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The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

2013; Wiley; Volume: 12; Issue: 5 Linguagem: Inglês

10.1111/acel.12113

1474-9726

Sophie D. Lefevre, Carlo W.T. van Roermund, Ronald J. A. Wanders, Marten Veenhuis, Ida J. van der Klei,

Peroxisome Proliferator-Activated Receptors

Aging CellVolume 12, Issue 5 p. 784-793 Original ArticleOpen Access The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae Sophie D. Lefevre, Sophie D. Lefevre Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The NetherlandsSearch for more papers by this authorCarlo W. van Roermund, Carlo W. van Roermund Departments of Pediatrics and Clinical Chemistry, Laboratory of Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The NetherlandsSearch for more papers by this authorRonald J. A. Wanders, Ronald J. A. Wanders Departments of Pediatrics and Clinical Chemistry, Laboratory of Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The NetherlandsSearch for more papers by this authorMarten Veenhuis, Marten Veenhuis Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The NetherlandsSearch for more papers by this authorIda J. van der Klei, Corresponding Author Ida J. van der Klei Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The Netherlands Correspondence Professor Dr. Ida J. van der Klei, Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, the Netherlands. Tel.: +31 (0) 50 363 2179; fax: +31 (0) 50 363 2348; e-mail:i.j.van.der.klei@rug.nlSearch for more papers by this author Sophie D. Lefevre, Sophie D. Lefevre Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The NetherlandsSearch for more papers by this authorCarlo W. van Roermund, Carlo W. van Roermund Departments of Pediatrics and Clinical Chemistry, Laboratory of Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The NetherlandsSearch for more papers by this authorRonald J. A. Wanders, Ronald J. A. Wanders Departments of Pediatrics and Clinical Chemistry, Laboratory of Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The NetherlandsSearch for more papers by this authorMarten Veenhuis, Marten Veenhuis Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The NetherlandsSearch for more papers by this authorIda J. van der Klei, Corresponding Author Ida J. van der Klei Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, The Netherlands Correspondence Professor Dr. Ida J. van der Klei, Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, P.O. Box 11103, 9700CC Groningen, the Netherlands. Tel.: +31 (0) 50 363 2179; fax: +31 (0) 50 363 2348; e-mail:i.j.van.der.klei@rug.nlSearch for more papers by this author First published: 11 June 2013 https://doi.org/10.1111/acel.12113Citations: 22AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary We studied the chronological lifespan of glucose-grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome-deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β-oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β-oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β-oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β-oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions. Introduction Yeast are attractive eukaryote model organisms in aging studies, both for replicative lifespan and chronological lifespan (CLS) analysis. This is exemplified by the fact that several conserved factors involved in aging were first identified in yeast. In particular, the role of mitochondria has been extensively studied (Seo et al., 2010; Breitenbach et al., 2012). However, in aging studies, relatively little attention has been paid to the function of another important class of oxidative cell organelles, namely peroxisomes. Like mitochondria, peroxisomes produce reactive oxygen species (ROS), compounds which have been implicated to play an important role in aging. Peroxisomes are single membrane-bound organelles