a grant from the Fondation Santé awarded to C.G. F.F. is a member of the DFG Heisenberg program (FR 3647/4-1) and is supported by SFB1557. We acknowledge support of this work from the project "The Greek Research Infrastructure for Personalised Medicine (pMED-GR)" (MIS 5002802), which is implemented under the action "Reinforcement of the Research and Innovation Infrastructure," funded by the operational program "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). A.H.M. was supported by a doctoral fellowship co-financed by Greece and the European Union (European Social Fund-ESF) through the operational program "Human Resources Development, Education and Lifelong Learning" in the context of the act "Enhancing Human Resources Research Potential by undertaking a Doctoral Research" sub-action 2: IKY Scholarship Programme for PhD candidates in the Greek Universities, and by an EMBO Scientific Exchange grant (9533). We dedicate this article to the memory of Dr. G. Thireos.

## AUTHOR CONTRIBUTIONS

A.H.M. and C.G. designed the project. A.H.M. performed and analyzed most of the experiments. B.M.E. contributed in performance and analyses of targeted (oxi)lipidomics and ubiquinol measurements. A.A. contributed in confocal
microscopy and developed scripts for image analysis. D.K. performed and analyzed experiments about ferroptosis. M.M., V.L., M.S., and J.Z. performed measured and analyzed proteomics. M.S. and J.Z. designed proteomics. V.S. provided critical advice on experimental design and data interpretation. B.A. made critical conceptual reorientations of the project. F.F. designed, analyzed, and supervised targeted (oxi)lipidomics and proteomics. C.G. supervised ex-periments and analyzed data. A.H.M. and C.G. prepared the manuscript. All authors discussed the results, contributed to the manuscript, and agreed on the content of the paper.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: April 9, 2023

