3.1. Lipid metabolism is altered in N88S seipin-expressing cells
In this study, we employed comparative unbiased quantitative mass spectrometric proteomic analysis to examine changes in protein abundance and profile between the WT-VN WT-VC and N88S-VN N88S-VC strain at the post-diauxic shift (PDS) phase, when cells shifted from a fermentative (exponential phase) to aerobic utilization of non-glycolytic substrates (e.g. ethanol) by mitochondria. The goal was to define a set of differentially expressed proteins (DEPs - either up- or down-regulated) and sort into functional networks to highlight perturbed protein functions and signalling pathways contributing to disease-related phenotypes. We were able to identify 115 proteins with reduced abundance and 97 proteins with increased abundance in N88S seipin-expressing cells (Fig. 1A and Fig. S1A, Tables S3 and S4). A volcano plot (Fig. 1B) is shown to highlight the differences in protein levels. Using YEASTRACT+ (74), a Gene Ontology (GO) analysis and KEEG pathway analysis for DEPs in biological processes revealed an enrichment in proteins related to ion transport (GO:0006811; p-value = 0.001380), phospholipid biosynthetic process (GO:0008654; p-value = 2.920 × 10− 9) and lipid metabolic process (GO:0006629; p-value = 1.562 × 10− 6) (Fig. S1B-C and Fig. 1C).
Notably, among the protein identified, eight showed reduced abundance, including seven key players in the phospholipid biosynthetic pathway: Cho1p, Cho2p, Opi1p, Opi3p, Psd1p, Cpt1p and Cds1p (Table S3 and Fig. 1D). This suggests potential alterations in the lipid profile, particularly in phospholipid and neutral lipids content. To investigate these changes, we conducted a quantitative lipidomic analysis at the same growth phase (PDS phase) to measure the levels of major phospholipids and their derivatives, inositol phosphates, and neutral lipids along with their corresponding precursors. We observed a decrease in 46 lipid metabolites, while 41 lipid molecules levels were increased (Fig. S2A-B). The levels of fatty acids (Fig. S2C) and of major phospholipids (PC, PE, PS and PI) remained essentially unaltered (Fig. 2A), however the levels of PG, which is derived from CDP-DAG, were decreased (Fig. 2A). The amount of ceramide (Fig. S2D) and lysophospholipids (Fig. 2B) was increased, suggesting that the activity of lysophospholipid acyltransferases may be impaired in the mutant. Despite the fact of DG abundance was unchanged (Fig. 2C), the levels of PA and TG are increased (Fig. 2D-E), suggesting that PA and lipid precursors are being redirected towards neutral lipid synthesis.
3.2. Inositol metabolism is deregulated in cells expressing the mutant seipin
Genes encoding enzymes in both the CDP-DAG (CDS1, CHO1, PSD1, CHO2, OPI3) and Kennedy (CPT1) pathways contain a UASINO element in their promoters whose expression is transcriptionally repressed by Opi1p (31, 75). To evaluate if their lower protein abundance in N88S seipin-expressing cells correlates with reduced transcription, we measured the transcription of Opi1p-dependent genes in WT and mutant cells, using an INO1-LacZ transcriptional reporter during lifespan. We observed that INO1 expression remains unchanged in the WT, however there is a ~ 4-fold increase in N88S seipin-expressing cells at PDS and stationary phases (Fig. 3A). It was previously demonstrated that the accumulation of PA acts as the metabolic signal that triggers the derepression of INO1 (29, 76, 77). Therefore, a plausible explanation for lower Opi1p repressor activity is the retention of Opi1p in the ER potentiated by accumulation of PA as observed by lipidomic analysis (Fig. 2D), allowing the Ino2p-Ino4p heteromeric complex to stimulate the transcription of INO1. We then conclude that post-transcriptional mechanisms may operate to control the steady protein levels of these phospholipid biosynthetic enzymes in response to changes in the lipid profile observed in the mutant strain (Fig. 1D). More recently, it was shown that the levels of PA (34:1) is correlated with optimal expression of INO1 irrespective of total PA content (78). We observed that the amount of the PA (34:1) is increased by ~ 7-fold at PDS phase in mutant cells, which coincides with the increase in INO1 expression at the same phase (Fig. S2E).
