INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Peroxisomes, together with the glyoxysomes of plants and the glycosomes of trypanosomes, make up the microbody family of organelles (de Duve, 1996). In electron micrographs, peroxisomes appear spherical in shape, surrounded by a single unit membrane and containing a granular matrix and sometimes a paracrystalline core. The functions of peroxisomes vary depending on the organism, cell type, and physiological conditions. Functions that have been conserved from yeast to humans include the -oxidation of fatty acids and the decomposition of hydrogen peroxide by catalase (for reviews, see Lazarow and Fujiki, 1985; van den Bosch et al., 1992). Proteins that are required for the proper functioning and biogenesis of peroxisomes have collectively been termed peroxins (Distel et al., 1996).

The importance of peroxisomes for human growth and development is underscored by a group of genetic disorders known as the peroxisome biogenesis disorders (PBD), including Zellweger syndrome, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy, in which peroxisomes fail to assemble normally (for reviews, see Lazarow and Moser, 1994; Fujiki, 1997, 2000). A great deal of attention has been paid recently to defining the molecular bases of these diseases, in particular through the identification of the genes controlling peroxisome assembly. Much progress has been made in the identification of these so-called PEX genes with the use of various yeasts as model systems. To date, the genes of 23 peroxins have been cloned from yeast (Subramani 1997, 1998; Götte et al., 1998; Purdue et al., 1998; Titorenko et al., 1998; Koller et al., 1999; Brown et al., 2000; Subramani et al., 2000; Titorenko and Rachubinski, 2001), and of these, 13 human orthologues have been identified by the screening of databases of Expressed Sequence Tags. Eleven human peroxins have been shown to complement the deficiencies of peroxisome assembly in cells of PBD patients (Subramani, 1997, 1998; Fujiki, 2000; Gould and Valle, 2000; Subramani et al., 2000; Titorenko and Rachubinski, 2001).

Protein targeting to peroxisomes is compromised in pex mutants. Peroxisomal proteins are encoded in the nucleus and are synthesized on cytosolic polysomes (Lazarow and Fujiki, 1985; Subramani, 1993, 1998, 2000). Most soluble matrix proteins are targeted to peroxisomes by one of two types of peroxisomal targeting signals (PTS). PTS1 is a carboxyl-terminal tripeptide (SKL and conserved variants; Gould et al., 1987, 1989; Aitchison et al., 1991; Elgersma et al., 1996) found in the majority of matrix proteins, whereas PTS2 is an amino-terminal nonapeptide found in a very limited subset of matrix proteins (Swinkels et al., 1991; Glover et al., 1994; Waterham et al., 1994). Pex5p and Pex7p are the receptors for PTS1 and PTS2, respectively, and various peroxins, including Pex13p and Pex14p, form a docking complex at the peroxisomal membrane for these receptors (reviewed by Erdmann et al., 1997; Subramani, 1998, 2000; Titorenko and Rachubinski, 2001). The PTS1 and PTS2 pathways are apparently not independent, because there is convergence of the two targeting pathways either in the cytosol or at the level of the peroxisomal membrane (Fransen et al., 1998; Otera et al., 1998, 2000; Schliebs et al., 1999). The sorting of peroxisomal membrane proteins is much less well understood than the sorting of matrix proteins, although it appears that the two pathways are independent. Although most pex mutants fail to target proteins to the peroxisomal matrix, mislocalizing them to the cytosol, they do target peroxisomal membrane proteins to vestigial structures called "peroxisome ghosts" (Santos et al., 1988; Subramani, 1993, 1998, 2000).

The peroxin Pex19p has been isolated from a variety of organisms, including humans and Chinese hamster and the yeasts Saccharomyces cerevisiae and Pichia pastoris (Braun et al., 1994; James et al., 1994; Götte et al., 1998; Matsuzono et al. 1999; Snyder et al., 1999a). Pex19p has been shown to be primarily a cytosolic protein that can interact with a number of peroxisomal membrane proteins (Götte et al., 1998; Snyder et al., 1999a, 1999b, 2000; Hettema et al., 2000; Sacksteder et al., 2000). Every ortholog of Pex19p contains a consensus sequence for the addition of a lipid farnesyl moiety at its extreme carboxy terminus (for a review of protein prenylation, see Omer and Gibbs, 1994), although the importance of this addition to the function of Pex19p is uncertain. In the absence of a functional PEX19 gene, growth of yeast on carbon sources metabolized by peroxisomes, such as oleic acid, is compromised, matrix proteins are mislocalized to the cytosol, and both true peroxisomes and peroxisomal ghosts are absent. In P. pastoris pex19 mutants, small vesicles not found in wild-type cells are observed (Snyder et al., 1999a, 1999b). These vesicles have been proposed to be precursors of peroxisomes. The absence of peroxisome ghosts in pex19 mutants has led to the hypothesis that Pex19p acts at an early step in peroxisome biogenesis.