that show an unprecedented variety of functions. Conserved functions are hydrogen peroxide metabolism and β-oxidation of fatty acids (Veenhuis et al., 1987; McCammon et al., 1990). Peroxisomes are dynamic organelles whose number and function are continuously adapted to cellular needs. Evidence has been presented that induction of peroxisome proliferation positively affects the lifespan of mice (Coschigano et al., 2003; Zhang et al., 2012). Also catalase, a conserved peroxisomal antioxidant enzyme, has been shown to contribute to lifespan determination in several model systems. The initial data on the role of peroxisomal catalase suggested that the absence of catalase decreases the lifespan of cells (Petriv & Rachubinski, 2004; Koepke et al., 2007, 2008). Recent studies however indicate that the absence of catalase also can extend the lifespan of cells at conditions that peroxisomal ROS activate anti-aging enzymes (Mesquita et al., 2010; Titorenko & Terlecky, 2011; Kawałek et al., 2013). Here, we address the role of peroxisomes in chronological aging using the yeast Saccharomyces cerevisiae. In S. cerevisiae, peroxisome proliferation is induced during growth of cells on oleic acid. At these conditions, peroxisomes harbor enzymes of the β-oxidation cycle, acyl-CoA oxidase (Pox1), bifunctional enzyme (Fox2) and thiolase (Pot1), the key enzymes of the glyoxylate cycle citrate synthase (Cit2), malate dehydrogenase (Mdh3) and malate synthase (Mls1) as well as catalase A (Cta1) (van Roermund et al., 2003). Peroxisome biogenesis is controlled by a unique set of proteins termed peroxins, encoded by PEX genes (Nuttall et al., 2011). Deletion of PEX genes-encoding proteins involved in matrix protein import or formation of the peroxisomal membrane biogenesis results in the mislocalization of matrix proteins. As a consequence, S. cerevisiae pex mutants are unable to grow on oleic acid. However, these mutants normally grow on glucose, a carbon source that is not metabolized by peroxisomal enzymes. So far, only few reports appeared on the role of peroxisomes in yeast aging. The chronological lifespan of two peroxisome-deficient mutant strains [Δpex5 (Goldberg et al., 2010) and Δpex6 (Jungwirth et al., 2008)] has been analyzed albeit at different cultivation conditions. Jungwirth et al. (2008) showed that deletion of PEX6 resulted in a decrease in survival of stationary phase cultures grown on synthetic complete (SC) media containing 2% glucose (Jungwirth et al., 2008). In cells lacking Pex6, all peroxisomal matrix proteins are mislocalized to the cytosol, but peroxisomal membrane proteins (PMPs) are correctly sorted to peroxisomal membrane remnants. When yeast cells are grown on 2% glucose, the acetic acid produced by the cells is a major cause of cell death. Acetic acid induces a mitochondrion-dependent apoptosis pathway (Ludovico et al., 2001; Burtner et al., 2009; Guaragnella et al., 2011). Why PEX6 deletion resulted in a decreased lifespan and enhanced acetic acid sensitivity remained speculative (Jungwirth et al., 2008). Goldberg et al. (2010) reported that deletion of PEX5 reduces the CLS especially when low glucose concentrations (0.2 or 0.5%) were used (Goldberg et al., 2010). These authors used complex YP medium (1% yeast extract, 2% peptone) instead of SC medium. Most peroxisomal matrix proteins contain a peroxisomal targeting signal 1 (PTS1), a tripeptide at the protein extreme C-terminus. Only few peroxisomal matrix proteins possess a peroxisomal targeting sequence 2 (PTS2). This signal is located at the N-terminus and has the consensus sequence (R/K)(L/V/I)X5(Q/H)(L/A/I). Pex5 and Pex7 are the receptor proteins for the PTS1 and PTS2, respectively. Deletion of PEX5 leads to mislocalization of PTS1 proteins to the cytosol, whereas PTS2 proteins are still properly sorted to peroxisomes. Conversely, in Δpex7 mutants, PTS2 proteins remain in the