Revised: November 2, 2023

Accepted: November 22, 2023

Published: December 12, 2023

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse anti-GFP, monoclonal (1:10000)</td><td>Roche</td><td>RRID:AB_390913</td></tr><tr><td>Mouse anti-mCherry, monoclonal (1:10000)</td><td>Abcam PLC</td><td>RRID:AB_11133266</td></tr><tr><td>Mouse anti-actin (1:1000)</td><td>Santa Cruz Biotechnology</td><td>RRID:AB_626632</td></tr><tr><td>Anti-mouse IgG, HRP-linked (1:1000-10000)</td><td>Cell Signaling technology</td><td>RRID:AB_2940774</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>E. coli DH5α</td><td>CGSC</td><td>14231</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>(+)-α-Tocopherol</td><td>Sigma-Aldrich</td><td>T3634</td></tr><tr><td>Oleic acid</td><td>Sigma-Aldrich</td><td>O1383</td></tr><tr><td>Linoleic acid</td><td>Sigma-Aldrich</td><td>L1012</td></tr><tr><td>Linolenic acid</td><td>Sigma-Aldrich</td><td>L2376</td></tr><tr><td>Stearic acid</td><td>Sigma-Aldrich</td><td>S4751</td></tr><tr><td>Bathophenanthroline disulfonic acid disodium salt hydrate (BPS)</td><td>Sigma-Aldrich</td><td>146617</td></tr><tr><td>Percoll</td><td>GE Healthcare, Chicago, Illinois, US</td><td>GE17-0891-02</td></tr><tr><td>Iodixanol</td><td>Stemcell technologies, Vancouver, Canada</td><td>07820</td></tr><tr><td>μ-Slide 8 Well</td><td>Ibidi, Gräfelfing, Germany</td><td>80826</td></tr><tr><td>Complete EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11-697-498-001</td></tr><tr><td>Trypsin; proteomics grade</td><td>Sigma-Aldrich</td><td>T7575</td></tr><tr><td>Ceramide, 17:0-18:1</td><td>Avanti Polar Lipids, Boston, USA</td><td>860517</td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 16:0-18:1 PE</td><td>Avanti Polar Lipids, Boston, USA</td><td>850757</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>GOD/PAP kit</td><td>Biotechnological Applications L.T.D., Athens, Greece</td><td>N/A</td></tr><tr><td>Ethanol measurement kit</td><td>Abcam PLC, Cambridge, UK</td><td>ab272531</td></tr><tr><td>iST protein preparation kit</td><td>Preomics, Planegg/Martinsried, Germany</td><td>P.O.00001</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>MS raw data of Figure 1</td><td>ProteomeXchange</td><td>PXD041191</td></tr><tr><td>MS raw data of Figure 3</td><td>ProteomeXchange</td><td>PXD041123</td></tr><tr><td>Workflow for lipidomics, Table S8</td><td>LSI</td><td>N/A</td></tr><tr><td>Original data</td><td>Mendeley data</td><td>https://doi.org/10.17632/jbzdh6vhnw.1</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>S. cerevisiae: Strain background: Sigma1278b</td><td>André laboratory</td><td>MATα</td></tr><tr><td>23344c</td><td>André laboratory</td><td>[Sigma1278b] ura3</td></tr><tr><td>AM029</td><td>This study</td><td>[23344c] ura3::PIL1-4A-GFP-URA3 LSP1-mCHERRY-KANMX6 nce102Δ::HPHMX4 pil1Δ ura3</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>AM042</td><td>This study</td><td>[23344c] PIL1-GFP-CaURA3 LSP1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM043</td><td>This study</td><td>[23344c] LSP1-mCHERRY-KANMX6 pil1Δ ura3</td></tr><tr><td>AM045</td><td>This study</td><td>[23344c] ura3::PIL1-4A-GFP-URA3 LSP1-mCHERRY-KANMX6 pil1Δ ura3</td></tr><tr><td>AM101</td><td>This study</td><td>[23344c] fmp45Δ::HPHMX4 PIL1-mCHERRY-KANMX6 URA3</td></tr><tr><td>AM105</td><td>This study</td><td>[23344c] pst2Δ::HPHMX4 PIL1-mCHERRY-KANMX6 URA3</td></tr><tr><td>AM115</td><td>This study</td><td>pun1Δ::KiURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM117</td><td>This study</td><td>[23344c] pil1Δ URA3</td></tr><tr><td>AM119</td><td>This study</td><td>[23344c] nce102Δ::KiURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM121</td><td>This study</td><td>[23344c] PIL1-mCHERRY-KANMX6 URA3</td></tr><tr><td>AM129</td><td>This study</td><td>[23344c] Lsp1-GFP-CaURA3 pil1Δ ura3</td></tr><tr><td>AM139</td><td>This study</td><td>[23344c] LSP1-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM145</td><td>This study</td><td>[23344c] ILV3-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM164, AM165</td><td>This study</td><td>[23344c] lsp1Δ::HPHMX4 pil1Δ ura3</td></tr><tr><td>AM170</td><td>This study</td><td>[23344c] lsp1Δ::HPHMX4 PST2-GFP-CaURA3 pil1Δ ura3</td></tr><tr><td>AM174</td><td>This study</td><td>[23344c] YNL194C-mCHERRY-KANMX6 LSP1-GFP-URA3 pil1Δ ura3</td></tr><tr><td>AM186</td><td>This study</td><td>[23344c] lsp1Δ::HPHMX4 pil1Δ URA3</td></tr><tr><td>AM203</td><td>This study</td><td>[23344c] lsp1Δ::HPHMX4 ILV3-GFP-CaURA3 pil1Δ ura3</td></tr><tr><td>AM220</td><td>This study</td><td>[Sigma1278b] lsp1Δ::HPHMX4</td></tr><tr><td>AM237</td><td>This study</td><td>[23344c] nce102Δ::HPHMX4 PIL1-GFP-CaURA3 LSP1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM238</td><td>This study</td><td>[Sigma1278b] nce102Δ::HPHMX4</td></tr><tr><td>AM254</td><td>This study</td><td>[23344c] atp1Δ::HPHMX4 ILV3-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM282</td><td>This study</td><td>[23344c] rho0 ILV3-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM288</td><td>This study</td><td>[23344c] cox7Δ::HPHMX4 ILV3-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM290</td><td>This study</td><td>[23344c] cbs1Δ::HPHMX4 ILV3-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM292</td><td>This study</td><td>[23344c] atp1Δ::HPHMX4 ura3</td></tr><tr><td>AM294</td><td>This study</td><td>[Sigma1278b] atp1Δ::HPHMX4</td></tr><tr><td>AM296</td><td>This study</td><td>[23344c] PST2-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>AM317</td><td>This study</td><td>[23344c] gpx1Δ::KANMX6 gpx3Δ::KiURA3 gpx2Δ rfs1Δ ycp4Δ::NATMX4 pst2Δ::HPHMX4 ura3</td></tr><tr><td>AM323</td><td>This study</td><td>[23344c] gpx3Δ::KiURA3 gpx1Δ gpx2Δ rfs1Δ ycp4Δ::NATMX4 pst2Δ::HPHMX4 ura3</td></tr><tr><td>AM325</td><td>This study</td><td>[23344c] cqd1Δ::KANMX6 gpx3Δ::KiURA3 gpx1Δ gpx2Δ rfs1Δ ycp4Δ::NATMX4 pst2Δ::HPHMX4 ura3</td></tr><tr><td>AM345</td><td>This study</td><td>[23344c] cqd1Δ::HPHMX4 gpx2Δ::KiURA3 gpx1Δ::KANMX6 gpx3Δ::NATMX4 ura3</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>AM349</td><td>This study</td><td>[Sigma1278b] LSP1-GFP-KANMX6<br>nce102Δ::HPHMX4</td></tr><tr><td>CG116</td><td>Gournas et al., 2018<sup>23</sup></td><td>[23344c] lsp1Δ::HPHMX4 PIL1-mCHERRY-KANMX6<br>can1Δ gap1Δ ura3</td></tr><tr><td>CG210</td><td>This study</td><td>[23344c] ynl194cΔ::KiURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>CG254</td><td>This study</td><td>[23344c] PAB1-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>CG271</td><td>This study</td><td>[23344c] rfs1Δ::KANMX6 ycp4Δ::NATMX4<br>pst2Δ::KANMX6 URA3</td></tr><tr><td>CG272</td><td>This study</td><td>[23344c] RFS1-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>CG277</td><td>This study</td><td>[Sigma1278b] coq3Δ::HPHMX4</td></tr><tr><td>CG280</td><td>This study</td><td>[23344c] rfs1Δ::KANMX6 ycp4Δ::NATMX4<br>pst2Δ::HPHMX4 ura3</td></tr><tr><td>CG281</td><td>This study</td><td>[23344c] YCP4-GFP-KiURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>CG282</td><td>This study</td><td>[23344c] RFS1-GFP-KANMX6 lsp1Δ::HPHMX4 pil1Δ<br>URA3</td></tr><tr><td>CG309</td><td>This study</td><td>[23344c] YCP4-GFP-CaURA3 lsp1Δ::HPHMX4 pil1Δ<br>ura3</td></tr><tr><td>CG317</td><td>This study</td><td>[23344c] FTR1-GFP-CaURA3 PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>CG319</td><td>This study</td><td>[23344c] FTR1-GFP-CaURA3 PIL1-mCHERRY-KANMX6 rfs1Δ ycp4Δ::NATMX4 pst2Δ::HPHMX4 ura3</td></tr><tr><td>CG371</td><td>This study</td><td>[Sigma1278b] gpx1Δ::HPHMX4 gpx3Δ::NATMX4<br>gpx2Δ:: KANMX6</td></tr><tr><td>GK022</td><td>This study</td><td>[23344c] PIL1-mCHERRY-KANMX6 ura3</td></tr><tr><td>SG035</td><td>Gournas et al., 2018<sup>23</sup></td><td>[23344c] PIL1-mCHERRY-KANMX6 can1Δ gap1Δ<br>ura3</td></tr></tbody></table>