Next, we decided to evaluate if higher INO1 expression was correlated with changes in Ino1p protein levels during lifespan. For that, we analyzed Ino1p-HA levels at the exponential and PDS phases by Western Blotting. We observed increased protein levels starting at the exponential phase, which remained elevated during the PDS phase (Fig. 3B), where INO1 expression is largely derepressed in mutant cells (Fig. 3A). Ino1p is essential for the de novo biosynthesis of inositol, which is incorporated into phosphatidylinositol (PI) for the synthesis of phosphatidylinositol phosphates (PIP). In N88S seipin-expressing cells, we also observed changes in PI-derived lipids, including decreased levels of phosphoinositides (PIP and PIP3). This indicates that PI metabolism is also deregulated (Fig. S2F).
We next tested whether cells expressing the seipin N88S mutation are responsive to changes in inositol levels. For that, cells expressing the INO1-LacZ reporter fusion were grown to exponential phase in the absence of inositol and then shifted to medium containing inositol. While WT cells adapted to inositol by reducing INO1 expression, the mutant was unable to adapt and failed to repress INO1 expression (Fig. 3C). Importantly, in the absence of inositol, INO1 expression was lower in the mutant compared to WT cells which is reminiscent of the behavior displayed by the mutant Opi1FFAT, in which the Opi1p-Scs2p interaction motif two phenylalanines (FF) in an acidic tract (FFAT) is absent, and the activity of Opi1p is mainly driven by the electrostatic interaction with PA is absent, and the activity of Opi1p is mainly driven by the electrostatic interaction with PA (79). Overall, we provide strong evidence that inositol and lipid metabolism is highly deregulated in the mutant.
In summary, the results suggest that increased PA levels may be linked to decreased flux of lipid precursors into phospholipid biosynthesis, due to lower protein levels of key enzymes in this pathway. Consequently, accumulation of PA levels causes Opi1p to be retained in the ER, allowing the transcription of INO1 to be derepressed, thus leading to elevated Ino1p protein levels. Additionally, PA may be redirected towards the synthesis of neutral lipids, thus explaining the higher amount of TG exhibited by the mutant.
3.3. PA and impaired inositol metabolism contribute to ER stress independently of protein misfolding in mutant cells
ER stress and UPR activation can influence cellular processes beyond ER protein folding, playing critical roles in lipid metabolism, as it constitutes a key site for lipid synthesis and storage (80). Previous studies showed that inositol depletion can trigger ER stress without causing a noticeable accumulation of unfolded proteins in the ER lumen (81–83). Instead, this stress response appears to activate the UPR via a membrane-based mechanism that functions independently of unfolded proteins (84). This offers an ideal framework to investigate whether deregulated lipid metabolism contributes to ER stress observed in the yeast model of N88S seipinopathy. We then evaluated how INO1 deficiency affects the activity of the UPRE-LacZ ER stress reporter. As previously described, N88S seipin-expressing cells displayed higher levels of ER stress (Fig. 3D) associated with elevated expression of the reporter fusion (45). Importantly, β-galactosidase activity further increased in mutant cells upon deletion of INO1, although to lower extent, indicating that misregulation of INO1 expression and impaired inositol metabolism also contribute to ER stress.
Notably, INO1 deletion is associated with reduced PI levels and accumulation of lipid precursors, including PA and CDP-DAG (31). This led us to investigate whether accumulation of PA is associated with higher induction of the UPR. When WT cells are grown in inositol-depleted conditions, PI levels remain low, leading to PA buildup (31). Importantly, under these conditions, N88S seipin-expressing cells exhibited higher UPR levels when grown in the absence of inositol in a INO1-dependent manner (Fig. 3E). In the presence of exogenous inositol, where PA is consumed as a precursor to support PI biosynthesis, the β-galactosidase activity of the ER stress reporter remained unchanged in WT cells regardless of inositol levels, but decreased in the mutant (Fig. 3E). This reinforces the hypothesis that the response to changing PA levels involves the induction of the UPR in cells expressing N88S mutant seipin.
