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The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p—now designated Pex30p, Pex31p, and Pex32p, respectively—together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.
Peroxisomes are highly responsive organelles, because their size, number, protein composition and biochemical functions vary dramatically depending on the organism, cell type, and environmental milieu. Peroxisomes are essential for normal human development and physiology, as demonstrated by the lethality of the peroxisome biogenesis disorders, a group of autosomal recessive diseases in which multiple peroxisomal metabolic pathways are dysfunctional because peroxisome biogenesis is compromised. Peroxisome assembly, division, and inheritance are controlled by at least 29 proteins termed peroxins that are encoded by the PEX genes. To date, mutations in 12 PEX genes have been shown to give rise to the peroxisome biogenesis disorders (reviewed in Subramani et al., 2000 ; Purdue and Lazarow, 2001 ; Titorenko and Rachubinski, 2001 ; Brosius and Gärtner, 2002 ; Matsumoto et al., 2003 ).
Peroxisomal proteins are encoded exclusively by nuclear genes, synthesized on cytosolic polysomes and posttranslationally targeted to the peroxisome. They are sorted to the peroxisome by specific peroxisome targeting signals (PTSs). PTSs are recognized in the cytosol by their cognate receptors, which direct the targeting and docking of proteins to the peroxisome membrane. Most peroxisomal matrix proteins are targeted by PTS1, an extreme carboxyl-terminal tripeptide, and its shuttling receptor, the peroxin Pex5p. Strikingly, Pex5p has been shown to enter the peroxisome matrix together with its cargo and to recycle back to the cytosol following dissociation from its cargo (Dammai and Subramani, 2001 ). A few matrix proteins are targeted by PTS2, an amino-terminal nonapeptide, and its cytosolic shuttling receptor, Pex7p. A limited number of matrix proteins are targeted by largely uncharacterized internal PTSs. The sorting of peroxisomal membrane proteins is much less understood and appears to be independent of matrix protein import. The cytosolic form of the peroxin Pex19p may act as a shuttling receptor for peroxisomal membrane proteins, whereas its peroxisome-associated form could function as a chaperone, assisting the assembly of multimeric complexes after their binding to the peroxisome membrane (reviewed in Subramani, 1998 ; Hettema et al., 1999 ; Subramani et al., 2000 ; Terlecky and Fransen, 2000 ; Purdue and Lazarow, 2001 ; Titorenko and Rachubinski, 2001 ).
In contrast to protein sorting to peroxisomes, much less is known about the mechanism of peroxisome proliferation and the proteins involved in this process. A few proteins have been implicated directly in regulating this process. Oversynthesis of Pex11p leads to the formation of small peroxisomes, whereas cells lacking Pex11p have fewer but larger peroxisomes than normal (Erdmann and Blobel, 1995 ; Marshall et al., 1995 ; Sakai et al., 1995 ; Li and Gould, 2002 ; Li et al., 2002 ). The dynamin-like protein Vps1p (Hoepfner et al., 2001 ) and the peroxins Pex25p (Smith et al., 2002 ) and Pex27p (Tam et al., 2003 ; Rottensteiner et al., 2003b ) of the yeast Saccharomyces cerevisiae have recently been shown to be required for the control of peroxisome size and number. Cells deleted for VPS1, PEX25, or PEX27 contain enlarged peroxisomes.
YlPex23p is a 48-kDa integral membrane protein of peroxisomes required for peroxisome assembly in the yeast Yarrowia lipolytica (Brown et al., 2000 ). A search of protein databases revealed that YlPex23p shares extensive sequence similarity with proteins encoded by the open reading frames (ORF) YLR324w, YGR004w, and YBR168w of the S. cerevisiae genome. Here we report that YLR324w, YGR004w, and YBR168w all encode peroxisomal integral membrane proteins, and we provide evidence for their role in controlling peroxisome size and number in S. cerevisiae.
The yeast strains used in this study are listed in Table 1. All strains were cultured at 30°C. Media components were as follows: YPD, 1% yeast extract, 2% peptone, 2% glucose; YPBO, 0.3% yeast extract, 0.5% peptone, 0.5% K2HPO4, 0.5% KH2PO4, 1% Brij 35, 1% (vol/vol) oleic acid; synthetic minimal (SM) medium, 0.67% yeast nitrogen base without amino acids, 2% glucose, 1× Complete Supplement Mixture (Bio 101, Carlsbad, CA) without histidine and/or leucine; sporulation medium, 1% potassium acetate, 0.1% yeast extract, 0.05% glucose, 2% agar; YNBD, 0.67% yeast nitrogen base without amino acids, 2% glucose, containing histidine, leucine, and uracil, each at 30 μg/ml.
