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© The Rockefeller University Press,
0021-9525/2000//489 $5.00
The Journal of Cell Biology, Volume 150, Number 3,
, 2000 489-498
Original Article |
Pex11p Plays a Primary Role in Medium-Chain Fatty Acid Oxidation, a Process That Affects Peroxisome Number and Size in Saccharomyces cerevisiae
h.f.tabak{at}amc.uva.nl
The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) β-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA β-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in β-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.
Key Words: peroxisome • β-oxidation • peroxin • organelle multiplication • morphology
© 2000 The Rockefeller University Press
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Introduction |
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Since S. cerevisiae contains only 1–2 small peroxisomes per cell under most conditions of growth, proliferation of peroxisomes (to 10–20/cell) and induction of the fatty acid β-oxidation machinery is required in order to grow on a fatty acid as sole carbon source. The heterodimeric transcription factor Pip2p (Rottensteiner et al. 1996)/Oaf1p (Rottensteiner et al. 1997; Karpichev and Small 1998) is required for fatty acid–induced peroxisome proliferation and regulates the expression of proteins required for fatty acid oxidation, i.e., the β-oxidation enzymes, proteins required for transport of metabolites across the peroxisomal membrane, and components of metabolite shuttles (Kal et al. 1999). Interestingly, 
oaf1/pip2 cells also fail to induce expression of the abundant peroxisomal membrane protein Pex11p, thereby implying that Pex11p is coregulated with the β-oxidation machinery. Previously, Pex11p has been implicated in the regulation of the number of peroxisomes (Erdmann and Blobel 1995; Marshall et al. 1995). Yeast mutants lacking the PEX11 gene are unable to increase the number of peroxisomes when grown on oleate-containing media and instead accumulate a few (4–5) giant peroxisomes. On the other hand, cells overexpressing Pex11p exhibit a large number of small peroxisomal structures (Erdmann and Blobel 1995; Marshall et al. 1995; Sakai et al. 1995). Proteins with a low amino acid sequence similarity (20%) have been found in a wide variety of eukaryotes. Overexpression studies of these homologues in Kinetoplastida, and mammals have also been shown to affect peroxisome abundance which suggests that all these proteins are orthologues (Lorenz et al. 1998; Passreiter et al. 1998; Schrader et al. 1998). Taken together, these results suggest that Pex11p is involved in a process leading to fission or vesiculation of preexisting peroxisomes. An interesting observation in support for such a role of Pex11p was made by Passreiter et al. 1998. Rat Pex11p
was shown to bind coatomer in vitro by virtue of its cytoplasmically exposed carboxy-terminal dilysine motif. Recruitment of coatomer by Pex11p has been proposed to initiate vesiculation of peroxisomes and thereby influence peroxisome proliferation (Passreiter et al. 1998). However, this dilysine motif is not conserved in other Pex11p homologues thereby raising doubt about the universality of the proposed mechanism for Pex11p-mediated peroxisome proliferation.
Here we report that S. cerevisiae Pex11p is primarily involved in the oxidation of fatty acids, a process that is restricted to peroxisomes. Fatty acid oxidation in S. cerevisiae is mediated via the peroxisomal matrix enzymes acyl-CoA oxidase (Fox1p), the bifunctional protein (Fox2p) and 3-ketoacyl-CoA thiolase (Fox3p; Kunau et al. 1995). The peroxisomal membrane has been shown to form a permeability barrier for substrates and metabolites, in vivo (van Roermund et al. 1995), which predicts the presence of specialized transport systems that facilitate transport of substrates and metabolites across the peroxisomal membrane. Substrates for β-oxidation have been shown to enter peroxisomes via two different pathways (see Fig. 1; Hettema et al. 1996). MCFAs enter peroxisomes as free acids and are subsequently activated via the peroxisomal acyl-CoA synthetase, Faa2p, whereas long-chain fatty acids (LCFA) are activated outside peroxisomes and are subsequently imported via the peroxisomal ABC transporter Pat1p/Pat2p (Shani et al. 1995; Hettema et al. 1996; Verleur et al. 1997).