We have cloned and characterized the PEX19 gene from the yeast Yarrowia lipolytica. Y. lipolytica cells lacking a functional PEX19 gene mislocalize the majority of their peroxisomal matrix proteins to the cytosol, as has been observed for pex19 mutants of other organisms. However, in contrast to the cells of these pex19 mutants, Y. lipolytica pex19 cells contain structures that resemble mature, wild-type peroxisomes morphologically. Some of these structures are of the same density as wild-type peroxisomes in subcellular fractionation, although their protein complement is different from that of wild-type peroxisomes. Interestingly, Pex19p is localized primarily to peroxisomes and not to the cytosol in Y. lipolytica.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Strains and Culture Conditions

The Y. lipolytica strains used in this study are listed in Table 1. Growth was at 30°C. Diploid strains were generated as described previously (Nuttley et al., 1993). Strains containing plasmids were grown on minimal medium (YND or YNO). All other strains were grown on rich medium (YEPD or YPBO). Media components were as follows: YND, 0.67% yeast nitrogen base without amino acids, Complete Supplement Mixture (Bio101, Vista, CA) minus the appropriate auxotrophic supplements at twice the manufacturer's recommended concentration (2× CSM), 2% glucose; YEPD, 1% yeast extract, 2% peptone, 2% glucose; YNO, 0.67% yeast nitrogen base without amino acids, 2× CSM, 0.05% (wt/vol) Tween 40, 0.1% (vol/vol) oleic acid; YPBO, 0.3% yeast extract, 0.5% peptone, 0.5% K₂HPO₄, 0.5% KH₂PO₄, 1% Brij 35, 1.1% (vol/vol) oleic acid. Escherichia coli was grown as described previously (Ausubel et al., 1994).

Cloning, Sequencing, and Integrative Disruption of the PEX19 Gene

The pex19-1 mutant strain was isolated from randomly mutagenized wild-type E122 cells as described previously (Nuttley et al., 1993). The PEX19 gene was isolated by functional complementation of the pex19-1 strain with a Y. lipolytica genomic DNA library in the autonomously replicating plasmid, pINA445 (Nuttley et al., 1993). Total DNA was isolated from colonies that recovered growth on YNO and was used to transform E. coli for plasmid recovery. Restriction fragments of the initial complementing insert were subcloned and tested for their ability to confer growth on YNO to the pex19-1 mutant strain. The smallest complementing fragment was sequenced in both directions.

Targeted disruption of the PEX19 gene in E122 and 22301-3 cells was performed with the URA3 gene of Y. lipolytica. Nucleotides 20-1025 of the PEX19 open reading frame (ORF) were excised by digestion with the restriction enzymes MfeI and BglII, and the resultant ends were made blunt with T4 DNA polymerase. A SalI fragment containing the URA3 gene was made blunt with T4 DNA polymerase and then ligated into the disrupted PEX19 ORF. A fragment consisting of the URA3 gene flanked by ~300-bp and 3-kb pairs of genomic sequence at the 5' and 3' ends of the insertion, respectively, was released by digestion with ApaI. This fragment was used to transform E122 and 22301-3 cells to uracil prototrophy. Ura⁺ transformants were selected and screened for the ole phenotype. Integration into the correct locus was confirmed by Southern blot analysis (Ausubel et al., 1994). Disruption strains and the original mutant strain were crossed with wild-type strains and with each other, and the resultant diploids were checked for growth on YNO agar.