The plasmids pDsRed-PTS1 (Smith et al., 2002 ) and pProtA/HIS5 (Rout et al., 2000 ) have been described. Genes to be overexpressed were amplified by PCR and cloned into the plasmid YEp13 (Broach et al., 1979 ). For overexpression, the YLR324w gene included 609 base pairs of upstream and 348 base pairs of downstream sequence, the YGR004w gene included 625 base pairs of upstream and 217 base pairs of downstream sequence, and the YBR168w gene included 592 base pairs of upstream and 381 base pairs of downstream sequence. PEX28 and PEX29 overexpression constructs have been described (Vizeacoumar et al., 2003 ).
Genes were genomically tagged with the sequence encoding Staphylococcus aureus protein A by homologous recombination using PCR-based integrative transformation into parental BY4742 haploid cells (Aitchison et al., 1995 ; Dilworth et al., 2001 ). The functionality of fusion proteins was confirmed by the ability of transformed strains to grow and to proliferate peroxisomes like the wild-type strain BY4742 in medium containing oleic acid as the sole carbon source.
Strains encoding protein A chimeras were transformed with the plasmid pDsRed-PTS1 were grown in SM medium for 12 h and then incubated in YPBO medium for 8 h. Cells were processed for immunofluorescence microscopy as described (Pringle et al., 1991 ; Tam and Rachubinski, 2002 ). Protein A chimeras were detected with rabbit antiserum to mouse IgG (ICN Biomedicals, Irvine, CA) and FITC-conjugated goat anti-rabbit IgG. Images were captured on a LSM510 META (Carl Zeiss MicroImaging, Thornwood, NY) laser scanning microscope or with a digital fluorescence camera (Spot Diagnostic Instruments, Sterling Heights, MI). Whole cells were processed for electron microscopy as described (Eitzen et al., 1997 ).
For each strain analyzed, electron microscopic images of 50 randomly selected cells at 17,000× magnification were captured with a digital camera (Soft Imaging System, Lakewood, CO), and the areas of individual cells and of individual peroxisomes were determined by the program analySIS 3.1 (Soft Imaging System). To determine the average area of a peroxisome, the total peroxisome area was calculated and divided by the total number of peroxisomes counted. To quantify peroxisome number, the numerical density of peroxisomes (number of peroxisomes per μm3 of cell volume) was calculated by the method of Weibel and Bolender (1973 ) for spherical organelles. First, the total number of peroxisome profiles was counted and reported as the number of peroxisomes per cell area assayed (NA). Next, the peroxisome volume density (VV) was calculated (total peroxisome area/total cell area assayed). Using the values VV and NA, the numerical density of peroxisomes was determined (Weibel and Bolender, 1973 ).
Subcellular fractionation and isolation of peroxisomes were done essentially as described (Smith et al., 2002 ; Vizeacoumar et al., 2003 ). Briefly, cells grown overnight in YPD medium were transferred to YPBO medium and incubated for 8 h. Cells were harvested, washed, and converted to spheroplasts by digestion with Zymolyase 100T (1 mg/g of cells) in 50 mM potassium phosphate, pH 7.5, 1.2 M sorbitol, and 1 mM EDTA for 1 h at 30°C. Spheroplasts were lysed by homogenization in buffer H (0.6 M sorbitol, 2.5 mM MES, pH 5.5, 1 mM KCl) containing 1 mM EDTA and 2× complete protease inhibitor cocktail (Roche, Nutley, NJ). The homogenate was subjected to centrifugation for 10 min at 2000 × g to yield a postnuclear supernatant (PNS) fraction. The PNS fraction was subjected to further differential centrifugation at 20,000 × g for 30 min to yield a supernatant (20KgS) fraction enriched for cytosol and a pellet (20KgP) fraction enriched for peroxisomes. The 20KgP fraction was resuspended in buffer H containing 11% Nycodenz and PINS (0.5 mM benzamidine, 2 μg leupeptin/ml, 2 μg aprotinin/ml, 1 μg pepstatin A/ml, 3 μg antipain/ml, and 0.5 mg Pefabloc/ml), and a volume containing 5 mg of protein was overlaid onto a 30-ml discontinuous gradient consisting of 17, 25, 35, and 50% (wt/vol) Nycodenz in buffer H containing PINS. Organelles were separated by centrifugation at 100,000 × g for 90 min in a VTi50 rotor (Beckman Coulter, Fullerton, CA). Fractions of 2 ml were collected from the bottom of the gradient.
