SFA alter the morphology of the trans-Golgi network
We have recently developed a yeast-based assay that brings together most of the trademarks of the lipid-induced ER-stress observed in pancreatic β-cells . This system is based on a knock-out mutant of the Δ-aminolevulinate (ALA) synthase (hem1Δ; Figure S1A). This strain can synthesize haem only if the medium is supplemented with ALA. Since haem is required in yeast as the prosthetic group of Ole1p, the fatty acid desaturase, as well as several enzymes of the ergosterol (ERG) pathway, the hem1Δ strain suffers double-lipid depletion when grown in the absence of ALA, i.e. loss of both ERG and unsaturated fatty acids (UFA) [16, 17]. When transferred from ALA-supplemented to YPD medium supplemented with or without an exogenous source of ergosterol (YPD + ERG and YPD, respectively), hem1Δ cells stop growing as early as 5 h after the shift . This arrest correlates with an accumulation of SFA [mainly myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids] at the expense of unsaturated forms [UFA; palmitoleic (C16:1) and oleic (C18:1) acids] .
As with pancreatic β-cells, SFA accumulation is associated with significant impacts on the ER function and morphology, in a process that can be synergized by ERG : endogenous SFA accumulation results in the induction of the UPR and, if ERG and long-chain SFA (namely palmitate) are supplied as exogenous sources, in a swelling of the ER that appears as clefts expanding throughout the cytosol.
In the later secretory pathway, we have also demonstrated that SFA result in the preclusion of the delivery of a model plasma membrane transporter, the uracil permease Fur4p, to its final destination . Under SFA accumulation and in a way independent of the addition of an exogenous ERG source, Fur4p fails to exit the Golgi and is rather diverted to the vacuole for degradation (Figure S1A).
These two processes are paralleled by an increase in the saturation rate of PL and results in an ordering of the membrane bilayers  (Figure S1B). Interestingly, cell growth can be fully recovered, the UPR alleviated and trafficking to the plasma membrane restored if selective UFA, such as the mono-unsaturated fatty acid oleic acid, are added to the medium (Figure S1B; ). Under these conditions, exogenous UFA are efficiently incorporated within PL and restore membrane properties to normal .
In this study, we hypothesized that preclusion of Fur4p in the late secretory pathway could be related to general effects of SFA on organelle function/integrity. Indeed, to account for permease diversion from the Golgi to the vacuole, a simple explanation would be that SFA could have a general impact on vesicle formation at the trans-Golgi Network. To evaluate this hypothesis, we used a strain bearing a thermosensitive mutation in the SEC6 gene, encoding a protein required for optimal targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane . As shown in Figure 1A, as compared to control conditions, a threefold decrease in the number of accumulating vesicles was observed under endogenous SFA accumulation at the non-permissive temperature. Moreover, under these conditions, many vesicles displayed an intriguing polygonal shape (Figure 1C–E), which appeared very different from the normal, characteristic, round morphology (Figure 1B). Intriguingly, some of the accumulating structures under SFA accumulation were delineated by two distinct membranes (Figure 1C–F). Overall, these polygonal structures represented 66% of the total vesicle population (estimated from 30 independent cells) within the hem1Δ, sec6ts strain under SFA accumulation. In order to address whether these structures conserved some ability to be efficiently targeted to and fused with the plasma membrane, such as regular secretory vesicles, their number was compared between hem1Δ and hem1Δ, sec6ts cells. In this aim, cells were grown for 7 h in YPD at 28°C before transfer to 39°C for 2 h, and the number of polygonal structures was determined as described in the legend of Figure 1. Interestingly, the average number of polygonal structures per cell was found to be 19.7 ± 2.1 in hem1Δ, sec6ts and 2.5 ± 0.3 in hem1Δ cells, respectively (n = 30; p < 0.0001). This observation strongly suggests that the polygonal structures/vesicles induced by SFA accumulation can be efficiently targeted and tethered to active sites of exocytosis at the plasma membrane in the non-sec6ts background.