Oligonucleotides

Included in Table S8

N/A

Recombinant DNA

<table><tr><td>pCG118</td><td>Gournas et al., 2018<sup>23</sup></td><td>CEN-ARS NCE102-5GA-GFP-URA3</td></tr><tr><td>pCG140</td><td>This study</td><td>CEN-ARS CAN1-(K5R/K13R/K42R/K45R/K47R/<br>K85R/K89R)-GFP-URA3</td></tr><tr><td>pFL038</td><td>Gournas et al., 2017<sup>79</sup></td><td>CEN-ARS-URA3</td></tr><tr><td>pAM3</td><td>This study</td><td>CEN-ARS-pMET25-PRESU9-GFP-pADH1-<br>PRECOX4-mCHERRY-URA3</td></tr><tr><td>pAM1</td><td>This study</td><td>CEN-ARS PIL1-URA3</td></tr><tr><td>pCG142</td><td>This study</td><td>CEN-ARS LSP1-5GA-GFP-URA3</td></tr><tr><td>pGFP-Atg8 URA3</td><td>Laboratory collection</td><td>CEN-ARS GFP-ATG8-URA3</td></tr></table>

Software and algorithms

<table><tr><td>Prism 8</td><td>GraphPad</td><td>https://www.graphpad.com/scientific-software/prism</td></tr><tr><td>FlJ1</td><td>NIH</td><td>https://imagej.nih.gov</td></tr><tr><td>Inkscape</td><td>Inkscape developers</td><td>https://inkscape.org</td></tr><tr><td>Perseus</td><td>Tyanova et al., 2016<sup>90</sup></td><td>https://maxquant.net/perseus/</td></tr><tr><td>MaxQuant</td><td>Cox and Mann, 2011<sup>81</sup></td><td>www.maxquant.org</td></tr><tr><td>VolcaNoseR2</td><td>Goedhart and Luijsterburg,<br>2020<sup>82</sup></td><td>https://goedhart.shinyapps.io/VolcaNoseR/</td></tr><tr><td>PANTHER</td><td>Mi et al., 2019<sup>83</sup></td><td>https://geneontology.org</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Excel</td><td>Microsoft, New Mexico, USA</td><td>https://www.microsoft.com</td></tr><tr><td>SciexOS</td><td>Sciex, Toronto, Canada</td><td>https://sciex.com/products/software/sciex-os-software</td></tr><tr><td>DIA-NN</td><td>Demichev et al., 2020<sup>84</sup></td><td>https://github.com/vdemichev/DiaNN</td></tr></tbody></table>

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christos Gournas (cgournas@spfb.brussels).

### Materials availability

All unique/stable reagents generated in this study are available from the Lead Contact.

### Data and code availability

* The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE Archives: PXD041191 and PXD041123. Source data for gels, images and graphs can be found in Mendeley Data: https://doi.org/10.17632/jbzdh6vhnw.1.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Saccharomyces cerevisiae strains used in this study are listed in the key resources table. They were grown and treated as described in method details below. Cloning procedures were carried out in E. coli DH5a.