Peroxisomes were extracted as described (Fujiki et al., 1982 ). Essentially, organelles in the 20KgP fraction (50 μg of protein) were lysed by incubation in 10 volumes of Ti8 buffer (10 mM Tris-HCl, pH 8.0) containing 3× PINS on ice for 1 h and separated into pellet (Ti8P) and supernatant (Ti8S) fractions by centrifugation at 245,000 × g for 1 h at 4°C in a TLA120.2 rotor (Beckman Coulter). The Ti8P fraction was resuspended in Ti8 buffer to a final protein concentration of 0.5 mg/ml, and a portion of the resuspended fraction was extracted with 0.1 M Na2CO3, pH 11.3, for 1 h on ice and then separated into supernatant (CO3S) and pellet (CO3P) fractions by centrifugation at 245,000 × g in a TLA120.2 rotor at 4°C for 1 h. Proteins in the Ti8S, Ti8P, CO3S, and CO3P fractions were precipitated by addition of trichloroacetic acid, and the precipitates were washed with acetone. Proteins in equal portions of each fraction were separated by SDS-PAGE and analyzed by immunoblotting.
Strains harboring different double deletions of the YLR324w, YGR004w, YBR168w, PEX28, and PEX29 genes were constructed in the manner described below for a strain deleted for the YLR324w and YGR004w genes.
The homozygous deletion diploid strain ylr324Δ-HD (Giaever et al., 2002 ) was sporulated, and the tetrads were dissected to select for the haploid MATa strain. This strain was mated to the haploid MATα deletion strain ygr004Δ by replica plating to obtain a heterozygous diploid strain harboring deletions for both the YLR324w and YGR004w genes. The diploid strain was sporulated, and tetrads from 10 heterozygous diploids were dissected by micromanipulation. All spores were grown in YPD medium, and DNA was extracted. Haploid strains carrying deletions in both the YLR324w and YGR004w genes were confirmed by PCR analysis. In this way, nine strains containing the following double gene deletions were constructed: ylr324Δ/ygr004Δ (DK1), ylr324Δ/ybr168Δ (DK2), ygr004Δ/ybr168Δ (DK3), pex28Δ/ylr324Δ (CD1), pex28Δ/ygr004Δ (CD2), pex28Δ/ybr168Δ (CD3), pex29Δ/ylr324Δ (CD4), pex29Δ/ygr004Δ (CD5), and pex29Δ/ybr168Δ (CD6).
The gene conferring kanamycin resistance used to disrupt the YBR168w gene in the MATa strain of ybr168Δ was replaced with the gene encoding resistance to the drug nourseothricin (Werner BioAgents, Jena, Germany) from the plasmid pHN15 (Krügel et al., 1993 ). This nourseothricin-resistant MATa strain deleted for YBR168w was crossed with the MATα strain DK1 (YLR324Δ/YGR004Δ), and the resultant diploid was sporulated. The strain TKO deleted for the YBR168w, YLR324w, and YGR004w genes was selected on agar plates containing both kanamycin and nourseothricin, and deletion of the three genes in this strain was confirmed by PCR analysis.
Physical interactions between Ylr324p, Ygr004p, Ybr168p, Pex28p, and Pex29p were detected using the Matchmaker Two-Hybrid System (Clontech, Palo Alto, CA). Chimeric genes were generated by amplifying the ORFs of the YLR324w, YGR004w, YBR168w, PEX28, and PEX29 genes by PCR and ligating them in-frame and downstream of the DNA encoding the transcription-activating domain (AD) and the DNA-binding domain (DB) of the GAL4 transcriptional activator in the plasmids pGAD424 and pGBT9, respectively. Cells of S. cerevisiae strain SFY526 were transformed simultaneously with a pGAD424-derived plasmid and a pGBT9-derived plasmid. Transformants were grown on SM medium lacking tryptophan and leucine and tested for activation of the integrated lacZ construct using a β-galactosidase filter assay (Smith and Rachubinski, 2001 ).
Antibodies to the carboxyl-terminal SKL tripeptide, thiolase, and Sdh2p have been described (Vizeacoumar et al., 2003 ). FITC-conjugated anti-rabbit IgG and rhodamine-conjugated anti-guinea pig IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) were used to detect primary antibodies in immunofluorescence microscopy. Rabbit antibodies to glucose-6-phosphate dehydrogenase (G6PDH) of S. cerevisiae were obtained from Sigma-Aldrich (St. Louis, MO).
Extraction of nucleic acid from yeast lysates and manipulation of DNA were performed as described (Ausubel et al., 1994 ). Immunoblotting was performed using a wet transfer system (Ausubel et al., 1994 ), and antigen-antibody complexes in immunoblots were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). Protein concentration was determined using a commercially available kit (Bio-Rad Laboratories, Richmond, CA) and bovine serum albumin as standard.