Construction of Mutant PEX19 Genes

Two mutant versions of the PEX19 gene were engineered to study the effects of deleting or modifying the putative farnesylation site of Pex19p. A mutant PEX19 coding for Pex19p lacking its four extreme carboxyl-terminal amino acids, -CNQQ, was constructed by PCR amplification of plasmid p19/NcA-5Zf, which contains the minimal pex19 complementing fragment, with the use of oligonucleotides A and B (Table 2) as primers. Primer B introduces a premature stop codon into the PEX19 gene immediately upstream of the last four codons of its ORF. The PCR product and p19/NcA-5Zf were digested with BglII and MfeI. This digestion removed nucleotides 16-972 of the ORF of PEX19, along with 53 nucleotides immediately downstream of the ORF, including the original PEX19 stop codon. The digested PCR product was then substituted for the deleted nucleotides to give a mutated PEX19 gene coding for a Pex19p lacking the four amino acids at its carboxy terminus. The mutant PEX19 construct was cloned into the vector pINA445 and tested for its ability to complement the ole phenotype of a pex19 disruption strain. The second mutant of PEX19 was engineered so as to change the codon encoding the putatively farnesylated cysteine residue 321 to one encoding a serine residue, which cannot be farnesylated. PCR amplification with the use of oligonucleotides A and C (Table 2) yielded a mutated PEX19 gene with a cytosine residue substituting for the guanosine residue at position 962, yielding a codon for serine in place of one for cysteine. The PCR product and plasmid p19/NcA-5Zf were digested and ligated as described above to yield a PEX19 gene encoding a serine at position 321 in place of a cysteine. As before, this mutant PEX19 gene was cloned into pINA445 and tested for its ability to complement the ole phenotype of a pex19 disruption strain.

Antibodies

Antibodies to Pex19p were increased in guinea pig and rabbit against a maltose-binding protein-Pex19p fusion. The ORF of the PEX19 gene lacking the 12 nucleotides encoding the putative farnesylation site of Pex19p was amplified by PCR with the use of oligonucleotides D and E (Table 2). The product was digested with EcoRI and HindIII and cloned into the vector pMAL-c2 (New England Biolabs, Beverly, MA) in-frame and downstream of the gene encoding maltose-binding protein.

Antibodies to the carboxyl-terminal SKL-tripeptide, thiolase (THI), isocitrate lyase (ICL), acyl-CoA oxidase (AOX), malate synthase (MLS), Pex2p, and Pex16p have been described (Szilard et al., 1995; Eitzen et al., 1996, 1997). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and HRP-conjugated goat anti-guinea pig IgG secondary antibodies (Amersham Pharmacia Biotech, Baie d'Urfé, Quebec, Canada) were used to detect primary antibodies in immunoblot analysis. Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG and rhodamine-conjugated anti-guinea pig IgG secondary antibodies were used to detect primary antibodies in immunofluorescence microscopy.

Microscopic Analysis

Electron microscopy (Goodman et al., 1990) and indirect immunofluorescence microscopy (Szilard et al., 1995) were performed as described.

Subcellular Fractionation and Peroxisome Isolation

Cell were grown for 16 h in glucose-containing YEPD medium, transferred to oleic acid-containing YPBO medium, and incubated for 8 h in YPBO medium. Fractionation of cells was performed as described (Szilard et al., 1995) and included the differential centrifugation of homogenized spheroplasts at 1000 × g for 8 min 4°C in a JS13.1 rotor (Beckman, Fullerton, CA) to yield a postnuclear supernatant (PNS) fraction. The PNS fraction was subjected to further differential centrifugation at 20,000 × g for 30 min 4°C in a JS13.1 rotor to yield a pellet (20KgP) fraction enriched for peroxisomes and mitochondria and a supernatant (20KgS) fraction enriched for cytosol. Peroxisomes were purified from the 20KgP fraction by isopycnic centrifugation on a discontinuous sucrose gradient (Titorenko et al., 1996). The 20KgS fraction was subjected to centrifugation at 200,000 × g for 30 min at 4°C in a TLA120.2 rotor (Beckman) to yield a pellet (200KgP) fraction enriched for small vesicles and a supernatant (200KgS) fraction highly enriched for cytosol (Titorenko et al., 1998).

View larger version (34K): [in this window] [in a new window]   Figure 1.   Cloning and analysis of the PEX19 gene. (A) Complementing activity of inserts, restriction map analysis, and targeted gene deletion strategy for the PEX19 gene. Recombinant 19O1 contains the original complementing insert DNA. Solid line, Y. lipolytica genomic DNA; boxes, vector DNA. The ORFs of the PEX19 and URA3 genes and their directionality are indicated by the arrows. Ability (+) and inability () of an insert to confer growth on oleic acid to strain pex19-1. A, ApaI; C, ClaI; N, NdeI; Nc, NcoI; S, SphI. (B) Nucleotide sequence of the PEX19 gene and deduced amino acid sequence of Pex19p. The putative farnesylation site is highlighted in bold. These sequence data have been deposited in the DDBJ/EMBL/GenBank databases under accession number AF346486.