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Materials and Methods |
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, leu2, trp1, ura3-251, prb1-1122, pep4-3, gal2; Jones 1977). The faa2 and pat1 mutants have been described before (Hettema et al. 1996). Yeast transformants were selected and grown on minimal medium containing 0.67% yeast nitrogen base without amino acids (YNB-WO; Difco), supplemented with 0.3% glucose and amino acids (20 µg/ml) as needed. Liquid rich media used to grow cells for DNA isolation, subcellular fractionation, β-oxidation assays, immunogold electron microscopy and enzyme assays were composed of 0.5% potassium phosphate buffer, pH 6.0, 0.3% yeast extract, 0.5% peptone, and either 3% glycerol or 0.12% oleate/0.2% Tween 40. Before shifting to these media, the cells were grown on minimal 0.3% glucose or 2% glucose medium for at least 24 h. Minimal oleate medium contains YNB-WO supplemented with all amino acids and 0.12% oleate/0.2% Tween 40.
Disruption of the FOX1 and PEX11 Genes
The fox1 and pex11 deletion mutants were generated by one-step PCR-mediated gene disruption using the kanMX4 (Wach et al. 1994) as selectable marker. The PCR-derived disruption constructs comprised of the kanMX4 gene flanked by short regions of homology (50 bp) corresponding to the FOX1 and PEX11 5' and 3' noncoding region. The resulting PCR fragments were introduced into S. cerevisiae BJ1991 cells. G418-resistant clones were selected by growth on YPD plates containing 200 mg/liter G418 (Geneticin; Wach 1996).
Subcellular Fractionation and Nycodenz Gradient Equilibrium Density Analysis
Subcellular fractionation was performed as described by (Van der Leij et al. 1992). Organelle pellets (25,000 g) were layered on top of 15–35% Nycodenz gradients (12 ml), with a cushion of 1.0 ml 50% Nycodenz solutions containing 5 mM MES, pH 6.0, 1 mM EDTA, 1 mM KCl, and 8.5% sucrose. The sealed tubes were centrifuged for 2.5 h in a vertical rotor (MSE 8 x 35 ml) at 19,000 rpm at 4°C. Gradients fractionated into 14 fractions were analyzed for enzyme activity of various marker enzymes as described below.
Electron Microscopy
Oleate-induced cells were fixed with 2% paraformaldehyde (wt/vol) and 0.5% glutaraldehyde (wt/vol). Ultra-thin sections were prepared as previously described (Gould et al. 1990).
Enzyme Assays
β-Oxidation assays in intact cells were performed as previously described (van Roermund et al. 1998). Cells were grown overnight in media containing oleate to induce fatty acid β-oxidation. The β-oxidation activity in wild-type cells in each experiment was taken as reference (100%) and is expressed as the sum of CO2 and water-soluble β-oxidation products produced. The activities of oleate-, palmitate-, myristate-, laureate-, and octanoate-β-oxidation in oleate-grown wild-type cells were 12.1 ± 1.5; 1.0 ± 0.3; 1.9 ± 0.2; 2.7 ± 0.6; 8.1 ± 1.7 nmol/h/mg protein, respectively. The β-oxidation activity in lysates measured with octanoate as substrate was 6.4 ± 0.5 nmol/min/mg protein.
3-Hydroxyacyl-CoA dehydrogenase activity was measured on a Cobas-Fara centrifugal analyzer by monitoring the acetoacetyl-CoA–dependent rate of NADH consumption at 340 nm (Wanders et al. 1992). Fumarase activity was measured on a Cobas-Fara centrifugal analyzer monitoring the APADH production at 365 nm. The reaction was started with 10 mM fumarate in an incubation mixture of 100 mM Tris (pH 9.0), 0.1% Triton X-100, 4 U/ml malate dehydrogenase (Boehringer) and 1 mM APAD for 5 min at 37°C. Acyl-CoA synthetase activity was measured essentially as described (Knoll et al. 1994). Acyl-CoA synthetase activity in wild-type cells measured with octanoate as substrate was taken as reference (100%) and corresponds to 120 pmol/min/mg protein in intact cells and 3,560 pmol/min/mg protein in lysates. Protein concentrations were determined by the bicinchonic acid method (Smith et al. 1985).