YlPex23p is an integral peroxisomal membrane protein that has been shown to be required for peroxisome assembly in the yeast Y. lipolytica (Brown et al., 2000 ). A search of protein databases with the GENEINFO(R)BLAST Network Service of the National Center for Biotechnology Information revealed three proteins encoded by the ORFs YLR324w, YGR004w, and YBR168w of the S. cerevisiae genome that exhibit extensive sequence similarity to YlPex23p (Figure 1). YlPex23p and Ylr324p exhibit 36.3% amino acid identity and 20.9% amino acid similarity (at positions of nonidentity), YlPex23p and Ygr004p exhibit 35.6% amino acid identity and 19.3% amino acid similarity, and YlPex23p and Ybr168p exhibit 17.6% amino acid identity and 24.4% amino acid similarity, whereas Ylr324p, Ygr004p, and Ybr168p exhibit 9.3% amino acid identity and 20.7% amino acid similarity among themselves. Ylr324p is predicted to be a protein of molecular weight 59,461 and to have two transmembrane helices at amino acids 3-22 and 191-209 (http://www.cbs.dtu.dk/services/TMHMM-2.0/; Krogh et al., 2001 ). Ygr004p is predicted to be a protein of molecular weight 52,942 and to have four transmembrane helices at amino acids 93-111, 110-129, 178-195, and 226-244. Ybr168p is predicted to be a protein of molecular weight 48,578 and to have six transmembrane helices at amino acids 45-62, 69-86, 102-121, 178-197, 205-224, and 230-249. Partial functional redundancy among Ylr324p, Ygr004p, and Ybr168p (discussed below) may have prevented them from being identified as being involved in peroxisome biogenesis in S. cerevisiae by procedures involving random mutagenesis and negative selection for growth of yeast on oleic acid-containing medium.
The culturing of yeast cells in oleic acid-containing medium elicits a dual cellular response in that it promotes the proliferation of peroxisomes and induces the expression of many genes encoding peroxisomal proteins. Genomically encoded protein A chimeras of Ylr324p, Ygr004p, and Ybr168p were monitored to analyze the expression of YLR324w, YGR004w, and YBR168w, respectively, under the control of their endogenous gene promoters. Yeast strains synthesizing Ylr324p-prA, Ygr004p-prA, and Ybr168p-prA were grown in glucose-containing YPD medium and then shifted to oleic acid-containing YPBO medium. Aliquots of cells were removed at various times after the shift to YPBO medium, and their lysates were analyzed by SDS-PAGE and immunoblotting (Figure 2). Ylr324p-prA, Ygr004p-prA, and Ybr168p-prA were all detected in glucose-containing YPD medium at the time of transfer. The levels of Ylr324p-prA and Ybr168p-prA increased with time of incubation of cells in YPBO medium, but not as dramatically as the levels of Pot1p (peroxisomal thiolase). The levels of Ygr004p-prA did not show any apparent increase with time of incubation of cells in YPBO medium. It is noteworthy that the promoter regions of YLR324w, YGR004w, and YBR168w contain sequences that resemble the canonical sequence CCGN3TNAN8-12CGG of the oleic acid response element (ORE; Table 2; Rottensteiner et al., 2002 ; 2003a ), which acts to increase gene transcription in S. cerevisiae in the presence of oleic acid as a carbon source through the binding of the transcription factors Pip2p and Oaf1p (Rottensteiner et al., 1996 ; Karpichev et al., 1997 ). Whether these sequences actually do function as OREs remains to be determined.
A carboxyl-terminal PTS1 is sufficient to direct a reporter protein to peroxisomes. A fluorescent chimera between Discosoma sp. red fluorescent protein (DsRed) and the PTS1 Ser-Lys-Leu has been shown to target to peroxisomes of S. cerevisiae (Wang et al., 2001 ; Smith et al., 2002 ). Genomically encoded protein A chimeras of Ylr324p, Ygr004p, Ybr168p, and the peroxisomal matrix protein Pot1p were localized in oleic acid-induced cells by indirect immunofluorescence microscopy combined with direct fluorescence of DsRed-PTS1 to identify peroxisomes (Figure 3A). Ylr324p-prA, Ygr004pprA, Ybr168p-prA, and Pot1p colocalized with DsRed-PTS1 to small punctate structures characteristic of peroxisomes by confocal microcopy.