Analytical Procedures

Whole cell lysates were prepared as described (Eitzen et al., 1997). Enzymatic activities of the peroxisomal marker catalase (Luck, 1963) and of the mitochondrial marker fumarase (Tolbert, 1974) were performed as described. SDS-PAGE (SDS-PAGE) (Laemmli, 1970), and immunoblotting with the use of a semidry electrophoretic transfer system (Model ET-20; Tyler Research Instruments, Edmonton, Alberta, Canada; Kyhse-Andersen, 1984) were performed as described. Antigen-antibody complexes in immunoblots were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). Protein concentration was measured as described by Bradford (1976) with the use of a commercially available kit (Bio-Rad, Mississauga, Ontario, Canada) and bovine serum albumin as a standard. Proteins were precipitated by addition of trichloroacetic acid to 10%, followed by washing of the precipitate with chilled 80% acetone. Total nucleic acid was isolated from yeast cells by disruption with glass beads, followed by phenol extraction. Oligonucleotides were synthesized on an Oligo 1000 M Synthesizer (Beckman). DNA sequencing was performed on ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and Characterization of the PEX19 Gene

The pex19-1 strain (Table 1) was identified from among randomly mutagenized wild-type Y. lipolytica E122 cells by its inability to grow on medium containing oleic acid as the sole carbon source (ole phenotype). Subsequent morphological and biochemical analyses (data presented below) demonstrated that this strain was affected in peroxisome assembly. The PEX19 gene was isolated from a library of Y. lipolytica genomic DNA by functional complementation, that is, restored growth on oleic acid-containing medium (the ole⁺ phenotype), of the pex19-1 strain. DNA was isolated from the complemented strain, and the complementing plasmid was recovered by transformation of E. coli. The complementing fragment was mapped by restriction endonuclease digestion (Figure 1A), and various restriction fragments were subcloned and introduced by transformation into the pex19-1 strain to define the minimal fragment of complementation. The minimal complementing fragment 19/NcA (Figure 1A) was sequenced in both directions and shown to contain an ORF of 972 nucleotides encoding a protein of 324 amino acids and having a predicted molecular weight of 34,822 (Figure 1B).

A search of protein databases with the use of the GENEINFO(R) BLAST Network Service of the National Center for Biotechnology Information showed that the protein encoded by the ORF of the minimal fragment 19/NcA that functionally complemented the pex19-1 mutant strain showed 29% identity and 43% similarity to Pex19p from Chinese hamster, 26% identity and 41% similarity to human Pex19p, 32% identity and 48% similarity to Pex19p from P. pastoris, and 26% identity and 41% similarity to Pex19p from S. cerevisiae (Figure 2). Therefore, the complementing gene was designated as YlPEX19 and its encoded protein as YlPex19p. As in all other reported Pex19 proteins, Pex19p of Y. lipolytica has a putative farnesylation signal, CNQQ, at its extreme carboxy terminus.

View larger version (79K): [in this window] [in a new window]   Figure 2.   Sequence alignment of Yarrowia lipolytica Pex19p with Pex19p peroxins of other organisms. The sequence of Y. lipolytica Pex19p was aligned with the sequences of Pex19p of Chinese hamster (Cl), human (Hs), Pichia pastoris (Pp), and S. cerevisiae (Sc). Amino acid sequences were aligned with the use of the ClustalW program (EMBL, Heidelberg, Germany). Identical residues (black) and similar residues (gray) in three or more proteins are shaded. Similarity rules: G = A = S; A = V; V = I = L = M; I = L = M = F = Y = W; K = R = H; D = E = Q = N; and S = T = Q = N. Dashes represent sequence gaps. The conserved cysteine of the farnesylation consensus sequence found in all Pex19p peroxins is marked with an asterisk.