cytosol, whereas PTS1 proteins are correctly imported into peroxisomes. Peroxisomal acyl-CoA oxidase (Pox1), Fox2, Cit2, Mdh3, Mls1, and Cta1 contain a PTS1, whereas Pot1 has a PTS2. Hence, deletion of PEX5 results in the mislocalization of most, but not all, enzymes of the β-oxidation pathway to the cytosol. Goldberg and colleagues suggested that the physical separation of the PTS2 protein Pot1 from the other β-oxidation enzymes in Δpex5 cells caused a block in this metabolic pathway, which could contribute to the reduced CLS (Goldberg et al., 2010). PEX5 and PEX6 deletions give rise to different peroxisomal phenotypes (Nuttall et al., 2011), but it is unknown whether they have the same effect on yeast CLS. This led us to address the CLS of these deletion strains as well as of Δpex3 and Δpex7, grown at the same conditions, namely mineral media containing 0.5% glucose. Δpex3 cells completely lack peroxisomal membranes, and hence, in these cells, all matrix proteins as well as PMPs are mislocalized. Our data indicate that β-oxidation is important for survival during chronological aging of S. cerevisiae. Results Peroxisome deficiency reduces the CLS of glucose-grown S. cerevisiae cells We first analyzed the CLS of a Δpex3 strain as this mutant shows the most severe peroxisome biogenesis defect. We did not observe significant differences in CLS (Fig. 1A,B, Table 1), upon growth of Δpex3 and wild-type (WT) control cultures on mineral media containing 2% glucose. At these conditions, the CLS of S. cerevisiae is mainly determined by acetic acid toxicity (Burtner et al., 2009). However, when Δpex3 cells were grown on mineral media containing 0.5% glucose, both the mean and maximal lifespan of the Δpex3 cultures were reduced (10.5 ± 2.98 days/22.9 ± 2.43 days) relative to the WT control (16 ± 2.52 days/26.6 ± 3.33 days) (Fig. 1C,D, Table 1). We therefore performed all subsequent CLS experiments with media containing 0.5% glucose. Table 1. Mean and maximal lifespans Strain Mean CLS (days) Maximal CLS (days) 2% glucose WT 13 ± 1.41 23.5 ± 0.71 Δpex3 12.5 ± 0.71 20.5 ± 0.71 0.5% glucose WT 16 ± 2.52 26.6 ± 3.33 Δpex3 11.1 ± 2.98 22.9 ± 2.43 Δpex5 6.7 ± 2.33 9.93 ± 2.07 Δpex6 7.7 ± 0.35 21.5 ± 0.71 Δ3'pex5 7.5 ± 2.15 13.4 ± 1.63 Δpot1 11.1 ± 1.44 20.4 ± 3.42 Δtgl3 14.9 ± 1.27 24.5 ± 0.71 Δpex3Δpot1 8.6 ± 1.44 14.2 ± 2.17 Δpex7 10.9 ± 0.14 20 ± 0.07 Δatg1 15.5 ± 0.71 22.7 ± 0.35 Δatg1 Δpex3 12 ± 0.01 19 ± 0.71 Oleic acid WT 36.5 ± 7.78 47 ± 2.83 The mean chronological lifespan is defined as the time point where 50% of the cells are viable. The maximum lifespan is the time point where 10% of the cells are viable. Figure 1Open in figure viewerPowerPoint Peroxisome-deficient cells have a shorter lifespan than wild-type cells. (A) Chronological lifespan of WT and Δpex3 cells grown on 2% glucose. (B) The mean chronological lifespans of WT and Δpex3 cells calculated from the data in panel A. (C) Chronological lifespan of WT, Δpex3, Δpex5, and Δpex6 cells grown on 0.5% glucose. (D) The mean chronological lifespans of WT, Δpex3, Δpex5, and Δpex6 cells calculated from the data in panel C. Data represent mean ± SEM from at least three independent experiments. *P < 0.05; ***P < 0.005. Next, we analyzed two other pex mutants with other peroxisome biogenesis defects. In Δpex6 cells, PTS1 and PTS2 proteins are mislocalized to the cytosol, but peroxisomal membrane proteins (PMPs) are still correctly sorted to remnant peroxisomal membrane structures. In Δpex5 cells, only PTS1 proteins are mislocalized to the cytosol in conjunction with PMPs and PTS2 proteins localized to peroxisomes. As a consequence, the PTS1 and PTS2 enzymes of the β-oxidation pathway become physically separated in Δpex5 cells. As shown in Fig. 1C, the CLS of these two mutants is also shortened relative to the WT control upon growth on 0.5% glucose (Fig. 1C). Remarkably, Δpex5 cells displaying the mildest peroxisome