Figure 1. SFA impact the late secretory pathway. hem1Δ, sec6ts cells were grown in YPD or YPD + ALA during 7 h. The accumulation of secretory vesicles was induced by incubating the cells at 39°C for 2 h. Cells were harvested and prepared for electron microscopy observation as described in the Materials and Methods. A) The number of secretory vesicles per cell was determined in at least 30 different cells. The p value was calculated by a two-tailed t-test, using the Graphpad Prism 5 software. ***: p < 0.0001. Means ± SEM are indicated. Characteristic electron micrographs of the vesicles are showed for cells grown in either YPD + ALA (B) or YPD (C–F). Eighty-seven percent of 31 random sections of hem1Δ, sec6ts cells grown in YPD, from three independent experiments, exhibited polygonal structures/vesicles at non-permissive temperature (C–F). In marked contrast, hem1Δ, sec6ts cells grown in YPD + ALA did not display any such structures, but rather round vesicles, with a bright content delineated by a single membrane (B).
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In summary, we concluded from these experiments that SFA affect the organizations of the trans-Golgi network/late secretory pathway, by decreasing the number of secretory vesicles en route to the plasma membrane. This process could account for altered delivery of proteins destined to the cell surface, including Fur4p, to their final destination.
Genome wide transcription profiling reveals intense remodeling of specific lipid biosynthetic pathways under SFA accumulation
A possible explanation to account for SFA alterations of the Golgi morphology could be their indirect impacts on the regulation of genes encoding tethers, coats and/or accessory proteins of the Golgi vesicular machinery, such as members of the COPI complex .
To check this hypothesis, we conducted a genomic analysis on hem1Δ cells by comparing their transcriptomic profiles under growth in complete medium supplemented with ergosterol and oleate (YPD + ERG + OLE), or with ergosterol alone (YPD + ERG; SFA accumulation). Differential expression analysis, conducted with a cut-off of 1.7 for fold change (FC) and of 0.0025 for the p value, identified a total of 92 genes with dysregulated expression under SFA accumulation, 71 being overexpressed and 21 being downregulated (Table S2).
However, among these genes, none of them appeared to be related to the COPI machinery and, more generally, to vesicle formation (Table S2). Instead, 16 showed direct connections to lipid homeostasis (Table 1), including sterol, fatty acid and sphingolipid metabolisms, some of which displaying strong functional similarities with mammalian homologues known to be related to T2D.
Table 1. SFA-dysregulated genes related to lipid metabolism
|Gene name||p value||Fold Change||Description|
|ERG5||2.53E−06||−1.74467734||C-22 sterol desaturase, a cytochrome P450 enzyme that catalyzes the formation of the C-22(23) double bond in the sterol side chain in ergosterol biosynthesis; may be a target of azole antifungal drugs|
|YPC1||2.88E−06||−1.73308228||Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and synthesis of phytoceramide; overexpression confers fumonisin B1 resistance|
|SCS7||0.00107647||−1.80540177||Sphingolipid alpha-hydroxylase, functions in the alpha-hydroxylation of sphingolipid-associated very long-chain fatty acids, has both cytochrome b5-like and hydroxylase/desaturase domains, not essential for growth|
|FAA1||0.00227896||−2.25801109||Long-chain fatty acyl-CoA synthetase, activates imported fatty acids with a preference for C12:0-C16:0 chain lengths; functions in long-chain fatty acid import; accounts for most acyl-CoA synthetase activity; localized to lipid particles|
|INO1||1.55E−06||2.7454811||Inositol-3-phosphate synthase, involved in synthesis of inositol phosphates and inositol-containing phospholipids; transcription is coregulated with other phospholipid biosynthetic genes by Ino2p and Ino4p, which bind the UASINO DNA element|