## METHOD DETAILS

### Strains, plasmids, and growth conditions

The yeast strains used in this study (key resources table) come from strain Sigma1278b. Only prototrophic strains were used. Cells were grown at 29 °C on a minimal buffered medium pH 6.1,²³ with glucose 3% w/v or lactic acid 0.5% v/v as the carbon source and ammonium in the form of (NH₄)₂SO₄ (10 mM) as the nitrogen source. Quiescent cells were not formed in non-buffered YNB medium (unpublished results). For liquid cultures, 10⁴ cells/ml were inoculated from precultures in the same medium, for avoiding the lag phase and obtaining high repetitive measurements. Buffered, pH 6.1, YNB was used in the experiments shown in Figure S1E, as Pil1(4A) is not epistatic over the deletion of NCE102 in our medium.²³ NCE102-5xGA-GFP was expressed from a plasmid in a NCE102⁺ strain, since the tagged protein could not complement the eisosome disassembly phenotype of the nce102Δ strain specifically in quiescent cells (data not shown). For drop tests, cells from OD₆₀₀=8-10 [OD₆₀₀=1-2 in experiments with the gpxΔflpΔ, which has a slower growth rate and slows down growth earlier than the other strains, ending up having low cell densities in stationary phase (Figure S4E)], were pelleted, washed in water, diluted to OD₆₀₀=0.3, ternary diluted 3-fold, and 5 µL from the dilutions were spotted on agarose plates. The final concentrations of substances added to solid or liquid media were ethanol 1,18% w/v, glycerol 2% v/v, CCCP (30µM), stearic acid (S4751, 0.1 and 0.3 mM), Oleic acid (O1383, 0.3 mM, 1 mM, and 6 mM), Linoleic acid (L1012, 0.1-10 mM), Linolenic acid (L2376, 0.1-1 mM), α-tocopherol (T3634, 0.5, 1 mM), bathophenanthrolinedisulfonic acid (146617, BPS) 100 µM, unless otherwise stated. Chemicals were purchased from Sigma Aldrich (St. Louis, Missouri, USA) or Cayman Chemical Company (Ann Arbor, Michigan, USA). For survival of the gpxΔflpΔ upon linoleic acid (LA) treatment, 10 mM linoleic acid was added to exponentially growing cultures at an OD₆₀₀=3, in the presence or not of 1 mM α-tocopherol or 100 µM BPS. Knockout, GFP- and mCherry-tagged versions of all genes were generated by insertion of cassettes, amplified from plasmids with the oligonucleotides of Table S8, as previously reported,⁸⁵,⁸⁶ and verified by PCR with external primers and primers specific to the ORF of the respective gene and the selection marker. Plasmids used in this study are listed in the key resources table. rho0 cells were isolated by treating cells in liquid YPD medium with 10 µg/ml ethidium bromide for 24h at 30 °C, streaking in YPD and testing isolated colonies for growth in YPG (3% v/v Glycerol). rho0 cells were verified by staining of fixated cells with DAPI (1 µg/ml in PBS) for 5-10 min in the dark and washout with PBS prior to epifluorescence microscopy.
Cell density measurements were performed by measuring the OD₆₀₀ of the cultures, properly diluted to fit the linear range of a standard curve, which was calculated from PGD cultures of the *wt* by plotting cell numbers counted in a Neubauer slide with OD₆₀₀ values within the linear range (OD₆₀₀ < 0.5) of the spectrophotometer. Statistical significance was assessed using the repeated-measures two-way ANOVA analyses for matched values spread across a row, coupled to Holm-Sidak's multiple-comparisons test. (Prism, Graphpad, San Diego, CA, USA).

Glucose measurements were performed using the GOD/PAP kit (Biotechnological Applications L.T.D., Athens, Greece). Supernatant from cultures was diluted 15-fold in ultrapure water, and 10 µL was mixed with 200 µL reagent in a 96-well plate. Concentrations were normalized to a standard curve and plotted to the OD of the culture. The equation deriving from the scatter plot (Figure S1A) was used to calculate glucose concentrations of the average ODs per timepoint at Figure 1B.

Ethanol measurements were performed with the ab272531 kit (Abcam PLC, Cambridge, UK), according to the manufacturer's in-
structions, for 100 µL of supernatants deproteinated with TCA, from at least 4 independent biological replicate PGD cultures. OD at
580 nm was measured using an Infinite M200 plate reader (Tecan Group Ltd), and the ethanol concentrations were calculated from the
standard curve, after subtracting the background of ethanol-depleted culture supernatants. Statistical significance was assessed
using the repeated-measures two-way ANOVA analyses for matched values spread across a row, coupled to Holm-Sidak's multi-
ple-comparisons test. (Prism, Graphpad, San Diego, CA, USA).