Subcellular fractionation and organelle extraction were used to establish if Ylr324p, Ygr004p, and Ybr168p are associated with peroxisomes and to determine their suborganellar locations. Ylr324p-prA, Ygr004p-prA, and Ybr168ppA, like Pot1p, preferentially localized to the 20KgP fraction enriched for peroxisomes (Figure 3B). Peroxisomes were isolated from the 20KgP fractions of each of the strains expressing Ylr324p-prA, Ygr004p-prA, or Ybr168p-prA. The gradients were fractionated, and equal portions of each fraction were analyzed by immunoblotting (Figure 3C). Ylr324pprA, Ygr004p-prA, and Ybr168p-prA coenriched with the peroxisomal matrix protein thiolase (Pot1p) and not with the mitochondrial protein, Sdh2p. Therefore, both microscopic analysis and subcellular fractionation showed Ylr324p, Ygr004p, and Ybr168p to be peroxisomal proteins. Some amount of Ylr324p-prA was always present in the lighter fractions during the gradient isolation of peroxisomes, whereas Ygr004-prA consistently enriched in fraction 9 at a density lighter than that of peroxisomes. Whether there is a selective liberation of a soluble form of Ylr324p-prA during the isolation of peroxisomes or there are vesicular elements containing either Ylr324p or Ygr004p remains to be determined. It is also noteworthy that Ygr004p-prA consistently migrated as two distinct molecular species in SDS-PAGE (see Figure 3C). The reason for this heterogeneity is unknown but could be due to some form of posttranslational modification, e.g., phosphorylation, of Ygr004p-prA.
Peroxisomes were hypotonically lysed by incubation in dilute alkali Tris buffer and subjected to centrifugation to yield a supernatant (Ti8S) enriched for matrix proteins and a pellet (Ti8P) enriched for membrane proteins (Figure 3D). The chimeras of Ylr324p, Ygr004p, and Ybr168p localized primarily to the Ti8P fraction, as did the chimeras of the peripheral peroxisomal membrane protein Pex17p (Huhse et al., 1998 ) and the integral peroxisomal membrane protein Pex3p (Höhfeld et al., 1991 ). The soluble peroxisomal matrix protein Pot1p was found almost exclusively in the Ti8S fraction. The reproducible presence of some Ylr324p-prA in the Ti8S fraction may again be representative of a soluble form of this protein that is selectively liberated from peroxisomes during their isolation (see Figure 3, B and C). The Ti8P fractions were then extracted with alkali sodium carbonate and subjected to centrifugation (Figure 3D). This treatment releases proteins associated with, but not integral to, membranes (Fujiki et al., 1982 ). Under these conditions, Ylr324p-prA, Ygr004p-prA, and Ybr168p-pA fractionated with Pex3p-prA to the pellet fraction enriched for integral membrane proteins, whereas Pex17p-prA fractionated to the supernatant fraction enriched for soluble proteins, including peripheral membrane proteins. These data suggest that Ylr324p, Ygr004p, and Ybr168p are primarily integral peroxisomal membrane proteins, as has been shown for YlPex23p (Brown et al., 2000 ).
Immunofluorescence analysis of oleic acid-incubated wild-type BY4742 cells with antibodies to the carboxyl-terminal PTS1 tripeptide Ser-Lys-Leu (SKL) or to the PTS2-containing enzyme Pot1p showed a pattern of small punctate structures characteristic of peroxisomes (Figure 4). In contrast, the majority of cells of the ylr324Δ strain showed increased numbers of punctate structures, whereas cells of the ygr004Δ and ybr168Δ strains showed a reduced number of often enlarged punctate structures. There was no evidence of increased cytosolic immunofluorescence with either anti-SKL or anti-Pot1p antibodies in cells deleted for one or more of the YLR324w, YGR004w, and YBR168w genes, suggesting that these genes do not encode proteins required for the import of matrix proteins into peroxisomes (Figure 4).
In electron micrographs, wild-type cells grown in oleic acid-containing medium contained characteristic peroxisomes 0.3 to 0.5 μm in diameter that were well separated from one another (Figure 5, A and I). In contrast, cells of the ylr324Δ strain (Figure 5B) showed increased numbers of peroxisomes (Table 3) that were similar in size to peroxisomes of wild-type cells (Figure 5I; Table 3). Cells of the ygr004Δ (Figure 5C) and ybr168Δ (Figure 5D) strains exhibited similar numbers of peroxisomes to wild-type cells (Table 3), but these peroxisomes were noticeably larger than wild-type peroxisomes, particularly in the case of ybr168Δ cells (Figure 5I; Table 3). Cells of strain DK1 (Figure 5E) carrying deletions in the YLR324w and YGR004w genes exhibited a mixed phenotype of increased numbers of peroxisomes (Table 3) of normal to enlarged size (Figure 5I; Table 3). Cells of strain DK2 (Figure 5F) carrying deletions in the YLR324w and YBR168w genes showed increased numbers of peroxisomes (Table 3) of normal to enlarged size (Figure 5I), some of which exhibited clustering, whereas cells of strain DK3 (Figure 5G) deleted for the YGR004w and YBR168w genes also contained greatly enlarged peroxisomes (Figure 5I; Table 3). Cells of the strain TKO carrying deletions in all three genes showed an approximately fivefold increase in the average number of peroxisomes per cell, but the size distribution of peroxisomes was similar to that of wild-type cells (Figure 5I). Our results suggest that Ylr324p acts primarily as a negative regulator of peroxisome number, whereas Ygr004p and particularly Ybr168p act as negative regulators of peroxisome size.