Next, we tested how the overexpression of CDS1 affects the ER stress response. Cds1p is responsible for the synthesis of CDP-DAG from PA. Cds1p consumes the PA pools and channels the CDP-DAG pool towards PI biosynthesis under conditions of inositol surplus (85). However, in the absence of inositol, where PI biosynthesis is essentially halted, CDP-DAG derived from PA is channeled into the phospholipid biosynthetic pathway. Under inositol starvation, we observed that the UPR is significantly increased in WT cells, and even more induced in N88S seipin-expressing cells upon overexpression of CDS1 (Fig. 4A). Higher UPR induction in the mutant might be linked to reduced enzymatic capacity to promote phospholipid biosynthesis under these conditions, as the overall phospholipid biosynthesis rate is likely reduced due to decreased protein levels of key enzymes involved in the pathway (Fig. 1D). So, we tested whether stimulation of phospholipid biosynthesis by overexpressing CHO1 in WT and N88S mutant cells induces ER stress under the same conditions (in the absence of inositol). In this case, we observed no changes in the induction of the LacZ reporter fusion for all strains tested (Fig. 4B). Notably, overexpression of CDS1, in contrast with CHO1 counterparts, leads to inositol auxotrophy and significant viability loss in the absence of inositol (Fig. 4C). Overall, the data support a model in which the accumulation of PA, whether due to a reduced rate of phospholipid biosynthesis or in response to changes in lipid flux upon inositol availability (Fig. 1D, 3C-E, and 4A-B), is a key feature of the ER stress response in N88S seipin mutant cells. This is particularly significant, as it demonstrates that lipid imbalance also contributes to the ER stress response beyond protein misfolding of seipin caused by the N88S mutation. In agreement with this idea, we found that INO1 deletion actually reduced IB formation by ~ 50% in N88S seipin-expressing cells, as monitored by fluorescence microscopy and flow cytometry (Fig. 4D-E), despite the earlier observation that the UPR is more strongly induced in cells grown under the same conditions (Fig. 3D).
Finally, we tested the relationship between impaired lipid metabolism and ROS production in the mutant strain. For this purpose, cells were grown to stationary phase, and ROS levels were detected using the DHE probe. The results showed that INO1 deficiency reduced ROS levels by ~ 30% in WT cells, but did not affect ROS levels in mutant cells (Fig. S3A). This indicates that oxidative damage is not directly linked to defects in membrane lipid metabolism in N88S seipin-expressing cells.