Density gradient separation of cells was performed two ways, using either Percoll (GE Healthcare, Chicago, Illinois, US)² or Iodixanol (Stemcell technologies, Vancouver, Canada).⁸⁷ Briefly, 1.75 mL of Percoll solution (9V Percoll + 1V NaCl 1.5 M) was centrifuged in 2 mL tubes at 19,320 g, 20 °C with an angle rotor for 15 min to form continuous densities gradient (1.00-1.31 g/mL). 100 µL sample (2 ml form PGD cultures, precipitated, washed, and resuspended in 1 M Tris pH 7.4) was carefully overlaid onto the preformed continuous density gradient, and centrifuged at 400 g for 60 min in an angle rotor tabletop centrifuge at 20 °C. For iodixanol, 500 µL of cells were laid on top of 500 µL of a 26 % solution in 10 mM Tris-HCl pH 7.5, 150 mM NaCl, in 2 mL tubes. Tubes were centrifuged at 2500 g for 2.5 min in an angle rotor tabletop centrifuge at 20 °C, rotated by 180 degrees and re-centrifugated. The fractions were collected, washed once with 500 µL Tris buffer (pH = 7.4), pelleted and resuspended in Tris buffer or conditioned medium for subsequent assays or sterile water.

Confocal microscopy, quantifications, and statistics

Images in Belgium were acquired on a Zeiss LSM710 microscope equipped with a 100× differential interference contrast, numerical aperture (NA) 1.46 Alpha-Plan-Apochromat objective, Airyscan module and ZEN 2.1 SP2 software. Airyscan processing was done using the default settings of the ZEN software. The final pixel size was 38.5 nm. Images in Athens were acquired on a Leica TCS SP8 MP with AOBS (acousto optical beam splitter), equipped with HC Plan APO 63x, NA 1.40 oil immersion objective. The final pixel size was 25.8 nm. GFP and mCherry fluorescence were excited with the 488-nm line of the argon laser and a 594 nm solid-state laser, respectively. Appropriate filters and beam splitters were used. In all conditions, gain percentage and laser intensities were kept to levels of non-saturated signal acquisition. The same laser intensities and other settings of the confocal microscope were used for all image acquisition in a single timepoint. For comparison between timepoints with important differences in signal, only laser intensity or gain were changed, and the ratios of the change were considered during quantifications. Images were smoothed, corrected for optimal brightness and contrast, cropped and merged using FIJI software (National Institutes of Health, Bethesda, MD, USA),⁸⁸ and annotated with Inkscape (https://inkscape.org). In each figure only a few cells representative of the whole cell population, observed in at least one preliminary experiment on epifluorescence microscopy and two independent biological replicate experiments on confocal, are shown.

For quantifications, images were analyzed with custom-made FIJI macros, calculating the normalized mean fluorescence intensity, the MCC-to-total surface area or total-to-internal fluorescence intensity, as previously.²³,⁸⁹ Briefly, for total-to-internal fluorescence intensity, two homocentric ellipses outlining the whole cell or the whole cells excluding the plasma membrane were manually drawn in middle-section images. For the other parameters, a mask was first created in the image of the MCC marker, in surface-section images. A 3×3 median filter (despeckle function) was applied, the ImageJ default thresholding method was applied, and then overlapping MCCs were separated using the watershed function. The intensities of the channels of interest were measured within manually selected cell outlines, while the median of the fluorescence intensity in the whole image was subtracted as background. The MCC-to-total surface area was defined as the ratio of the area of MCC-positive objects to the area of the outlined region. The Pearson's correlation coefficient was obtained using a custom-made macro based on the coloc2 plugin of FIJI. Due to the much higher number of MCCs at PGD timepoints, our custom-made script sometimes tended to recognize closely localized MCCs as one, leading to a slight overestimation of the MCC/total surface ratio. All parameters were calculated from at least two independent biological replicates for each condition. The values for single cells are presented in box-and-whisker plots. After verification that two independent biological replicates gave statistically nonsignificant differences in mean values, the values of the two experiments were merged. Prism software (Graphpad, San Diego, CA, USA), one-way ANOVA with the nonparametric Kruskal-Wallis test, and Dunn's multiple-comparison post hoc analyses were used to assess the significance of the value differences of all measurements.

Determination of the relationship between eisosome induction and the morphology of mitochondria

4 independent biological replicate cultures of a Pi1-mCherry- and llv3-GFP-expressing strain were imaged by confocal microscopy at different timepoints PGD (n total = 5028). Cells were qualitatively sorted in different categories, by 2 experienced investigators,
regarding the mitochondrial morphology and the eisosome expansion/assembly. For mitochondrial morphology the distribution of
llv3-GFP was used and the three following categories were considered, according to Laporte et al. 2018⁶: 1) cells with no green fluo-
rescence, reportedly dead 2) cells with only 4 or less aggregates of green fluorescence, which were considered senescent, and 3) the
rest of the cells which displayed several vesicles, which were considered possessing cortical mitochondria and thus, quiescent.
Regarding eisosome expansion, cells were first manually sorted in two categories: 1) cells with induced Pil1, and 2) cells without
Pil1 induction. The validity of this manual classification was verified by measuring Pil1 intensity in representative cells from selected
timepoints of 1 biological replicate (Figure S2C). An additional criterion of classification about eisosome assembly was the topology
of Pil1-mCherry, which was either mainly at the periphery of the cells, or cytoplasmic or an intermediate situation. This eventually led
us to adopt a total of 6 categories of eisosomal states, summarized in Figure 2A and for which representative images are shown in
Figure 2B. Results in Figure 2C show percentages of these cell categories at different timepoints PGD, from 4 biological replicates.
The physiological significance of all these categories was not further investigated, and only the main categories regarding induction of
Pil1 or not have been analyzed further. Cell numbers in all categories were summed (Table S2) and the probability of a cell with or
without eisosome induction to be Quiescent, senescent, or dead (Table S3) was calculated and expressed as pie charts. Chi-squared
tests for each timepoint were performed to determine the statistical significance of independence of the values. Chi-squared values
and p-values for each time point are shown in Table S2.