biogenesis defect show the shortest CLS (6.7 ± 2.33 days/9.9 ± 2.07 days) (Fig. 1D, Table 1). Moreover, Δpex3 cells showing the strongest peroxisome biogenesis defect had a longer mean CLS relative to Δpex6 cells (7.8 ± 0.35 days vs. 10.5 ± 2.98 days), while the maximal lifespan is similar (21.5 ± 0.71 days vs. 22.9 ± 2.43 days) (Table 1). Unlike cultures of Δpex3 and Δpex6 cells, Δpex5 cultures showed a partial growth defect on glucose. Analysis of the genomic region of PEX5 revealed that PRP28 and MNN10 are flanking the PEX5 gene (Fig. 2A). PRP28 encodes an essential RNA helicase involved in RNA isomerization at the 5′ splice site (Strauss & Guthrie, 1991; Staley & Guthrie, 1999). MNN10 encodes a subunit of a Golgi mannosyltransferase complex the deletion of which affects growth (Karpova et al., 1993; Mondesert & Reed, 1996). In the used Δpex5 strain of the Euroscarf collection, the PEX5 gene is deleted from start to stop codon. The short distance between PRP28/MNN10 and PEX5 does not exclude that this deletion strategy may have shortened the promoter region of one or both of the flanking genes and affect their transcription. To test this, we complemented the Euroscarf PEX5 deletion strain by reintroducing the deleted PEX5 gene on a plasmid (Δpex5/PEX5 strain). As shown in Fig. 2B, reintroduction of the PEX5 gene did not result in restoration of the glucose growth defect. We also constructed a new PEX5 deletion strain preserving at least 500 bps in front of the start codon of PRP28 and MNN10 to avoid truncation of the promoter regions of these two genes (Fig. 2A). This strain is named Δ3'pex5. Figure 2Open in figure viewerPowerPoint Deletion of PEX5 by the Euroscarf strategy affects neighboring genes. (A) Genomic region of the PEX5 gene. The distance between PEX5 and PRP28 or MNN10 is 277 and 397 bps, respectively. Via the Euroscarf strategy, each gene was deleted from start to stop codon irrespectively of the genomic environment. A new strategy was designed to delete PEX5 from +250 to +1700 leaving 557 and 546 bps for the PRP28 and MNN10 promoters, respectively. (B) Growth curves of different yeast strains on media containing 0.5% glucose. The optical density was determined as absorbance at 600 nm. Data represent mean ± SEM, from at least two experiments. (C) Chronological lifespan of Δpex5 and Δ3'pex5 cells using WT as control. Data represent mean ± SEM from at least three experiments. (D) The mean and maximal chronological lifespans of Δpex5 and Δ3'pex5 cells calculated from the data shown in panel C. **P < 0.01. Growth experiments using different Δ3'pex5 clones showed that the defect observed in growth of Δpex5 cells on glucose is abolished in all Δ3'pex5 clones tested (Fig. 2B). This suggests that the transcription of either PRP28 or MNN10 or both was altered in the Euroscarf strain leading to an earlier entry into the stationary phase. Subsequently, we tested both Δpex5 and Δ3'pex5 mutants for chronological aging. The maximal lifespan of the Δ3'pex5 mutant is extended compared to the Euroscarf mutant (13.4 ± 1.63 days vs. 9.9 ± 2.07 days) indicating that the CLS of the Euroscarf strain is a result of a combination of the PEX5 deletion in conjunction with other deficiencies (Fig. 2C, D, Table 1). Nevertheless, the lifespan of Δ3'pex5 cells is still clearly reduced relative to WT cells and the shortest among all pex mutants under study, indicating that the defect in PTS1 protein import has a strong negative effect on chronological aging. Changes in autophagy do not explain the reduced lifespan of pex mutants Increased autophagy during chronological aging enhances yeast chronological lifespan (Alvers et al., 2009; Eisenberg et al., 2009; Morselli et al., 2009, 2010; Aris et al., 2013). One explanation of the reduced lifespan in pex mutants may be that these mutations affect autophagy. To test this, we performed chronological aging experiments