|IZH4||4.36E−06||5.1719371||Membrane protein involved in zinc ion homeostasis, member of the four-protein IZH family, expression induced by fatty acids and altered zinc levels; deletion reduces sensitivity to excess zinc; possible role in sterol metabolism|
|RTA1||6.40E−06||4.8023256||Protein involved in 7-aminocholesterol resistance; has seven potential membrane-spanning regions; expression is induced under both low-heme and low-oxygen conditions; member of the fungal lipid-translocating exporter (LTE) family of protein|
|IZH2||1.44E−05||2.2640908||Plasma membrane protein involved in zinc homeostasis and osmotin-induced apoptosis; transcription regulated by Zap1p, zinc and fatty acid levels; similar to mammalian adiponectins; deletion increases sensitivity to elevated zinc|
|FAA4||2.17E−05||2.0488887||Long-chain fatty acyl-CoA synthetase, activates imported fatty acids with a preference for C12:0-C16:0 chain lengths; functions in long-chain fatty acid import; important for survival during stationary phase; localized to lipid particles|
|ALE1||2.47E−05||2.0267613||Broad-specificity lysophospholipid acyltransferase, part of MBOAT family of membrane-bound O-acyltransferases; key component of Lands cycle; may have role in fatty acid exchange at sn-2 position of mature glycerophospholipids|
|ICT1||3.42E−05||2.2208792||Lysophosphatidic acid acyltransferase, responsible for enhanced phospholipid synthesis during organic solvent stress; null displays increased sensitivity to Calcofluor white; highly expressed during organic solvent stress|
|ATF1||6.97E−05||2.4021553||Alcohol acetyltransferase with potential roles in lipid and sterol metabolism; responsible for the major part of volatile acetate ester production during fermentation|
|OLE1||7.84E−05||4.4421267||Delta(9) fatty acid desaturase, required for mono-unsaturated fatty acid synthesis and for normal distribution of mitochondria|
|MGA2||0.00021876||1.7750116||ER membrane protein involved in regulation of OLE1 transcription, acts with homolog Spt23p; inactive ER form dimerizes and one subunit is then activated by ubiquitin/proteasome-dependent processing followed by nuclear targeting|
|FAS2||0.00095185||1.7266125||Alpha subunit of fatty acid synthetase, which catalyzes the synthesis of long-chain saturated fatty acids; contains the acyl-carrier protein domain and beta-ketoacyl reductase, beta-ketoacyl synthase and self-pantetheinylation activities|
|HES1||0.00113412||1.7001732||Protein implicated in the regulation of ergosterol biosynthesis; one of a seven member gene family with a common essential function and non-essential unique functions; similar to human oxysterol binding protein (OSBP)|
For example, SFA accumulation resulted in the upregulation of Ole1p, a homologue of the human stearoyl CoA desaturase 1 (SCD1). SCD1 levels have been demonstrated to be strictly related to sensitivity to SFA in humans. Indeed, enhanced expression of SCD1 protects β-cells from lipoapoptosis , whereas low hepatic SCD1 activity is associated with fatty liver and insulin resistance in obese individuals .
Particularly pertinent hits from our genomic screen were also two members of the IZH [implicated in zinc homeostasis; ] family (IZH2 and IZH4). IZH are homologues of mammalian adiponectin receptors  which mediate antidiabetic metabolic effects . It therefore appears that a core, basic pathway could have been conserved from yeast to human, for cells to adapt lipotoxicity, via the conserved IZH/AdipoRQ membrane sensors/receptors.
Another interesting observation was the upregulation of two genes encoding broad-specificity lysophospholipid acyltransferases, namely ALE1 and ICT1. Ale1p and Ict1p have been shown to display a strong preference for unsaturated acyl-CoA substrates [24, 25]. One may therefore assume that, under conditions of UFA scarcity, cells may try to counter saturated PL accumulation by specifically targeting UFA to PL. Interestingly, Ale1p displays human homologues, belonging to the membrane-bound O-acetyltransferases (MBOAT), namely MBOAT 1, 2, 5 and 7, all of them showing a strong preference for unsaturated substrates . These proteins, which could constitute potential targets for future therapies, will deserve particular attention.