Epifluorescence microscopy

images were obtained with an Axioplan 2 (Carl Zeiss, Inc) microscope equipped with Plan-Apochromat x100 1.40 NA oil immersion objective lens, and appropriate fluorescence light filter sets. Images were captured with a Retiga3 (QImaging, Surrey, BC, Canada) digital camera and Occular 2.01 acquisition software (Teledyne Photometrics, Tucson, AZ, USA). Images were processed with FIJI⁸⁸ and annotated with Inkscape (https://inkscape.org). Staining of dead cells with methylene blue was performed by mixing an equal volume of cell culture with a 0.1% methylene blue, 2% sodium citrate solution. For visualization of stress granules, cells were incubated at 37 °C for 30 min, as previously,²⁷ and manually measured from 2 independent experiments. Cells from liquid cultures were laid on a thin layer of 1% agarose supplemented with appropriate nutrients and observed at room temperature. In each figure, a few representative cells, observed in at least two independent biological replicate experiments, are shown.

Western blotting

Total cell protein extracts were prepared and analyzed by SDS-PAGE as previously.⁸⁹ Proteins were transferred to a PVDF mem-
brane (Immobilon, Macherey Nagel, Düren, NW, Germany) and probed with a mouse monoclonal anti-GFP (RRID:AB_390913,
Roche, Basel, Switzerland), mouse monoclonal anti-mCherry (RRID:AB_11133266, ab125096, Abcam PLC, Cambridge, UK) or
anti-actin (RRID:AB_626632, sc47778, Santa Cruz Biotechnology, Dallas, TX, USA). Primary antibodies were detected by enhanced
chemiluminescence (Millipore, Burlington, MA, USA) after treatment with horseradish-peroxidase-conjugated anti-mouse immuno-
globulin (Ig) G secondary antibody (RRID:AB_2940774, Cell Signalling, Danvers, MA, USA). Signals were detected with ImageQuant
LAS 4000 mini (FujiFilm, Tokio, Japan), and images were corrected for optimal brightness and contrast with FIJI and annotated with
Inkscape. Relative semi-quantitative amounts of total proteins were estimated from 1-4 biological replicates of non-saturated expo-
sures using the gel analyzer tool of FIJI.⁸⁸ Each band was selected by using rectangular ROI selection and “Gels” analyzer, followed
by quantification of the peak area of obtained histograms. Data were acquired as area values. In each graph, the ratios of signal/actin
are plotted in scatter dot plots, normalized to the ratio of EXP which is set as 1. Statistical significance was assessed using the
repeated-measures two-way ANOVA analyses for matched values spread across a row, coupled to Holm-Sidak's multiple-compar-
isons test. (Prism, Graphpad, San Diego, CA, USA).

Estimation of cell survival

Cells from cultures at different timepoints were normalized to OD₆₀₀=0.9, diluted decimally 3-fold, and 30 µL were spread on YPD plates, incubated for 2-3 days at 30 °C and Colony Forming Units (CFUs) were counted. 2 technical replicates were performed for each condition and averaged. CFUs are expressed as percentage of total cells in the sample, which was estimated by cell density measurements. The number of total cells calculated were verified by CFU counting of wt cells from the GD timepoint, considering that at this stage 10% of the cells are senescent or dead (Figure 2C). Independent biological replicates are shown in box and whiskers plots. Unpaired two tailed students T-tests at indicated timepoints were used to calculate the significance of the value differences.

Proteomics

For determination of the proteome of EXP vs PGD cells, 3 biological replicates of cultures of the wt strain in minimal buffered medium were inoculated, and samples of at least 10⁸ cells were collected at an OD₆₀₀=1, and 30 h later (OD₆₀₀=18), washed three times with PBS and stored at -80 °C. Samples were homogenized in FASP lysis buffer (4% SDS, 0.1M DTE, 0.1M Tris-HCI pH 7.6). Protein concentration was determined by Bradford assay. Protease inhibitors (Roche, Basel, Switzerland) were added at a final concentration of 3.6% and samples were stored at -80°C until further use. Protein extracts (200 µg/sample) were processed using filter aided sample preparation (FASP) as described previously,⁹⁰ with minor modifications.⁹¹ Briefly, buffer exchange was performed in Amicon Ultra Centrifugal filter devices (0.5 mL, 30 kDa MWCO; Merck) at 14,000 rcf for 15 min at room temperature. The protein extract was mixed with urea buffer (8M urea in 0.1M Tris-HCI pH 8.5) and centrifuged. The concentrate was diluted with urea buffer and centrifugation
<figure><img src="image_52.png" /></figure>