3.4. N88S seipin expressing cells are sensitive to iron deficiency conditions
The proteomic analysis revealed that proteins involved in iron ion homeostasis, including Fit1p, Arn1p, Arn2p and Hmx1p, were present at lower levels in N88S seipin expressing cells (Fig. 1C and Table S3). This prompted us to investigate whether there were alterations in cellular iron levels during lifespan imparted by the N88S seipin mutation. The results revealed that cells expressing the N88S seipin mutant accumulated iron (Fe) at the exponential phase (Fig. 5A). Nevertheless, there was a significant decrease in Fe levels from the exponential to PDS phase in the mutant, suggesting that cells experience iron deficiency. It is known that the demand for iron increases during the diauxic shift, and Aft1p is responsible for the regulation of proteins involved in capturing, internalizing, and mobilizing Fe to meet the needs of mitochondria, DNA repair, and other cellular processes (86–88). Using a LacZ reporter where the Aft1p binding sequence from the CTH2 promoter is fused to the LacZ gene, we found that Aft1p transcriptional activity boosted ~ 15-fold in WT cells grown from exponential phase to PDS phase. In contrast, in N88S seipin-expressing cells, the increase was approximately 7-fold, about half of the one observed in WT cells (Fig. 5B). This is in agreement with decreased protein levels of Fit1p, Arn1p, Arn2p and Hmx1p observed in these cells at PDS phase as identified by proteomic analysis (Table S3), which collectively correspond to proteins whose genes are transcriptionally activated by Aft1p/Aft2p in response to iron deficiency (37). To test if this effect is related to impaired ability to activate the iron regulon, cells were grown to the exponential phase and treated with the iron chelator bathophenanthrolinedisulfonate (BPS), which limits iron availability and causes an iron deprivation condition. As expected, β-galactosidase activity was significantly increased in WT cells as an adaptive response to BPS-induced iron depletion, but the induction was lower by 50–60% in the mutant strain compared to WT cells (Fig. 5C). To further address how Aft1p deregulation affects the iron deficiency response in cells expressing mutant seipin, WT and N88S seipin-expressing cells were subjected to transcriptomic analysis under BPS-induced iron deprivation (Fig. S4A-D). As expected, bioinformatic analysis revealed that GO functional categories were enriched among differentially expressed genes (DEGs) involved in iron-regulated processes and mitochondrial-related functions, namely tricarboxylic acid cycle and mitochondrial electron transport chain, iron-sulfur cluster assembly, sterol biosynthetic process and aminoacid metabolism (Fig. S4A-D). The results revealed that 197 and 250 genes had lower and higher expression in the mutant respectively, when compared to WT cells (Tables S5 and S6). We thus focused our attention on transcripts that are transcriptionally regulated by Aft1p/Aft2p upon BPS treatment, as defined in a previous study (49). Of the DEGs, 10 out of 28 genes previously reported to be upregulated during Fe starvation in WT cells (49) are downregulated in N88S seipin-expressing cells, including members of the Fe regulon: CCC2, ARN1, FIT1, FTH1, OLE1, FIT3, FET4, ATX1, SIT1 and FTR1 (Fig. S4E and Table S6). Conversely, 13 (ISA1, COR1, PYC2, LEU1, RNR4, BIO2, CYC1, HAP4, NFU1, CCP1, CYT1, QCR2 and RIP1) out of 34 genes with reduced expression in WT cells under similar conditions (49) are increased in the mutant strain (Fig. S4E and Table S5).
Overall, these results suggest that the N88S mutation renders cells unable to effectively activate Aft1p-mediated transcription of the iron regulon in response to changes in iron levels during lifespan and in response to iron deficiency. We then propose that this defective response may contribute to the increased ROS generation and oxidative damage previously reported for the mutant strain (45). To test this, cells were incubated with BPS at different growth phases and then allowed to grow in medium for 48 hours. When treated with the iron chelator at the exponential phase, where iron accumulation was observed in mutant cells (Fig. 5A), both WT and N88S seipin-expressing cells exhibited an acute growth defect (Fig. 5D). Although an increase in ROS content was noted in BPS-treated WT cells, the mutant displayed similar levels of ROS regardless of the presence of BPS in the medium (Fig. 5D). When cells were incubated with BPS at the diauxic shift, we observed no changes in growth and ROS content in WT and mutant cells (Fig. 5E). Next, we evaluated the sensitivity of WT and mutant cells to the oxidants t-BOOH and H2O2, and determined if this was altered by the addition of BPS to the medium (Fig. 5F). We observed that N88S seipin-expressing cells were slightly more sensitive to the tested oxidants than WT cells, but their sensitivity was not significantly altered by BPS when compared to WT cells (Fig. 5F). In the absence of any oxidative stress but in the presence of BPS, N88S seipin-expressing cells also displayed a mild growth defect as previously reported for cth2Δ cells (89), which is in agreement with lower Aft1p transcriptional activity and impaired response to iron deficiency (Fig. 5F). Overall, the data suggest that disruption of iron homeostasis is not the primary cause of oxidative damage in the yeast model of N88S seipinopathy.