using Δatg1 and Δatg1Δpex3 cells, strains in which autophagy is blocked. Upon deletion of PEX3 in Δatg1 cells, both the mean and maximum lifespan decreases, indicating that also in the absence of autophagy, peroxisome deficiency reduces yeast lifespan (Fig. S1). Peroxisomes proliferate and the β-oxidation pathway is induced during chronological aging In order to elucidate the principles of the shorter CSL of pex mutants relative to the WT control, as well as the mutual differences between the pex mutants studied, we investigated the fate of peroxisomes during chronological aging in WT cells. The growth curve of S. cerevisiae on 0.5% glucose starts with a short lag phase followed by logarithmic growth (2–10 h/0.4 days) and progresses through the diauxic shift (10–13 h/0.4–0.55 days) to the postdiauxic (13–48 h/0.55–2 days) and stationary phase (Fig. 3A). Using GFP-SKL as peroxisomal matrix marker, we observed that peroxisomes proliferate from 2.08 ± 0.87 per cell to 4.07 ± 1.29 per cell between 24 h (D1) and 48 h (D2), that is, during the late postdiauxic phase (Fig. 3B). Interestingly, in stationary phase cells, peroxisomes appear clustered in some cells, but remain detectable by fluorescence until 16 days (Fig. 3B). Figure 3Open in figure viewerPowerPoint Peroxisome proliferation and β-oxidation during chronological aging of WT cells. (A) Growth curve of WT cells on 0.5% glucose. The data represent mean ± SEM from at least six independent experiments. L, logarithmic phase; D, diauxic phase; PD, postdiauxic phase; ST, stationary phase. (B) Fluorescence microscopy images of WT cells producing GFP-SKL to mark peroxisomes at different time points of a chronological aging experiment. The cell contours are indicated in blue. D-day. (C, D) Western blots, using total crude extracts, decorated with anti-Pex5 (C) or anti-Pot1 (D) antibodies showing the levels of these proteins during chronological aging. Antibodies against mitochondrial porine (Por1) were used as a loading control. The quantification reflects the relative amount of protein. The value at day 15 (D15) was set to 1. Differences in loading were corrected using quantification of the Por1 bands. Values were calculated from two different gels. (E) β-oxidation activities during chronological aging measured as the release of 14C-CO2 by a suspension of whole cells in the presence of 14C-lauric acid. The data represent mean ± SEM, n = 3. We subsequently analyzed the levels of two proteins during chronological aging: Pex5 and Pot1, which are important for peroxisome biogenesis and function, respectively. Western blot analyses revealed that Pex5 is detectable at similar levels from day 1 (24 h) until day 15 (Fig. 3C). In contrast, Pot1 was very low during the growth phase (16 h after the start of the cultures the Pot1 level was 4% of that detected at day 15). This level rapidly increased during the postdiauxic phase (24 h after inoculation the pot level was 36% of the level detected at day 15 h). The highest level was detected at day 10, after which the levels slightly decreased (Fig. 3D). In order to further analyze whether β-oxidation is induced during chronological aging of glucose-grown cells, we determined the activity of this pathway by the detection of CO2 production from radiolabelled lauric acid. These assays revealed that indeed, the β-oxidation pathway is induced in the early stationary phase (from day 3) (Fig. 3E), but drops to a low, basal level at day 7, although Pot1 protein levels were still relatively high at this stage. Peroxisomal β-oxidation supports the lifespan The β-oxidation pathway converts activated fatty acids (fatty acyl-CoA) into NADH and acetyl-CoA that will subsequently be transferred to mitochondria to produce ATP. As sufficient ATP is essential to maintain cell viability, we tested whether peroxisomal β-oxidation contributes to survival of chronologically aging