was repeated. Alkylation of proteins was performed with 0.05M iodoacetamide in urea buffer for 20 min in the dark followed by a centrifugation at 14,000 rcf for 10 min at RT. Additional series of washes were conducted with urea buffer (2 times) and ammonium bicarbonate buffer (50 mM NH₄HCO₃ pH 8.5, 2 times). Tryptic digestion was performed overnight at RT in the dark, using a trypsin to protein ratio of 1:100. Peptides were eluted by centrifugation at 14000 rcf for 10 min, lyophilized and stored at -80°C until further use. The peptides were purified using a modified Sp3 clean up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated and the peptide concentration was determined through absorbance at 280nm measurement using a nanodrop instrument. Samples were analyzed on a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup consisting of a Dionex Ultimate 3000 nanoRSLC coupled in line with a Thermo Q Exactive HF-X Orbitrap mass spectrometer. Peptidic samples were directly injected and separated on an 25 cm-long analytical C18 column (PepSep, 1.9µm3 beads, 75 µm ID) using an one-hour long run, starting with a gradient of 7% Buffer B (0.1% Formic acid in 80% Acetonitrile) to 35% for 40 min and followed by an increase to 45% in 5 min and a second increase to 99% in 0.5min and then kept constant for equilibration for 14.5min. A full MS was acquired in profile mode using a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer, operating in the scan range of 375-1400 m/z using 120K resolving power with an AGC of 3x 106 and maximum IT of 60ms followed by data independent acquisition method using 8 Th windows (a total of 39 loop counts) each with 15K resolving power with an AGC of 3x 105 and max IT of 22ms and normalized collision energy (NCE) of 26. Each sample (e.g. three biological replicas) was analyzed in three technical replicas. Orbitrap raw data were analyzed in DIA-NN 1.8.1 (Data-Independent Acquisition by Neural Networks) through searching against the *Saccharomyces cerevisiae* Reference Proteome (downloaded from Uniprot, 6052 proteins entries, downloaded 30/5/2022) using the library free mode of the software, allowing up to two tryptic missed cleavages and a maximum of three variable modifications/peptide. A spectral library was created from the DIA runs and used to reanalyse them (double search mode). DIA-NN search was used with oxidation of methionine residues and acetylation of the protein N-termini set as variable modifications and carbamidomethylation of cysteine residues as fixed modification. The match between runs feature was used for all analyses and the output (precursor) was filtered at 0.01 FDR and finally the protein inference was performed on the level of genes using only proteotypic peptides. The proteomics data were processed in Perseus 1.6.15.⁰⁰ Values were log(2) transformed, a threshold of 70% of valid values in at least one group was applied and the missing values were replaced from normal distribution. For statistical analysis, Student's t-test was performed and permutation-based FDR was calculated. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁹² partner repository with the dataset identifier PXD041191.

For determining the changes in the proteomes of *mccΔ* and *flpΔ* mutants compared to *wt* cells, label free proteomics were performed. Triplicates of 2 OD units of *wt* and *mccΔ* cells were collected by centrifugation, 2 days PGD. The pellets were resuspended in lysis buffer of the commercially available proteomics kit (iST Kit, preomics) and peptides were prepared according to the manufacturer's recommendations. A mixture of trypsin and LysC was used to perform protein digestion. Reversed-phase chromatography was performed using a Thermo Ultimate 3000 RSLCnano system coupled with a Q ExactivePlus mass spectrometer (Thermo) via a nano-electrospray ion source, as previously described with some adjustments. Peptides were eluted from a 50 cm C30 column using a gradient of acetonitrile from 1-12 % for 45 min and from 12-35% in 0.1% formic acid for 70 min, followed by 35-60% for 20 min and 60-90% for 10 min at a constant flow rate of 200 nl/min. The 10 most intense multiply charged ions (z = 2-5) from the survey scan were selected with an isolation width of 1.4 m/z. Sequenced peptides were dynamically excluded for 20 s. The resulting spectra were analyzed with MaxQuant (version 2.1.4.0).⁸¹ Label free analysis was performed with Perseus.⁸⁰ The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁹² partner repository with the dataset identifier PXD041123 and 10.6019/PXD041123.

Volcano-plots were created using VolcaNoseR2 online tool.⁸² Each plot summarizes only the significant differences [log₂(fold-change)] in protein abundance in the x-axis and the -log₁₀(p-value) of the Student's t-test performed from replicates in the y-axis. The colored dots correspond to upregulated (purple) or downregulated (blue) proteins and the gray to the unaffected proteins based on the cut-off set in each plot. For Figure 1C, the cut-off of difference was set at 1.9 and the significance of Student's t-test at p<0.05. For Figures 3F, and S3H, the cut-off of difference was set at 0.5 and the significance of Student's t-test at p<0.05.