Because cells deleted for one or more of the YLR324w, YGR004w, and YBR168w genes are compromised in their regulation of peroxisome size and number, we investigated the effects of overexpression of these genes on the overall peroxisome phenotype in cells harboring various combinations of deletions of these genes. Overexpression of YLR324w, YGR004w, and YBR168w (Figure 6A) in their respective deletion backgrounds led to restoration of the wild-type peroxisomal phenotype. Overexpression of YLR324w in cells deleted for one or both of YGR004w and YBR168w did not lead to the complementation of the peroxisomal phenotype observed in cells deleted for either or both of YGR004w and YBR168w (Figure 6B). Similarly, overexpression of YBR168w in cells deleted for one or both YLR324w and YGR004w did not lead to complementation of the peroxisomal phenotype seen in cells deleted for either or both of the YLR324w and YGR004w genes (Figure 6C). In contrast, overexpression of the YGR004w gene in cells deleted for one or both of the YLR324w and YBR168w gene led essentially to the restoration of the wild-type peroxisomal phenotype, although evidence of peroxisomal clustering was still observed in cells deleted for both the YLR324w and YBR168w genes (Figure 6, D and E). Taken together, these data suggest that Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial, but not complete, functional redundancy with Ylr324p and Ybr168p.
A limited yeast two-hybrid screen was performed to identify physical interactions between Ylr324p, Ygr004p, and Ybr168p and between these proteins and Pex11p, Pex25p, Pex28p, Pex29p, and Vps1p, which have also been implicated in the control of peroxisome size and number (Erdmann and Blobel, 1995 ; Marshall et al., 1995 ; Hoepfner et al., 2001 ; Smith et al., 2002 ; Vizeacoumar et al., 2003 ). Others have used this methodology to detect interactions between peroxins (for examples, see Girzalsky et al., 1999 ; Smith and Rachubinski, 2001 ; Sichting et al., 2003 ). Chimeric genes were made by ligating the ORFs of YLR324w, YGR004w, YBR168w, PEX11, PEX25, PEX28, PEX29, and VPS1 in-frame and downstream of sequences encoding one of the two functional domains (AD or DB) of the GAL4 transcriptional activator. All possible combinations of plasmid pairs encoding AD and DB fusion proteins were transformed into S. cerevisiae strain SFY526, and β-galactosidase filter detection assays were performed. Interactions were detected between Ylr324p and Ygr004p, Pex29p, and itself (Figure 7). Ygr004p also interacted with itself, whereas weak interactions were detected between Ybr168p and Ylr324p and between Ybr168p and Pex28p (Figure 7). No interactions were detected between Ylr324p, Ygr004p, or Ybr168p and any of Pex11p, Pex25p, Pex29p, and Vps1p (unpublished data).
Because yeast two-hybrid analysis provided evidence of interaction between the S. cerevisiae homologues of YlPex23p and the S. cerevisiae homologues of YlPex24p and because cells of deletion mutants of the genes encoding these proteins are affected in the control of peroxisome size and number (data herein and Vizeacoumar et al., 2003 ), we investigated the hierarchical organization of these genes. Strains containing systematic pairwise gene deletions of the S. cerevisiae homologues of YlPEX23 and YlPEX24 were made, namely pex28Δ/ylr324Δ (CD1), pex28Δ/ygr004Δ (CD2), pex28Δ/ybr168Δ (CD3), pex29Δ/ylr324Δ (CD4), pex29Δ/ygr004Δ (CD5), and pex29Δ/ybr168Δ (CD6). Electron microscopy analysis (Figure 8) showed that the majority of cells containing double deletions of a YlPEX23 gene homolog and a YlPEX24 gene homolog exhibited the clustered peroxisomal phenotype typical of pex28Δ and pex29Δ cells (Vizeacoumar et al., 2003 ), although cells of the CD2 and CD3 strains (Figure 8, B and C, respectively) often exhibited enlarged peroxisomes that were well separated one from another. Together our data suggest that YLR324w, YGR004w, and YBR168w function downstream of PEX28 and PEX29.