cultures and is responsible for the reduced CLS of glucose-grown cultures of pex strains. First, we deleted the third enzyme of the β-oxidation pathway (3-ketoacyl-CoA thiolase, Δpot1), which completely prohibits β-oxidation (see below; Fig. 5A,B). The mean and the maximal lifespan of Δpot1 cells are shortened (11.1 ± 1.44 days/20.4 ± 3.42 days) relative to WT controls (16 ± 2.52 days/26.6 ± 3.33 days; Fig. 4A,B, Table 1), indicating that β-oxidation is important for chronological aging. Figure 4Open in figure viewerPowerPoint The significance of β-oxidation in chronological aging. (A) Chronological lifespan of Δpot1 and Δtgl3 cells relative to WT control cells. Cells were grown on 0.5% glucose. The data represent mean ± SEM from at least two experiments. (B) The mean chronological lifespans calculated from the data presented in panels A and D. *P < 0.05; **P < 0.01; ***P < 0.005. (C) Accumulation of free fatty acids (FFA) in WT, Δpex3, Δ3'pex5, and Δpot1 cells at day 3 of a chronological aging experiment. *P < 0.05. (D) Chronological lifespans of WT cells grown on 0.5% glucose or 0.1% oleic acid. Data represent mean ± SEM from at least three experiments. (E) Transmission electron microscopy images showing the high content of lipid bodies in WT cells grown on oleic acid during chronological aging. During glucose growth, fatty acids are stored in lipid bodies as triacylglycerol (TG) and steryl esters (SE) (Henry et al., 2012). TG lipases and SE hydrolases are needed to mobilize them from these lipid bodies. Tgl3 is a TG lipase present at lipid bodies that converts TG to diacylglycerol (DG) plus free fatty acids (FFA) and also DG to monoacylglycerol (MG) plus FFA (Kurat et al., 2006). FFA can serve as substrates for peroxisomal β-oxidation (Henry et al., 2012). Deletion of TGL3 slows down the mobilization of neutral lipids and hence the levels of FFA released from lipid bodies. As shown in Fig. 4A, the mean and maximum CLS of Δtgl3 cells are reduced (14.9 ± 1.27 days/24.5 ± 0.71 days) relative to WT cells (16 ± 2.52 days/26.6 ± 3.33 days), but the CLS decrease is less severe than in Δpot1 cells (11.1 ± 1.44 days/20.4 ± 3.42 days; Fig. 4A,B, Table 1). The accumulation of FFA induces necrotic cell death (Rockenfeller et al., 2010). To test whether enhanced FFA levels in pex mutants explain their reduced lifespans, we determined FFA levels in the different mutant strains. FFA levels were measured at day 3 of the aging experiment, which corresponds to a time point of high β-oxidation activity in WT cells (Fig. 3E). As shown in Fig. 4C, FFA levels were similar in Δpex3 and WT cells. Hence, the reduced lifespan of Δpex3 cells cannot be explained by enhanced FFA levels. Moreover, the FFA levels in Δ3'pex5 and Δpot1 are similar, but the lifespan of Δpex5 cells is much shorter relative to that that of Δpot1 cells. These data suggest that lipid toxicity had no major effect on the lifespan of the cultures under the experimental conditions used. To test whether indeed β-oxidation enhances yeast CLS, we performed aging experiments using WT cells grown on oleic acid, a substrate that induces enzymes of the β-oxidation pathway. As shown in Fig. 4B,D and Fig. S2, culturing WT cells on oleic acid significantly extends both the mean and maximum lifespan (see also Table 1). In these cells, massive amounts of lipid bodies accumulate (Fig. 4E), which can supply substrates for peroxisomal β-oxidation during chronological aging. However, the CLS of mutants showing strong defects in β-oxidation is extremely short upon growth in the presence of oleic acid (Fig. S2). This is associated with a strong increase in FFA levels (Fig. S2B), which could lead to lipotoxicity. The peroxisomal β-oxidation pathway is functional in Δpex3 and Δpex6 cells but not in Δpex5 cells The above data suggest that β-oxidation may be important to support the lifespan; alterations in the activity of