The putative PEX19 gene was disrupted by targeted integration of the Y. lipolytica URA3 gene to make the strains pex19KOA and pex19KOB in the A (E122) and B (22301-3) mating types, respectively (Table 1). These strains were unable to grow on oleic acid-containing medium and displayed the same morphological, biochemical, and protein targeting defects as the original pex19-1 strain (see below). The diploid strains D1-19 and D3-19 (Table 1) from the mating of strains pex19-1 and pex19KOA to wild-type strain 22301-3, respectively, could grow on oleic acid-containing medium, demonstrating the recessive nature of the original pex19-1 mutation and of the PEX19 gene deletion. The diploid strain D4-19 made by mating the original pex19-1 strain to pex19KOB (Table 1) was unable to grow on oleic acid-containing medium, demonstrating that the authentic PEX19 gene was cloned and that the ability to use oleic acid as the sole carbon source required at least one intact copy of the PEX19 gene.

pex19 Cells Preferentially Mislocalize Peroxisomal Proteins to the Cytosol but Still Contain Morphologically Identifiable Peroxisomal Structures

In electron micrographs, normal peroxisomes of Y. lipolytica incubated in oleic acid-containing medium appear as round vesicular structures, 0.2-0.5 µm in diameter, with an homogeneous granular matrix and a single delimiting unit membrane (Figure 3A; P). Organellar structures readily identifiable as peroxisomal are also seen in cells of both the original mutant strain pex19-1 (Figure 3B; Ps) and of the disruption strain pex19KOA (Figure 3C; Ps).

View larger version (67K): [in this window] [in a new window]   Figure 3.   Ultrastructure of wild-type and of pex19 mutant cells. E122 (A), pex19-1 (B), and pex19KOA (C) strains were grown in glucose-containing YEPD medium for 16 h, transferred to oleic acid-containing YPBO medium, and incubated for an additional 8 h. Cells were fixed in 1.5% KMnO4 and processed for electron microscopy. M, mitochondrion; N, nucleus; P, peroxisome; Ps, peroxisomal structure; V, vacuole. Bar, 1 µm.

Immunofluorescence analysis of oleic acid-incubated wild-type E122 cells with anti-SKL antibodies and antibodies to the peroxisomal matrix proteins thiolase (THI), acyl-CoA oxidase (AOX), and isocitrate lyase (ICL) showed a punctate pattern of staining characteristic of peroxisomes (Figure 4). In contrast, pex19-1 and pex19KOA cells stained with the same antibodies showed a more diffuse, generalized pattern of fluorescence characteristic of a cytosolic localization. The strain P19TR transformed with the PEX19 gene showed characteristic peroxisomal punctate staining with the four antibodies, indicating the ability of this gene to rescue the import of these matrix proteins.

View larger version (84K): [in this window] [in a new window]   Figure 4.   pex19 mutant cells mislocalize peroxisomal matrix proteins to the cytosol. Wild-type strain E122, mutant strains pex19-1 and pex19KOA, and transformed strain P19TR were grown in YEPD medium for 16 h, transferred to YPBO medium (YNO medium for strain P19TR), and incubated for an additional 8 h. Cells were processed for immunofluorescence microscopy with antibodies to the PTS1 tripeptide SKL (SKL), thiolase (THI), acyl-CoA oxidase (AOX), and isocitrate lyase (ICL). Rabbit (SKL, AOX, ICL) and guinea pig (THI) primary antibodies were detected with rhodamine-conjugated secondary antibodies.

Cells of the wild-type strain E122 and of the mutant strains pex19-1 and pex19KOA were grown in glucose-containing YEPD medium for 16 h, shifted to oleic acid-containing YPBO medium for an additional 8 h, and subjected to subcellular fractionation to yield a postnuclear supernatant (PNS) fraction, a 20,000 × g pellet (20KgP) fraction enriched for peroxisomes and mitochondria, and a 20,000 × g supernatant (20KgS) fraction enriched for cytosol. As expected, the peroxisomal matrix proteins recognized by anti-SKL antibodies, as well as the matrix proteins THI, AOX, and ICL, were all found to be preferentially localized to the 20KgP of E122 cells (Figure 5A). In contrast to the results from wild-type cells and in agreement with the data from immunofluorescence, peroxisomal matrix proteins were preferentially localized to the 20KgS of pex19-1 and pex19KOA cells, although low levels of all these proteins were reproducibly detected in the 20KgP fraction of both pex19 mutant strains. In both the wild-type E122 strain and the mutant pex19-1 and pex19KOA strains, the mitochondrial marker enzyme fumarase was preferentially localized to the 20KgP (data not presented). Because in pex19 mutant strains all matrix proteins investigated mislocalized preferentially to the 20KgS enriched for cytosol and gave a general pattern of fluorescence characteristic of the cytosol, pex19 mutants are compromised in the import of PTS1 (anti-SKL proteins and ICL), PTS2 (THI), and nonPTS1, nonPTS2 proteins (AOX; Wang et al., 1999).