3.5. The MAPK Hog1p/p38 contributes to impaired iron metabolism in cells carrying the seipin N88S mutation
The regulation of Aft1p phosphorylation by the MAPK kinases Hog1p and Slt2p has been implicated in the control of iron homeostasis and associated stress response pathways (43, 44). In particular, Hog1p negatively regulates Aft1p transcriptional activity, which allows its export from the nucleus to the cytosol under iron sufficient conditions (37, 38, 43, 90). Based on this, we posit that reduced activation of the iron regulation by Aft1p might be associated with changes in Hog1p activation and/or Aft1p phosphorylation. Detailed proteomic analysis revealed increased levels of Sko1p (Table S4). Sko1p is a key transcription factor regulating osmostress-induced gene expression under the direct control of the Hog1p (91). Sko1p binds to cAMP-responsive element (CRE) sequences, and the expression analysis of CRE-driven reporter genes is used as a reporter of osmostress-activated expression, which depends solely on Sko1p and Hog1p proteins (92–94). Using a 2xCRE-LacZ reporter, we evaluated changes in Hog1p activation during lifespan. At the exponential phase, we observed no significant changes, however a higher activation of the reporter fusion was observed in N88S seipin-expressing cells at PDS phase, indicating higher Hog1p activation (Fig. 6A). This coincided with reduced Aft1p transcriptional activity at this phase (Fig. 5B). To evaluate changes in Aft1p phosphorylation, we analyzed the migration pattern of HA-tagged Aft1p using Western blotting at PDS phase (Fig. 6B). Although total Aft1p levels are slightly higher (but not statistically significant), no noticeable alterations in the phosphorylation mobility pattern of Aft1p were observed in the mutant compared to WT cells (Fig. 6B). As a result, we conclude that the Aft1p phosphorylation status is not a primary cause for defective activation of the iron regulon at PDS phase (Fig. 5B).
The transcriptional activity of Aft1p is governed by its localization in the nucleus, which is controlled by several regulatory mechanisms involving a range of proteins. These interactions can result in alterations in the activation of the iron regulon. We thus investigated the localization of GFP-Aft1p in WT and mutant cells using fluorescence microscopy. Under iron-rich conditions, around 40% of wild-type cells displayed nuclear Aft1p, whereas only 21% of cells expressing the N88S seipin variant showed nuclear localization of the transcription factor (Fig. 6C). These data suggest increased export of Aft1p to the cytosol, which could explain why full activation of the iron regulon is hampered in the yeast model of N88S seipinopathy.
We decided to extend our analysis of the contribution of Hog1p to the loss of iron homeostasis imparted by the N88S seipin mutation. Firstly, we measured iron levels during lifespan, and the results revealed that deletion of HOG1 in WT cells increased iron levels at the exponential phase, consistent with its role as a negative regulator of the iron regulon (Fig. 6D). Importantly, the absence of Hog1p suppressed the iron accumulation phenotype of the mutant, and restored its ability to maintain proper iron levels as observed in WT cells (Fig. 6D). Next, we analyzed the Aft1p transcriptional activity during lifespan and upon BPS treatment in cells expressing pCTH2-LacZ. At PDS phase, we observed that deletion of HOG1 had no significant alterations in the β-galactosidase reporter activity in both WT and N88S seipin-expressing cells (Fig. 6E). This indicates that during lifespan, the activation of the iron regulon by Aft1p/Aft2p is mediated by other signal transduction pathways in addition to Hog1p regulation (37, 38). However, under iron deprivation, CTH2-LacZ expression was induced in WT cells and reached higher levels in the corresponding hog1Δ mutant (Fig. 6F). Notably, the transcriptional activity of Aft1p in cells expressing the N88S mutation was restored to WT levels (Fig. 6F) in the absence of the MAPK, supporting the idea that Hog1p controls iron levels and the adaptive response to iron deficiency in the yeast model of N88S seipinopathy.