this pathway may modulate CLS of peroxisome-deficient cells. In Δpex3 and Δpex6 cells, all enzymes of the β-oxidation pathway are cytosolic, whereas in Δpex5 cells, only Pox1 and Fox2 are cytosolic while the PTS2 protein Pot1 is normally imported in peroxisomes. This leads to a physical partitioning of these enzymes. When cells were grown on glucose until the late exponential phase, β-oxidation activities were very low in all strains. In WT cells, β-oxidation was still detectable, but in the pex mutants, the activities of this pathway were undetectable or at the limit of detection (measured as production of [14C]-CO2 from 14C-lauric acid). Growth on oleic acid resulted in a strong increase in β-oxidation activity in WT cells. Growth in the presence of oleic acid also resulted in an increase in β-oxidation activities in Δpex3 and Δpex6 cells, but not in Δpex5 cells. Like in the Δpot1 control strain, the β-oxidation activity in Δpex5 cells was at the limit of detection (Fig. 5A). Figure 5Open in figure viewerPowerPoint β-oxidation occurs in Δpex3 cells and is an important factor in chronological aging. (A) β-oxidation activity was measured using whole cells by determining the release of [14C]-CO2 during incubation in the presence of 14C-lauric acid. Cells were grown until OD = 1 on 0.5% glucose or in medium containing 0.1% oleic acid. Data are expressed as percentage of the WT activities, arbitrarily set to 100%. (B) β-oxidation activities measured by determining the levels of released 14C-labeled acid soluble products (ASP) in the presence of 14C-lauric acid. Cells were grown as indicated at A. Data are expressed as percentage of the WT activities, arbitrarily set to 100%. (C) Chronological lifespans of WT, Δpex3Δpot1, and Δ3'pex5. Data represent mean ± SEM from at least three experiments. (D) The mean and maximal chronological lifespans calculated from the curves presented in panel C. To exclude that low β-oxidation activity, measured as production of [14C]-CO2 from 14C-lauric acid, was related to a defect in mitochondrial respiration, we also measured the production of 14C-labeled acid soluble products (ASP) from 14C-lauric acid. ASPs are direct products of the β-oxidation pathway. As shown in Fig. 5B, the production of ASPs followed the same trend as [14C]-CO2 production (Fig. 5B). Inactivation of β-oxidation in Δpex5 cells contributes to decrease cell viability To test whether the differences in β-oxidation in Δpex3 and Δpex5 cells explain the differences in chronological aging, we blocked the residual β-oxidation activity in Δpex3 creating a Δpex3Δpot1 double deletion strain. The CLS of Δpex3Δpot1 was reduced compared to Δpex3 (Fig. 5C, Table 1). Moreover, the mean and maximal lifespans of Δpex3Δpot1 are similar to that of Δ3'pex5. This suggests that indeed, the difference in CLS of Δpex3 and Δ3'pex5 cells is related to inactivation of the β-oxidation pathway in Δpex5 cells (Fig. 5D, Table 1). Deletion of PEX7 results in a decreased CLS The lifespan of Δpot1 cells (Fig. 4A) is longer compared to that of Δpex3Δpot1 or Δ3'pex5 cells (Table 1). Because in all three strains, the β-oxidation is blocked, additional processes most likely contribute to the reduction of the lifespan of peroxisome-deficient strains. In all three mutant strains, the initial oxidation of fatty acyl-CoA (catalyzed by Pox1) can still occur. This reaction results in the production of hydrogen peroxide, which is decomposed by peroxisomal catalase (Cta1) in WT cells. In Δpex3Δpot1, Δ3'pex5 and Δpex3, where both Pox1 and Cta1 are mislocalized to the cytosol, cells accumulate comparable levels of H2O2 during chronological aging as in Δpot1 cells, in which both enzymes are localized to peroxisomes (Fig. 6A). Hence, the observed differences in CLS of Δpot1 cells and the various pex mutants are unlikely due to differences in cellular H2O2 levels. Figure 6Open in figure viewerPowerP