View larger version (53K): [in this window] [in a new window]   Figure 5.   pex19 mutant strains mislocalize matrix proteins and the peroxisomal peripheral membrane peroxin Pex16p and exhibit greatly reduced levels of the peroxisomal integral membrane protein Pex2p. The wild-type strain E122, the original mutant strain pex19-1, and the gene deletion strain pex19KOA were grown for 16 h in glucose-containing YEPD medium, transferred to oleic acid-containing YPBO medium, incubated for an additional 8 h, and subjected to subcellular fractionation to yield a postnuclear supernatant (PNS) fraction, a 20KgP fraction enriched for peroxisomes and mitochondria, and a 20KgS fraction enriched for cytosol. Equal portions of the PNS, 20KgP, and 20KgS were analyzed by immunoblotting to the indicated matrix proteins and to Pex2p (A) and to Pex16p (B).

Like matrix proteins, the peroxisomal integral membrane protein Pex2p was preferentially localized to the 20KgP of E122 cells (Figure 5A). Interestingly, the levels of Pex2p were at the limit of detection in all fractions from both pex19-1 and pex19KOA cells, suggesting that this peroxisomal integral membrane protein exhibits instability in pex19 mutant strains. In contrast, Pex16p, an intraperoxisomal peripheral membrane protein, can still be detected in pex19 mutant strains, but its targeting is compromised (Figure 5B). Pex16p was preferentially localized to the 20KgP of wild-type E122 cells, but was detected almost exclusively in the 20KgS of pex19KOA cells.

Cells of pex19 Mutants Contain Peroxisomal Structures of Comparable Density to Wild-type Peroxisomes but with Different Protein Complements

The 20KgP fractions of the wild-type strain E122 and of the pex19-1 and pex19KOA mutant strains incubated in oleic acid-containing medium were subjected to isopycnic sucrose density gradient centrifugation. Immunoblot analysis of the gradient fractions of E122 cells with antibodies to various peroxisomal matrix proteins and to the peroxisomal integral membrane protein Pex2p revealed strong immunoreactivity for each antibody in fractions 3-5, with the strongest signals being found in fraction 4 (Figure 6). Fraction 4 has a density of sucrose of 1.21 g/cm³, which has previously been reported as the density of peroxisomes of Y. lipolytica in sucrose (Szilard et al., 1995; Titorenko et al., 1996; Brown et al., 2000). Weaker signals for each peroxisomal protein were observed in fractions of lighter density in the wild-type strain. These signals probably arise from vesicular structures of lighter buoyant density that contain peroxisomal proteins, which have been observed previously for Y. lipolytica (Eitzen et al., 1997; Brown et al., 2000). Whether these vesicular structures constitute the precursors of mature peroxisomes (Titorenko et al., 2000) is unknown.

View larger version (57K): [in this window] [in a new window]   Figure 6.   Peroxisomal proteins of pex19 cells localize in part to vesicular structures that are of the same density as wild-type peroxisomes. The 20KgP fractions of the wild-type strain E122 and of the mutant strains pex19-1 and pex19KOA incubated in oleic acid-containing medium were subjected to isopycnic centrifugation on discontinuous sucrose gradients, as previously described (Titorenko et al., 1996). Seventeen 2-ml fractions were collected from the bottom of each tube. Equal volumes of each fraction were analyzed by SDS-PAGE and subjected to immunoblotting with antibodies to the indicated peroxisomal proteins.

Immunoblot analysis of gradients of pex19-1 and pex19KO cells revealed the presence of vesicular structures containing SKL-reactive proteins, THI, AOX, and ICL that were of a density similar to that of wild-type peroxisomes. Again, vesicular elements of lower density, but containing peroxisomal proteins, could be isolated from the pex19 mutant strains. Analysis of the enzymatic activity of catalase showed a distribution in the pex19 strains similar to that of the wild-type E122 strain, whereas the mitochondrial marker fumarase localized in all strains primarily to fractions 9 and 10 with densities of sucrose of 1.17 and 1.16 g/cm³ (data not presented), as has previously been reported (Szilard et al., 1995; Titorenko et al., 1996; Brown et al., 2000).