Studies combining electron microscopy morphometry, pulse-chase analysis of peroxisomal protein trafficking in vivo, and the isolation and protein characterization of distinct peroxisomal subforms have shown that yeast peroxisomes do not grow and divide at the same time (Veenhuis and Goodman, 1990 ; Tan et al., 1995 ; Titorenko et al., 2000 ). There appears to be at least two different temporal patterns of peroxisome growth and division. In the yeast Candida boidinii (Veenhuis and Goodman, 1990 ), an initial event in peroxisome development is the extensive proliferation of immature peroxisomal vesicles containing only minor amounts of matrix proteins. This large increase in the number of immature peroxisomes by division precedes their growth through the import of membrane and matrix proteins and their conversion to mature organelles containing the complete set of peroxisomal proteins (Veenhuis and Goodman, 1990 ). The timing of events of peroxisome growth and division is different in Y. lipolytica. In this organism, the growth of immature peroxisomal vesicles, which is accomplished by the import of matrix proteins, and their development into mature peroxisomes occur before completely assembled mature peroxisomes undergo division (Titorenko et al., 2000 ). Similar temporal patterns of peroxisome growth and division have been observed for the yeast Hansenula polymorpha (Tan et al., 1995 ). In human cells, both immature peroxisomal vesicles and mature peroxisomes are proposed to be able to divide (Gould and Valle, 2000 ). However, the division of immature peroxisomes before their growth and maturation by peroxisomal protein import can be seen only in some peroxin-deficient fibroblasts following reactivation or reexpression of an originally defective peroxin-encoding gene (Matsuzono et al., 1999 ; South and Gould, 1999 ; Sacksteder and Gould, 2000 ). In normal human cells, in contrast, conversion of immature peroxisomal vesicles to mature peroxisomes by membrane and matrix protein import may occur before peroxisomes undergo division (Gould and Valle, 2000 ).
Members of the Pex11 family of peroxins, including Pex25p (Smith et al., 2002 ) and Pex27p (Tam et al., 2003 ; Rottensteiner et al., 2003b ) of S. cerevisiae, have been shown to effect peroxisome division in different organisms (Erdmann and Blobel, 1995 ; Marshall et al., 1995 ; Sakai et al., 1995 ; Li and Gould, 2002 ; Li et al., 2002 ). The dynamin-like protein Vps1p has also been implicated in this process (Hoepfner et al., 2001 ), and we recently showed that the peroxisomal integral membrane proteins Pex28p and Pex29p are also involved in controlling the number, size, and separation of peroxisomes in S. cerevisiae (Vizeacoumar et al., 2003 ). Here, we have identified three novel peroxisomal proteins encoded by the ORFs YLR324w, YGR004w, and YBR168w of S. cerevisiae and demonstrated that these proteins also act to control peroxisome size and number in this organism.
The identification of novel proteins required for peroxisome biogenesis in S. cerevisiae through their sequence similarity with known peroxins in other organisms enabled the identification of Pex28p and Pex29p (Vizeacoumar et al., 2003 ). YlPex23p is a peroxisomal integral membrane protein required for peroxisome assembly in Y. lipolytica that shares extensive sequence similarity to three proteins of unknown function and unknown localization encoded by the ORFs YLR324w, YGR004w, and YBR168w of the S. cerevisiae genome. Genomically encoded protein A chimeras of Ylr324p, Ygr004p, and Ybr168p were shown by a combination of confocal microscopy and subcellular fractionation to be peroxisomal proteins. In their response to extraction by different chaotropic agents, Ylr324p, Ygr004p, and Ybr168p act primarily as integral membrane proteins.
Ylr324p, Ygr004p, and Ybr168p are not required for peroxisome assembly per se, as cells harboring deletions for one, two, or all three of these genes still contain peroxisomes that are unaffected in their capacity to import PTS1- or PTS2-containing proteins. These peroxisomes are functional, at least to a degree, because the cells harboring deletions of these genes are able to grow in oleic acid-containing medium with essentially the same kinetics as wild-type cells (unpublished data). YLR324w, YGR004w, and YBR168w are also apparently not required for peroxisome inheritance, because all cells deleted for one or more of these genes still contained peroxisomes after numerous cell divisions. Also, if YLR324w, YGR004w, and YBR168w had a direct role in the inheritance of peroxisomes, one might expect that a loss of peroxisomes from cells over time resulting from the impaired segregation of peroxisomes into daughter cells would lead to inhibited growth in oleic acid-containing medium for the deletion strains as compared with the wild-type strain, which was not observed.