We also decided to investigate whether Hog1p signalling also modulates IB formation in the mutant. For that, we followed intracellular localization of IBs using the Venus signal (45). We observed a reduction of number of cells displaying IBs upon deletion of either HOG1 or SKO1 (Fig. S5A-B), indicating that Hog1p/p38 also promotes IB generation beyond iron imbalance in cells expressing mutant seipin.
Finally, we decided to investigate if regulation of ER stress response is linked to loss of iron homeostasis observed in the mutant. To test this hypothesis, we analyzed CTH2-LacZ reporter activity in cells shifted to SC-glucose medium with or without inositol, supplemented or not with BPS. In WT cells, shifting to inositol-containing medium did not result in measurable changes in the β-galactosidase activity. Notably, CTH2-LacZ expression was induced only in the presence of BPS, with no significant changes due to inositol alone in these cells (Fig. S5C). However, in N88S seipin-expressing cells, there was a subtle but consistent increase in Aft1p transcriptional activity when grown in inositol-containing medium supplemented with BPS, compared to cells grown without inositol plus BPS (Fig. S5C). This finding is particularly important as it reveals a previously unexplored functional relationship between inositol and iron metabolism. It indicates that under conditions where PA is consumed and the ER stress response is attenuated in the mutant (Fig. 3E), there is higher activation of the iron regulon by Aft1p/Aft2p in the yeast model of N88S seipinopathy.
3.6. FET3 expression is altered in cells carrying the seipin N88S mutation
The expression of FET3, a gene involved in iron uptake, is also regulated by the transcription factor Aft1p (37, 95, 96). To evaluate if Aft1p inhibition in N88S seipin-expressing cells also affects FET3 expression, we used a construct containing the FET3 promoter (-863 bp to + 1) fused to the LacZ reporter gene (YEplac181-FET3-LacZ) (51). In this construct, there is an identified functional Aft1p consensus site (96) located − 254 bp upstream of the ATG translation initiation codon, and a predicted Aft1p-binding site at position − 670 bp (GGCACCC) in the FET3 promoter (51). The impact of N88S seipin mutation on FET3-LacZ expression was firstly assessed by measuring the β-galactosidase activity of the reporter during lifespan (Fig. 7A). The results showed no differences in the reporter gene expression in WT and N88S seipin-expressing cells at the exponential phase. We observed an increase in FET3 expression in WT cells at PDS phase, but the induction was higher in the mutant (Fig. 7A), which contrasts with reduced Aft1p-controlled CTH2 expression observed at the same phase (Fig. 5B). This suggests that in mutant cells, Aft1p regulates FET3 expression differently compared to other Aft1p-regulated genes.
We also tested if this effect is also observed under BPS-induced iron deficient conditions. As expected, BPS treatment induced FET3 expression in WT cells, but the reporter fusion activity was even higher in N88S seipin expressing cells (Fig. 7B). Overall, the results indicate that CTH2 expression was reduced, whereas FET3 expression is increased at PDS phase and under iron-deficient conditions in cells expressing N88S mutant seipin.
3.7. In-silico analysis revealed the presence of a potential Msn2p/Msn4p binding site in the FET3 promoter
It was previously shown that FET3 expression is not entirely dependent on Aft1p, and that the transcription factor Ace1p regulates the response to copper overload by limiting the expression of FET3 (51). To delineate potential promoter regions responsible for Aft1p-independent activation of FET3, we performed an unbiased bioinformatic analysis of the FET3 promoter (-863 bp to + 1) to search for potential transcription factor binding sites using YEASTRACT+ (74). The results revealed more than 50 transcription factors that could potentially bind to the FET3 promoter (Table S7). The results identified the putative Aft1p/Aft2p binding sequence (-670 bp), the predicted Ace1p-like binding site (-783 bp) (51), and importantly a Msn2p binding sequence located upstream (-813 bp) on the FET3 promoter was now uncovered (Fig. 7C).