Sphingolipid metabolism could also be altered in yeast under SFA accumulation since two genes encoding enzymes involved in this pathway were found to be downregulated (YPC1 and SCS7; Table 1 and see below). Scs7p, which catalyzes the hydroxylation of ceramides/sphingolipids, has a direct functional homologue in mammalian cells, known as FA2H. Relevantly, FA2H has been connected to insulin resistance, since depletion of FA2H inhibits both basal and insulin-stimulated glucose uptake (ISGU) in adipocytes  (see below).
In more to this first observation, we focused in this study on the ceramide/sphingolipid metabolism for additional reasons. First, the late steps for complex sphingolipid synthesis take place in the Golgi apparatus . Second, sphingolipids have been shown to play critical roles in the sorting of specific sets of cargo proteins, en route to the plasma membrane, at the Golgi level . Third, implication of ceramides/sphingolipids in insulin resistance and lipotoxicity has been clearly demonstrated . These lipid species have been somehow connected to insulin signal transduction, chronic inflammation, lipotoxicity in several organs and nutrient transport [30, 31]. This latest process refers to the property of ceramides to downregulate nutrient permeases in yeast [32, 33] and mammalian cells , which could, in turn, lead to cell death by starvation. However, the mechanisms of action of ceramides/sphingolipids on permease biogenesis remain poorly understood. Consistently, we could observe the transcriptional downregulation of four different genes encoding nutrient transporters under SFA accumulation, namely the high-glucose affinity transporter Hxt4p (a strict functional homologue of the human glucose transporter GLUT4), the uracil permease Fur4p, the oligopeptide transporter Opt2p and the di-tripeptide transporter Ptr2p (Table S2).
Altogether, these observations prompted us to investigate in further detail the connections between permease biogenesis and the ceramide/sphingolipid pathway under SFA accumulation in our yeast model.
Ceramides/sphingolipids act at early steps of the secretory pathway
As already mentioned, among the two genes involved in ceramide/sphingolipid biosynthetic pathway that were shown to be downregulated under SFA accumulation, was the SCS7 gene, encoding a sphingolipid alpha-hydroxylase. Accordingly, SCS7 downregulation correlated with the accumulation of hypo-hydroxylated forms [twofold increase for complex sphingolipids (Figure 2A) and ninefold increase for ceramides (Figure S2)]. Sphingolipids are important components of the lipid rafts. Interestingly enough, accelerated diffusional mobility of raft-associated lipids has been shown to be increased under depletion of FA2H (the mammalian Scs7p homologue), a process that could account for enhanced degradation of both GLUT4 and the insulin receptor in adipocytes under these conditions . It therefore seemed important to test whether preclusion of sphingolipid hydroxylation could account for Fur4p mistargeting in our yeast model. In this aim, Fur4p trafficking was evaluated in a scs7 deleted strain. In such a strain, even if hydroxy-sphingolipids were hardly detectable (Figure 2C, right), Fur4p delivery to the plasma membrane was not fully abolished (Figure 2D, right; compare ERG and scs7Δ in Figure 2E). However, significant accumulation of intracellular Fur4p could be observed in this strain (Figure 2D, right; Figure 2E). This demonstrates that, if hydroxy-sphingolipid levels are clearly important for optimal permease delivery to the cell surface, the moderately decreased amounts observed under SFA accumulation (Figure 2A, right) cannot account, by themselves, for the complete block in Fur4p trafficking observed under these conditions (Figure 2B, right).