Pex19p Is Associated with Peroxisomes in Wild-type Cells

Polyclonal antibodies to Pex19p recognized a single polypeptide species of ~42 kDa in whole cell lysates prepared from the wild-type-strain E122 but not from the pex19KOA disruption strain (Figure 7). Subcellular fractions of wild-type E122 cells incubated in oleic acid-containing medium were subjected to immunoblot analysis with these anti-Pex19p antibodies (Figure 8A). The majority of Pex19p associated with structures pelletable at 20,000 × g (20KgP), whereas the Pex19p observed in the 20KgS fraction could be pelleted by centrifugation at 200,000 × g (200KgP). Double-label immunofluorescence analysis of wild-type E122 cells grown in oleic acid-containing medium with antibodies to SKL-containing proteins and anti-Pex19p antibodies revealed an almost exact colocalization of these proteins, indicating that Pex19p is associated mostly with peroxisomes (Figure 8B).

View larger version (31K): [in this window] [in a new window]   Figure 7.   Immunoblot analysis of whole cell lysates of the wild-type strain E122 and of the pex19KOA disruption strain probed with anti-Pex19p antibodies. Cells were grown for 16 h in glucose-containing YEPD medium, transferred to oleic acid-containing YPBO medium, and incubated for an additional 8 h. The more slowly migrating species detected in the pex19KOA lane is due to spillover of a maltose-binding protein-Pex19p fusion loaded in the adjacent lane at right.

View larger version (47K): [in this window] [in a new window]   Figure 8.   Pex19p is localized primarily to peroxisomes. (A) Wild-type E122 cells incubated in oleic acid-containing YPBO medium were subjected to differential centrifugation to yield postnuclear supernatant (PNS) and 20KgS and 20KgP fractions. The 20KgS fraction was subjected to centrifugation at 200,000 × g to yield a pellet (200KgP) fraction enriched for small vesicles and a supernatant (200KgS) fraction highly enriched for cytosol. Equal portions of each fraction were analyzed by SDS-PAGE and subjected to immunoblotting with antibodies to Pex19p. (B) Double-label immunofluorescence microscopy of wild-type E122 cells. Cells were incubated in YPBO medium for 8 h and processed for immunofluorescence microscopy with guinea pig antithiolase (THI) antibodies and rabbit anti-Pex19p antibodies. Primary antibodies were detected with rhodamine-conjugated anti-guinea pig and fluorescein-conjugated anti-rabbit secondary antibodies.

Pex19p Levels Are Not Increased by Incubation of Cells in Oleic Acid

Wild-type E122 cells grown in glucose-containing medium were transferred to oleic acid-containing YPBO medium and incubated for a further 7 h in this medium. Aliquots of cells were removed every hour, and total protein was recovered, analyzed by SDS-PAGE, and subjected to immunoblotting with various antibodies. Under these conditions, the levels of the peroxisomal matrix enzyme thiolase (THI) and of the integral membrane peroxin Pex2p increased with the time of incubation, whereas the levels of the cytosolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) remained unchanged under the same conditions (Figure 9). Pex19p levels also appeared not to change over the same time period.

View larger version (44K): [in this window] [in a new window]   Figure 9.   Synthesis of Pex19p is not induced by incubation of Y. lipolytica in oleic acid-containing medium. Wild-type E122 cells grown in glucose-containing YEPD medium for 16 h were transferred to and then incubated in oleic acid-containing YPBO medium. Samples were removed from the YPBO medium at the times indicated. At each time point, equal amounts of protein of total cell lysates were analyzed by SDS-PAGE, followed by transfer to nitrocellulose and immunoblotting with antibodies to the peroxisomal matrix protein thiolase (THI), the integral peroxisomal membrane peroxin Pex2p, the cytosolic enzyme glucose-6-phosphate dehydrogenase (G6PDH), and Pex19p.