In S. cerevisiae, the stress response involves a sophisticated network of sensing and signal transduction mechanisms, prominently featuring the transcriptional regulation of various genes. In yeast, the zinc-finger transcription factors Msn2p and Msn4p, which share 66% sequence homology, are key regulators of stress-responsive gene expression (97–102). They bind specifically to the stress response element (STRE) sequences 5′-AGGGG or 5′-GGGGA. Msn2p and Msn4p control the expression of over 90% of genes activated in response to heat stress, osmotic stress, and carbon starvation, and are essential for regulating chronological lifespan (97–102). Importantly, it was reported that overexpression of the plasma membrane receptor IZH2 inhibits FET3 expression via negative regulation of Msn2p/Msn4p transcriptional activation without requiring Aft1p-dependent induction of the iron-responsive element FeRE (103). Based on this study, we posit that overexpression of IZH2 should decrease the FET3-LacZ reporter gene expression if the latter relies on Msn2p/Msn4p activation. At the exponential phase, no significant differences were observed in all strains tested, but upon transition to PDS phase, we observed higher β-galactosidase activity of the FET3-LacZ reporter in cells expressing mutant seipin cells, which was reduced to WT levels upon overexpression of IZH2 (Fig. 7D). Importantly, we observed a reduction in FET3 expression in WT cells when IZH2 was overexpressed (Fig. 7D), which is consistent with a negative regulation of FET3 expression by Izh2p.
In keeping with a role for Msn2p/Msn4p in the regulation of FET3 expression, we further tested the expression of the pSTRE-LacZ reporter gene, which contains the STRE sequences from the CTT1 promoter regulated by these transcription factors (104). Here, we transformed both strains with the reporter, and β-galactosidase activity was measured during lifespan. We found that higher FET3 expression (Fig. 7A) paralleled the increased activation of Msn2p/Msn4p transcriptional activity at PDS phase in N88S seipin-expressing cells (Fig. 7E). In addition, a bioinformatic search for target genes with documented regulation by Msn2p/Msn4p identified genes encoding proteins whose levels were found to be higher at PDS phase by proteomic analysis (Table S4), including GRX2, PUT3, HXT1, HXT7, RAS2, DIP5, ARO10, MAL12 and ARO9 (Table S8). Altogether, the data strongly suggest that stress responsive Msn2p/Msn4p transcription factors are activated and may positively regulate FET3 expression at PDS phase, thus compensating for the reduced Aft1p transcriptional activity (as observed for CTH2-LacZ reporter) in N88S seipin-expressing cells (Fig. 5B). It is possible that FET3 expression acts as an adaptive response to partially counteract the reduced Aft1p-controlled CTH2 expression and lower protein levels of Fit1p, Arn1p, Arn2p, and Hmx1p. It is important to note that analysis of the CTH2 promoter used in the CTH2-LacZ reporter fusion (49) did not reveal any binding sites for Msn2p/Msn4p using YEASTRACT+ (data not shown).
3.8. The induction of FET3 expression is associated with a reduction in aconitase activity in cells expressing N88S mutant seipin
Cells respond to defective Fe-S cluster synthesis by accumulating iron both within the mitochondria and in the cell. It is known that reducing intracellular iron pool results in decreased activity of the Fe-S cluster enzyme aconitase and subsequently triggers FET3 expression (105, 106). Based on this, we measured aconitase activity of WT and N88S seipin-expressing cells grown to PDS phase, when FET3 expression was increased for the mutant. Aconitase activity was decreased by ~ 40% in the mutant (Fig. 7F), suggesting that mitochondrial Fe-S cluster synthesis/assembly may signal the upregulation of FET3 expression at PDS phase, while compensating for the reduced activation of the iron regulon by Aft1p, in an attempt to reestablish homeostatic intracellular iron levels. In agreement with this hypothesis, we observed that many genes involved in Fe-S cluster assembly were among the most affected DEGs in cells expressing the N88S seipin mutation under conditions of Fe deprivation (Fig. S4D).