Figure 2. Impacts of hypo-hydroxylated sphingolipids on Fur4p trafficking. A) hem1Δ cells were grown in YPD + ERG + OLE or YPD + ERG (SFA accumulation) for 7 h before lipid extraction and MS analysis in the negative ion mode. The mature sphingolipid forms IPC-C (m/z = 952 Da) and IPC-B/B′ (m/z = 936 Da), which differ from each other by one hydroxyl group, are indicated. A.U.: Arbitrary Units. B) hem1Δ (pFl38gF-GFP) cells were grown in YPD + ERG or YPD + ERG + OLE for 7 h and Fur4p-GFP synthesis was induced by adding galactose to the medium before visualization of GFP by confocal microscopy. Y10000 (pFl38gF-GFP) and scs7Δ (pFl38gF-GFP) cells were grown to the stationary phase in YPD, before shifting to early exponential phase (2 x 106 cells mL−1) in fresh YPRaff. Seven hours after the shift, lipids were extracted for MS analysis of the IPC species in the negative ion mode (C) and Fur4p-GFP was visualized after galactose addition (D). Bar: 1 µm. B) In order to normalize the relative expression of Fur4p-GFP at the plasma membrane, the plasma membrane/intracellular fluorescence ratio (Fur4p-GFP PM/Intra) was estimated as described in the Materials and Methods. A.U.: Arbitrary Units. The p value was calculated by a two-tailed t test, using the Graphpad Prism 5 software. *: p < 0.05. **: p < 0.01. ****: p < 0.0001. Means ± SEM are indicated.
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To get a broader view of the potential impacts of SFA on ceramide/sphingolipid synthesis, we reconsidered our microarray data and applied a hierarchical clustering on 27 yeast genes involved in this pathway (Table S3). This approach identified nine genes of the sphingolipid pathway with a highly significant deregulated expression (p < 0.02), seven of which being downregulated (IFA38, LAC1, SCS7, YPC1, LCB2, TSC13, LAG1), and two upregulated (ISC1 and SKN1) (Figure 3A). When considering these genes in the context of the overall sphingolipid pathway (Figure 3B), one may anticipate a global decrease of ceramide/sphingolipid synthesis due to a shutdown of the early steps. However, we have already reported that SFA accumulation results in complex sphingolipid accumulation in yeast [namely IPC, MIPC and M(IP)2C; ] and quantification of ceramides in this study revealed an overall increase of these species under the same conditions (Figure S2). The apparent discrepancies between expected and measured ceramide/sphingolipid amounts likely find their explanation in the fact that palmitate, which is the main SFA-accumulating species under our experimental conditions, is the substrate of serine palmitoyltransferase (SPT; under the form of palmitoyl-CoA), the first enzyme of the ceramide/sphingolipid pathway (Figure 3B). In reaction to this, knowing the deleterious effects of high ceramide/sphingolipid levels, cells may react by shutting down the overall pathway at the transcriptional level.
Figure 3. SFA-induced remodeling of the sphingolipid pathway. A) Hierarchical clustering of selected genes of the sphingolipid pathway. hem1Δ cells were grown to the stationary phase in YPD + ALA, before shifting to early exponential phase (2 x 106 cells mL−1) to YPD + ERG + OLE or YPD + ERG (SFA accumulation). After 7 h growth in the indicated conditions, differential expression analyses were driven on 4 independent biological samples (1–4), as described in the Materials and Methods. Among the 27 genes involved in the ceramide/sphingolipid pathway in yeast (Table S3), 9 showed significant variations in expression (FC > 1.2 and p value < 0.02) under SFA accumulation, 7 being downregulated (green) and 2 upregulated (red). B) Outline of the sphingolipid pathway in the yeast Saccharomyces cerevisiae. Intermediates of the sphingolipid pathway are shown in regular lettering and genes are in italics. Genes which are either down- (green) or upregulated (red) under SFA accumulation are also indicated. Adapted from Dickson .
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Since high ceramide amounts have been demonstrated to kill both yeast and mammalian cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation [31, 32, 35], we next questioned if these lipid species may contribute to the observed Fur4p trafficking defect under SFA accumulation. To test this hypothesis, cells were incubated with the serine palmitoyl transferases inhibitor myriocin (Figure 4A). As expected, treating SFA-intoxicated cells with myriocin resulted in decreased levels of ceramides (Figure S2). Surprisingly, however, myriocin failed to restore normal Fur4p trafficking (Figure 4A,B), demonstrating that high ceramide/sphingolipid levels are not, by themselves, responsible for impaired permease localization.