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Results |
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In human cells, a deficiency of one of the peroxisomal β-oxidation enzymes such as acyl-CoA oxidase (Poll-Thé et al. 1988) leads to a defect in the proliferation of this organelle. We tested whether a yeast β-oxidation mutant deficient in acyl-CoA oxidase (fox1) would also be disturbed in peroxisomal proliferation (see Fig. 1 for a simplified view of the peroxisomal β-oxidation system). Therefore, both wild-type and fox1 cells were grown in oleate-containing medium and peroxisomes were visualized by immunogold electron microscopy using 10-nm gold particles coupled to antibody raised against Fox3p (thiolase). This analysis revealed aberrant peroxisomal structures in fox1 cells, which were larger and frequently surrounded by multiple membranes (see Fig. 2, A–C). To follow the peroxisomal proliferation process during the transition from glucose- to oleate-containing medium, we used the GFP-based proliferation assay developed by Marshall et al. 1996 which allows visualization of peroxisomal structures in living S. cerevisiae cells. For this purpose, we expressed green fluorescent protein containing a peroxisomal targeting signal type 1 (GFP-PTS1) in fatty acid oxidation mutants, in which either the gene encoding acyl-CoA oxidase (fox1), the peroxisomal ABC transporter Pat1p (pat1) or the peroxisomal fatty acyl-CoA synthetase (faa2) was deleted. Previously, we have shown that Faa2p is specifically required for MCFA β-oxidation, whereas Pat1p is required for β-oxidation of long-chain fatty acids (Fig. 1). We found that the β-oxidation mutants faa2 and fox1, but not pat1 cells, showed less fluorescent structures per cell (Fig. 2 D). However, 4 h after the shift to oleate medium these structures seem to be larger and more intensely fluorescent (not shown).
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Pex11p Is Required for MCFA Oxidation
Since MCFA β-oxidation is required for peroxisome proliferation, the peroxisome fission defect observed in pex11 cells might be the consequence of a disturbance in β-oxidation.
To test this hypothesis we analyzed oxidation of the (1-14C)-labeled MCFA octanoate (Fig. 3 A). We found that oxidation of this MCFA is impaired in intact pex11 cells, but importantly, rates of fatty acid oxidation in lysates were unaffected (Fig. 3 B). These results show that the activity of the β-oxidation enzymes themselves was unaffected.
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pex11 cells are not disturbed in import of peroxisomal proteins (Erdmann and Blobel 1995; Marshall et al. 1995). The data in Fig. 4 A are in line with these results since virtually all β-oxidation activity in a homogenate of pex11 cells was present in the crude organellar pellet. Subsequent fractionation of the organellar pellet by density gradient centrifugation revealed that the β-oxidation activity cofractionated with the peroxisomal marker 3HAD (Fig. 4 B). These data show that all β-oxidation enzymes are present in peroxisomes of pex11 cells and strongly suggest that Pex11p is required for transport of β-oxidation metabolites/substrates across the peroxisomal membrane. Subsequently, we tested the β-oxidation capacity of pex11 cells using fatty acids of various chain length. As shown in Fig. 5 A, β-oxidation of MCFA was as deficient in pex11 cells as in fox1 cells (<1% of control), whereas only a partial deficiency was found with fatty acids of longer chain length. We conclude that Pex11p is specifically required for β-oxidation of MCFAs.
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pex11 cells suggests that Pex11p functions in the Faa2p-dependent pathway. We constructed double mutants lacking the PEX11 gene and either the FAA2 gene or the PAT1 gene and measured MCFA- and LCFA- β-oxidation activity in these double mutants to test this hypothesis (Fig. 5 B). Indeed, pex11/pat1 cells show a complete block in both MCFA and LCFA β-oxidation, whereas pex11/faa2 cells are specifically disturbed in MCFA β-oxidation. These results show that Pex11p functions in the same fatty acid entry pathway as Faa2p and in parallel to Pat1p. We conclude that Pex11p is required for β-oxidation of fatty acids that enter peroxisomes as free fatty acids.