Peroxisomes in cells deleted for the YLR324w, YGR004w, and YBR168w genes are not normal and show distinctive phenotypic differences from wild-type peroxisomes. ylr324Δ cells showed increased numbers of peroxisomes versus wild-type cells, whereas ygr004Δ and ybr168Δ cells contained not only greater numbers of peroxisomes but also enlarged peroxisomes (Figure 5). Cells deleted for two of the YLR324w, YGR004w, and YBR168w genes contained increased numbers of generally enlarged peroxisomes. Cells of the strain deleted for all three genes contained increased numbers of smaller to normally sized peroxisomes. Morphometric analyses and quantification revealed a fivefold increase in the numbers of peroxisomes on average per cell for the triple deletion strain than for the wild-type strain. Although an occasional enlarged peroxisome was evident in cells deleted for all three genes, the peroxisomal phenotype of these cells strongly resembled that of cells deleted for only the YLR324w gene. The characteristics of peroxisomes of cells of the deletion strains are consistent with a role for YLR324w, YGR004w, and YBR168w in the control of peroxisome size and number within S. cerevisiae cells. Our results suggest that Ylr324p acts primarily acts as a negative regulator of peroxisome number, whereas Ygr004p and particularly Ybr168p act as negative regulators of peroxisome size. Nevertheless, Ygr004p shares some redundancy of function with Ylr324p and Ybr168p, but not vice versa, as overexpression of YGR004w in cells deleted for one or both of the YLR324w and YBR168w genes results essentially in the reestablishment of the wild-type peroxisome phenotype, but in contrast to wild-type cells, there is some evidence of peroxisome clustering. The reason why peroxisomes cluster in these overexpressing cells is unknown.
It is interesting to note that Y. lipolytica cells deleted for the PEX23 gene also show evidence of abnormal peroxisomal divisional control. These cells lack mature peroxisomes but do accumulate small vesicular structures that contain both peroxisomal matrix and membrane proteins (Brown et al., 2000 ). However, these membrane structures do not function as peroxisomes, because pex23Δ cells cannot grow on medium containing oleic acid as the sole carbon source. Therefore, although YlPex23p, like Ylr324p, Ygr004p, and Ybr168p, likely has a role in the regulation of peroxisome division, YlPex23p probably does not function identically to Ylr324p, Ygr004p, or Ybr168p in this process.
Pex28p and Pex29p have been implicated in the control of peroxisome size and number (Vizeacoumar et al., 2003 ). A limited yeast two-hybrid screen revealed physical interactions among Ylr324p, Ygr004p, Ybr168p, Pex28p, and Pex29p. No interactions were detected between these five proteins and Pex11p, Pex25p, and Vps1p, which also have been shown to play a role in the control of peroxisome size and division. Further experimentation is required to determine whether these interactions are direct or bridged by other proteins. It is interesting to note that Ylr324p was shown to interact with Pex29p. Some amount of Ylr324p (Figure 3) and of Pex29p (Vizeacoumar et al., 2003 ) was always present in the 20KgS fraction in differential fractionation and in the less dense fractions during the gradient isolation of peroxisomes. Whether some portion of these proteins forms a complex and is localized to some compartment other than peroxisomes remains to be determined.
How might Ylr324p, Ygr004p, Ybr168p, Pex28p, and Pex29p act to control the abundance, size, and distribution of peroxisomes in the S. cerevisiae cell? Cells systematically deleted for one of the YLR324w, YGR004w, and YBR168w genes and one of the PEX28 and PEX29 genes exhibited clusters of peroxisomes typically observed in cells deleted for the PEX28 or PEX29 gene (Vizeacoumar et al., 2003 ). Our data suggest that Pex28p and Pex29p act upstream of Ylr324p, Ygr004p, and Ybr168p in controlling peroxisome abundance and size.
Organelles are highly dynamic structures that undergo fission and fusion to control their numbers and modify their morphology in response to intracellular and extracellular cues and to permit their correct segregation at cell division. As a consequence, the maintenance of compartmental integrity by the eukaryotic cell requires the tight coordination of mechanisms controlling these events. Many proteins, including those encoded by the genes YLR324w, YGR004w, and YBR168w of S. cerevisiae, are involved in controlling peroxisome number and size in the cell. Because of their role in the control of peroxisome size and number, we propose that YLR324w, YGR004w, and YBR168w be designated as PEX30, PEX31, and PEX32, respectively, and their encoded peroxins as Pex30p, Pex31p, and Pex32p. The challenge remains to understand how the increasing number of proteins shown to be involved in controlling peroxisome number and size interplay among themselves and signal to the cell how to control its peroxisome dynamics.
We thank Honey Chan for her expert assistance with electron microscopy, Richard Poirier for help with confocal microscopy, and Patrick Lusk for useful discussions. This work was supported by operating grant 39322 from the Canadian Institutes of Health Research to R.A.R. R.A.R. holds the Canada Research Chair in Cell Biology and is an International Research Scholar of the Howard Hughes Medical Institute.
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-09-0681. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-09-0681.
Abbreviations used: 20KgP, 20,000 × g pellet; 20KgS, 20,000 × g supernatant; DsRed, red fluorescent protein from Discosoma sp.; ORE, oleic acid response element; ORF, open reading frame; PNS, postnuclear supernatant; PTS, peroxisome targeting signal.