Figure 4. Physiological effects of myriocin under SFA accumulation. A) hem1Δ, end3Δ (pFl38gF-GFP) cells were grown to the stationary phase in YPD + ALA, before shifting to early exponential phase (2 x 106 cells mL−1) in fresh YPRaff + ERG + OLE or in YPRaff + ERG supplemented with myriocin at the indicated concentrations. Seven hours after the shift, Fur4p-GFP expression was induced by adding galactose, and GFP fluorescence was visualized 12 h later. Bar: 1 µm. B) The Fur4p-GFP PM/Intra ratio was estimated as described in the legend of Figure 2. A.U.: Arbitrary Units. The p value was calculated by a two-tailed t test, using the Graphpad Prism 5 software. ****: p < 0.0001. ns: non-significant. Means ± SEM are indicated. C and D) hem1Δ (pJC104) cells were grown to the stationary phase in YPD + ALA, before shifting to early exponential phase (2 x 106 cells mL−1) in fresh YPD + ALA or YPD + ERG + OLE and YPD + ERG supplemented with myriocin at the indicated concentrations. After 7 h following the shift to the indicated medium, cells were harvested to measure HXT4 expressions by RT-qPCR (C), or to quantify β-galactosidase activities to follow induction of the UPR (D), as described in Materials and Methods. A.U.: Arbitrary Units. RT-qPCR values are expressed as a percentage of ALG9 expression, used as a standard. Values are means ± SD of at least three independent determinations.
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We next questioned if ceramides/sphingolipids could act at earlier steps of permease biogenesis. SFA accumulation is associated to the transcriptional downregulation of nutrient permeases (Table S2). To evaluate the potential involvement of ceramides/sphingolipids in this process, the mRNA of the glucose tranporter HXT4 were quantified by RT-qPCR (Figure 4C) under myriocin treatment. As shown, myriocin restored mRNA amounts to levels very similar to what observed under non SFA-accumulating conditions. Interestingly, similar results were obtained for Fur4p, Opt2p and Ptr2p (data not-shown), showing that permease transcriptional downregulation is likely a ceramide/sphingolipid regulated process.
Finally, the involvement of ceramides/sphingolipids on the induction of the ER-stress under SFA accumulation was evaluated. SFA accumulation is known to result in ER-stress and induction of the UPR. Interestingly, myriocin abolished SFA-induced UPR (Figure 4D).
Taken together, this set of data shows that ceramides/sphingolipids are not the main actors of Fur4p trafficking defect at the Golgi exit but are important modulators of both SFA-induced UPR induction and permease transcriptional downregulation.
Saturated PL as key regulators of vesicle formation at the trans-Golgi network
A striking characteristic of the ER and the Golgi apparatus is that their membranes are constituted of highly unsaturated PL species, whereas saturated PL levels increase later secretory pathway, to reach a maximum at the plasma membrane . High levels of unsaturated PL result in loose lipid packing, a biophysical parameter that appears to be crucial for the recruitment of selected proteins required for optimal vesicular budding. Prime examples are the members of the Arf-GAP1 family, one of them being Gcs1p in yeast. Gcs1p has been shown to mediate both Golgi-ER and post-Golgi vesicle transport in yeast . Interestingly, deletion of the GCS1 gene and of its homolog AGE2 results in Golgi fragmentation and preclusion of post-Golgi transport .