We investigated whether the impaired oxidation of MCFA in pex11 cells is caused by a loss of Faa2p activity. Therefore, we measured acyl-CoA synthetase activity with an Faa2p-specific substrate (C8:0, octanoate) in both intact cells and lysates (Fig. 6A and Fig. B). In lysates prepared from pex11 cells C8:0-CoA synthetase activity was normal. However, intact pex11 cells are not able to produce C8:0-CoA in vivo.
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pex11 cells we performed immunogold electron microscopy on cryo sections of oleate-grown pex11 cells expressing NH-tagged Faa2p (Fig. 6 C). NH-Faa2p is fully active and is associated with inner leaflet of the peroxisomal membrane (Hettema et al. 1996). We found NH-Faa2p to be associated with the peroxisomal membrane in pex11 cells, showing that Faa2p reaches its normal subcellular localization independent of Pex11p. Alternatively, Pex11p is required for transport of substrates across the peroxisomal membrane. As peroxisomes are notoriously fragile upon isolation, we decided to test this possibility by an in vivo experiment. If Pex11p is required for transport of substrates, then cytosolically located Faa2p should be active in both the absence and presence of Pex11p, and the MCFAs activated in the cytosol should be oxidized dependent on the peroxisomal ABC transporter Pat1p/Pat2p. We expressed an Faa2p version that lacks its peroxisomal targeting signal in pex11 cells, pex11/pat1 cells, and wild-type cells and measured MCFA β-oxidation activity 4 h after the shift to oleate medium (Fig. 7A and Fig. B). Indeed, mislocalization of Faa2p to the cytosol partially rescues the MCFA β-oxidation defect observed in pex11 cells. The cytosolically-produced MCFA-CoA esters can now enter peroxisomes via the pathway for activated fatty acids as indicated by the inability of cytosolic Faa2p to rescue the MCFA β-oxidation defect in pex11/pat1 cells.
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Pex11p Is Primarily Involved in MCFA Oxidation, which Secondarily Affects Peroxisomal Proliferation
We performed a kinetic experiment where we transferred cells from glucose- to fatty acid–containing medium and followed β-oxidation after the transfer. Fig. 8 A shows that pex11 cells were not able to oxidize octanoate already shortly after the transfer. In parallel, peroxisome proliferation was followed using GFP-PTS1. In pex11 cells peroxisome number and size were indistinguishable from wild-type cells up to 2 h after the transfer to oleate-containing medium (Fig. 8 C). After prolonged incubation on oleate medium (>10 h) the typical morphology of giant peroxisomes became apparent. Initially, induction of oleate β-oxidation was slightly affected (Fig. 8 B), and only at later timepoints (24 h) oleate β-oxidation decreased (not shown). The defect in oleate oxidation of pex11 cells 24 h after the shift explains the retarded growth on oleate medium and probably reflects a failure to segregate giant peroxisomes to daughter cells (Erdmann and Blobel 1995).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Characterization of Pex11p in both yeast and in mammalian cells has led to the proposal that it is involved in fission of larger peroxisomes into smaller ones. Cells that lack Pex11p accumulate a few large peroxisomes in contrast to cells in which Pex11p is overproduced which contain many small peroxisomes. Furthermore, it was shown that rat Pex11p was capable of binding coatomer in vitro suggesting that vesiculation of peroxisomes made use of a more general combination of factors required for vesiculation of other organelles.