GO-term enrichment for biological processes and cellular components, for proteins with Student's T-test p-value <0.05 and the indicated difference, was performed with PANTHER⁸³ using the default sorting for fold-enrichment and hierarchical clustering of the overrepresented functional classes. In Figures 1D, S1B, S3F, and S3I only the most specific subclasses are shown. Mean fold-changes of the protein constituents of the yeast cellular components in Figure 1D were calculated in Microsoft Excel (using the SUMPRODUCT equation), for all the proteins of the dataset with T-test p-value <0.05. The proteins of the indicated yeast GO-Slim cellular components in Figure 1D were acquired from the *Saccharomyces* Genome Database.

## Fatty acid supplementation and targeted lipidomics for Phosphatidylethanolamine

For LC-MS/MS analysis of Phosphatidylethanolamine (PE) species, cells were grown at 29 °C on a minimal buffered medium, pH 6.1²³ with glucose 3% as carbon source and ammonium as nitrogen source. Cultures were supplemented or not with the indicated concentrations of Fatty acids. Fatty acids were purchased from Sigma [Oleic acid (O1383), Linoleic acid (L1012), Linolenic acid (L2376)]. Cell pellets (19 OD units) were resuspended in 0.75 ml of 150 mM ammonium formate and lysed at 4 °C with 500 µl of acid-washed glass beads using FastPrep Homogenizer (MP Biomedicals). The protein concentration of each sample was determined using the Bradford reagent (Bio-Rad). Lipids were extracted from lysed yeast cells according to 50 µg of protein by chloroform/methanol extraction.⁹³ Prior to extraction, ceramide (17:0/18:1 Avanti Polar Lipids) was spiked into each sample for
<figure><img src="image_53.png" /></figure>

normalization. Dried lipids were dissolved in a 65:35 mixture of mobile phase A (50:50 water/acetonitrile, including 10 mM ammonium formate and 0.1% formic acid) and mobile phase B (88:10:2 2-propanol/acetonitrile/H20, including 2 mM ammonium formate and 0.02% formic acid). HPLC analysis was performed on a C30 reverse-phase column [Accucore C30 LC column (150 mm x 2.1 mm 2.6 µm Solid Core; Thermo Fisher Scientific)] connected to aNexera XR HPLC (Shimadzu). A binary solvent system (mobile phases A and B) was used in a 6-min gradient at a flow rate of 0.4 ml/min and an injection volume of 1 µl. For the gradient 40% B for 0.1 min was used followed by its increase from 40% to 50% over 2 min. Afterwards, Buffer B was increased from 50% to 100% over 1 min. 100% B was kept for 1 min and decreased to 40% B for 0.1 min. 40% B was kept until the end of the gradient. Lipid samples were analyzed by a Qtrap 5500 (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI) probe. The MS was operated in negative ion mode using multiple reaction monitoring mode with 60-s detection windows. The predefined transitions for the PE species are provided in Table S6. Peaks were analyzed using the SciexOS software as described previously.⁹⁴ Peaks were defined through raw files, product ion and precursor ion accurate masses. From the intensities of the lipid standard (PE 16:0/18:1, Avanti Polar Lipids), absolute values for each lipid in pmol/µg protein were calculated. Data from 2-6 replicates are displayed as a percentage of each phospholipid PE species normalized to the total PE detected in each sample. The lipidomics workflow was reported in the Lipidomics Standards Initiative (LSI) platform (https://lipidomicstandards.org/).

## Determination of ubiquinol/ubiquinone ratios

For MS/MS analysis of ubiquinone and ubiquinol we used a one-step lipid extraction protocol using methanol as a solvent and isocratic elution conditions.⁹⁵ All steps were performed under nitrogen gas conditions. Cell samples (34 OD₆₀₀ units) were collected 1 day and 3 days PGD. Prior to extraction, ceramide (17:0/18:1 Avanti Polar Lipids) was spiked into each sample for normalization. For the extraction, cell pellets were mixed with 300 µL beats and 300 µL methanol. The mixture was vortexed for 5 min in the cold room and placed on ice for 15 min and then centrifuged at 12000 rpm at 4 °C for 10 min. Each supernatant was transferred into an auto-sampler vial and analyzed immediately. HPLC analysis was performed on a C30 reverse-phase column [Accucore C30 LC column (150 mm x 2.1 mm 2.6 µm Solid Core; Thermo Fisher Scientific)] connected to aNexera XR HPLC (Shimadzu). Mobile phase contained methanol with 2 mM ammonium acetate at the flow rate of 0.4 mL/min under isocratic elution conditions. The reduced ubiquinone (attached to ammonium) eluted at around 1.7 min while the oxidized form at around 2.3 min. Lipid samples were analyzed by a Qtrap 5500 (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI) probe. The MS was operated in positive ion mode using multiple reaction monitoring mode with 60-s detection windows. The predefined transitions for ubiquinone are provided in Table S7.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Prism software (Graphpad) has been used for statistical analyses. Details for the type of quantification and the statistical tests that have been used to define significance for each experiment are provided at the corresponding sections of the *method details* above, as well as at the figure legends. Generally, “n” refers to single-cells or measurements or biological replicates and is specified at the figure legends. Significance has been defined using Student’s test, one way or two-way ANOVA analyses, and employing the following annotation: \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001, ns p > 0.05.