Arf-GAP1 proteins respond to membrane curvature through the membrane absorption of a specific motif, referred to as ArfGAP1 Lipid Packing Sensor (ALPS; ). In fact, the ALPS motif does not recognize membrane curvature per se, that is, a curved geometry, but loose lipid packing, which is a consequence of membrane curvature . Since increased amounts of saturated PL under SFA accumulation have been shown to increase lipid packing , we then questioned if abnormal Golgi morphology, poor secretory vesicle formation and consequent Fur4p trafficking defect could be the consequence of direct impacts of saturated PL on Gcs1p recruitment to the trans-Golgi network. Indeed, if we could not succeed in determining the precise PL composition of the polygonal structures displayed in Figure 1, we have already demonstrated that SFA accumulate within the PL of microsomal fractions under our experimental conditions . This strongly suggests that PL composition is altered in a similar fashion within the organelles of the entire secretory pathway, including the trans-Golgi network and secretory vesicles, under SFA accumulation.
In a first step, we evaluated the recruitment of Gsc1p (Gcs1p-GFP; Figure 5) to the Golgi complex in vivo, by its colocalization with the trans-Golgi resident protein Sec7p (Sec7p-RFP; Figure 5; ). As shown in Figure 5, Gcs1p failed to colocalize with Sec7p under SFA accumulation, but rather appeared redistributed within the cytosol.
Figure 5. SFA accumulation impairs Gcs1p recruitment to the trans-Golgi network. hem1Δ, SEC7RFP cells transformed with the pRS315-GFP-GCS1 plasmid were grown for 7 h in YPD + ALA or YPD (SFA accumulation) and before visualization of the Gcs1p-GFP and Sec7p-RFP fusion proteins by confocal microscopy. Bar: 1 µm.
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To evaluate if saturated PL could account by themselves for altered Gcs1p recruitment to membranes, we used a flotation assay in which liposomes of defined PL compositions and associated proteins can be recovered at the top of a density gradient (Figure 6A; ). In this aim, liposomes were obtained from phosphatidylcholine (PC) species purified from hem1Δ cells grown either in the presence (ALA-PC) or in the absence (YPD-PC) of ALA (Figure S1). ALA-PC are mainly constituted of di-unsaturated species (i.e. bearing two mono-unsaturated fatty acyl chains; 55%), followed by mono-unsaturated species (one saturated chain and one mono-unsaturated chain; 40%) and saturated species (two saturated chains; 5%) and form highly disordered membranes [17, 18]. On the other hand, saturated forms raise up to 40% in YPD-PC (55% mono-unsaturated species and 5% di-unsaturated species) and result in highly ordered, packed membranes [17, 18]. As shown in Figure 6B, the binding of Gcs1p to ALA-PC liposomes strongly increased with membrane curvature, i.e. smaller liposome radius, in a very similar manner as what could be observed with liposomes obtained with a purified mono-unsaturated species, palmitoyl-oleyl PC (POPC; Figure S3). By contrast, Gcs1p poorly associated with YPD-PC liposomes, whatever their size (Figure 6C). Similar results could be obtained when the same experiments were conducted with dimyristoyl PC liposomes (a saturated species; DMPC, Figure S3). This experiment demonstrated that Gcs1p cannot bind YPD-PC liposomes, likely because of increased lipid packing related to high saturated PL amounts. This process could therefore account for Gcs1p mislocalization in vivo and may likely participate in the observed reduced vesicular budding at the trans-Golgi network.
Figure 6. SFA accumulation alters Gcs1p recruitment to vesicles. A) Purified Gcs1p was incubated without or with either ALA-PC or YPG-PC liposome of different radius and loaded at the bottom of a three steps sucrose gradient before centrifugation (see Materials and Methods for details). Bottom (B: 250 μL), middle (M: 150 μL) and top (T: 100 μL) fractions were collected and analyzed. B and C) Gcs1p recruitment to liposome was determined by analysis of top fractions on SDS–PAGE and comparison to 100% protein fraction (total loaded protein volume diluted into 100 μL final top volume). Typical results with ALA-PC liposome (B) or with YPG-PC liposome (C). D) Combined results from two lipid purifications with two independent extrusion each. Liposome size varied for each experiment and was here referred depending on the pore size of the polycarbonate filters used in each condition.
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