Here we have reevaluated this proposed role of Pex11p and suggest another function for this peroxin. Our interest was raised by observations suggesting that formation of large peroxisomes could be the result of abnormal fatty acid metabolism. For instance, deficiencies of acyl-CoA oxidase (Poll-Thé et al. 1988; Fan et al. 1996; Chang et al. 1999) or the multifunctional enzymes D- or L-bifunctional enzyme (Suzuki et al. 1997; Chang et al. 1999; Qi et al. 1999; van Grunsven et al. 1999) in mammalian cells results in a reduced number of peroxisomes with enlarged volume and in mouse hepatocytes overexpressing the multi-drug transporter MDR2 large numbers of peroxisomes are occasionally seen (Mauad et al. 1994). These observations suggest that peroxisome proliferation is under control of a signaling pathway influenced by certain metabolic ligands. We have explored these facts in a more systematic way in S. cerevisiae in combination with morphological analysis of peroxisomes using electron microscopy and fluorescence light microscopy. All our experiments point to the importance of MCFA oxidation for peroxisome proliferation to occur. On the basis of previous work Pex11p would be expected to act at a late execution point in this putative signaling cascade. Surprisingly, our experiments rather suggest that Pex11p acts at a much earlier stage. This is based on the fact that pex11 cells first show a deficiency in MCFA oxidation and much later develop abnormal peroxisomal structures when shifted from glucose to oleate-containing medium. These results provide strong evidence against the model that Pex11p is directly involved in a vesiculation process (at least in S. cerevisiae). We rather favor the idea that Pex11p is part of a chain of events leading to the production of a signaling molecule responsible for modulation of the peroxisome proliferation process.
Pex11p is involved either directly or indirectly in getting MCFAs across the membrane since a pex11 mutant shows latency characteristic of a membrane-mediated process: deficiency of MCFA oxidation when membranes are intact but full capacity for MCFA oxidation in a detergent lysate in which membranes are dissolved. Indeed, one of the substrates is not able to reach the fatty acid CoA synthetase Faa2p which is associated with the inside of the peroxisomal membrane, since no MCFA-CoA esters are formed in pex11 cells. These characteristics qualify Pex11p as a transporter either for MCFAs or for essential cofactors involved in β-oxidation such as ATP, CoA etc. Interestingly, Pex11p shows extensive amino acid sequence similarity to the ligand-binding domain (LBD) of the nuclear hormone receptors (Barnett et al. 2000). The greatest similarity is found with the LBDs of PPAR, which are able to bind fatty acids. In analogy, Pex11p might contain a binding site for fatty acids which suggests a role in fatty acid transport across the peroxisomal membrane.
Unfortunately, the properties of ScPex11p are not completely in line with being a straightforward metabolite transporter. The primary amino acid sequence does not reveal obvious membrane spanning regions that could form a pore-like structure. There is also some controversy about its peroxisomal sublocalization. Pex11p of S. cerevisiae has been reported to be an integral membrane protein by one group and was shown to be inside peroxisomes, tightly associated with the matrix side of the peroxisomal membrane by another group (Erdmann and Blobel 1995; Marshall et al. 1995, Marshall et al. 1996). In our hands, ScPex11p behaved as a peripheral membrane protein as it was extractable from membranes by carbonate treatment (data not shown). Mammalian Pex11p is considered to be an integral membrane protein with NH2 and COOH termini protruding into the cytoplasm (Passreiter et al. 1998). Some of these studies made use of epitope-tagged versions of Pex11p which might have influenced its position. Further work is required to settle this issue which is crucial for further delineation of the role of Pex11p in fatty acid β-oxidation.
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Acknowledgments |
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This work was supported by the Netherlands Foundation for Scientific Research (NWO).
Submitted: 16 December 1999
Revised: 8 June 2000
Accepted: 13 June 2000
Abbreviations used in this paper: GFP-PTS1, green fluorescent protein containing a peroxisomal targeting signal type 1; LBD, ligand-binding domain; LCFA, long-chain fatty acids; MCFA, medium-chain fatty acid; PPAR, peroxisomal proliferation activator receptors.
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) or 3-hydroxy-CoA dehydrogenase (•) activity in each gradient fraction is expressed as a percentage of the sum activity measured throughout the gradient. (M) Mitochondria and (P) peroxisomes indicate fractions with the highest fumarase or 3-hydroxy-CoA dehydrogenase activity, respectively. Fraction 1 is at the bottom of the gradient.