The Putative Farnesylation Site of Pex19p Is Dispensable for Pex19p Function

Pex19p has a farnesylation consensus sequence at its carboxy terminus (Figure 1B). Two mutants of Pex19p that are incapable of being farnesylated were made to determine the importance of this site to Pex19p function. These mutants either lacked the 4 carboxyl-terminal amino acids (CNQQ) of Pex19p or had a substitution of serine for the putatively farnesylated cysteine at position 321 (C321S). Expression of either of these mutant forms of Pex19p in the strain pex19KOA led to reestablished growth on oleic acid-containing medium, as did expression of the original complementing fragment 19/NcA (Figure 10A). Transformation of the pex19KOA strain with the parental vector pINA445 did not result in reestablished growth (Figure 10A). Immunoblot analysis showed that the levels of the mutant forms of Pex19p were similar to those of wild-type Pex19p when expressed from plasmid in the pex19KOA strain, and approximately five times higher than the endogenous expression of Pex19p in wild-type E122 cells (Figure 10B).

View larger version (34K): [in this window] [in a new window]   Figure 10.   Expression of Pex19p variants mutated in the putative farnesylation site leads to reestablished growth of the disruption strain pex19KOA on oleic acid-containing medium. (A) Growth profiles on oleic acid-containing YNO medium of the wild-type strain E122, of the untransformed pex19KOA strain, and of the pex19KOA strain transformed with vector alone (pINA445), vector expressing the original PEX19 minimal complementing fragment (19/NcA), vector expressing the Pex19p variant with a cysteine to serine substitution at position 321 (C321S), or vector expressing the variant lacking the four carboxyl-terminal amino acids of Pex19p (-CNQQ). (B) Levels of Pex19p and Pex19p variants. The strains reported in panel (A) were grown for 16 h in glucose-containing YND medium and then transferred to oleic acid-containing YNO medium for 8 h. Equal amounts of protein of total cell lysate from each strain were analyzed by SDS-PAGE and subjected to immunoblot analysis with antibodies to Pex19p.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Pex19p of Y. lipolytica is a 324-amino acid protein with a predicted molecular mass of 34,822 Da. Like all Pex19p peroxins already reported, Pex19p of Y. lipolytica contains a consensus sequence for farnesylation at its carboxy terminus. The addition of farnesyl moieties to proteins is predicted to promote protein-membrane interactions through their hydrophobicity (Omer and Gibbs, 1994). Human, Chinese hamster, and S. cerevisiae Pex19p peroxins have all been demonstrated to be farnesylated (James et al., 1994; Götte et al., 1998; Matsuzono et al., 1999); however, P. pastoris Pex19p is not farnesylated (Snyder et al., 1999a). Because farnesylation is a posttranslational modification, a protein that undergoes farnesylation is generally observed as a doublet on immunoblots, with the farnesylated species running with slightly increased electrophoretic mobility relative to the unmodified species due to the cleavage of three amino acids at the carboxy terminus during the process of farnesylation. Immunoblot analysis always revealed only one species of Y. lipolytica Pex19p, strongly suggesting that in Y. lipolytica, Pex19p is not farnesylated. Farnesylation of Pex19p is required for its function in S. cerevisiae, because the expression of unfarnesylated Pex19p constructs at levels comparable to that of the wild-type protein are able only to partially rescue the pex19 mutant phenotype in this yeast (Götte et al., 1998). In contrast, we have found that the putative farnesylation site of Y. lipolytica Pex19p is not required for its function, because deletion of this site or modification of the site to prevent any possible farnesylation did not affect the ability of these variants of Pex19p to confer reestablished growth on oleic acid-containing medium to a pex19 mutant strain.

Pex19p has been found to be primarily a cytosolic protein (Snyder et al., 1999a) having some association with the outer surface of the peroxisomal membrane (James et al., 1994; Götte et al., 1998). In contrast, we have found that the majority of Pex19p in Y. lipolytica cells fractionates to a 20,000 × g pellet (20KgP) enriched for peroxisomes. Double-labeling immunofluorescence studies have also shown that Pex19p colocalizes with the peroxisomal matrix protein thiolase to punctate structures characteristic of peroxisomes of wild-type cells. Together, these data show that in Y. lipolytica, Pex19p is primarily a peroxisomal protein.

Peroxisomal matrix proteins are preferentially mislocalized to the cytosol in Y. lipolytica pex19 mutants, although a small amount of each matrix protein can be reproducibly recovered in a 20KgP fraction after subcellular fractionation. S. cerevisiae and P. pastoris pex19 mutants also show defects in the import of matrix proteins (Götte et al., 1998; Snyder et al., 1999a). Importantly, electron micrographs of Y. lipolytica pex19 cells reveal the presence of subcellular structures that closely resemble wild-type peroxisomes in regards to size, shape, electron density, and number. No such structures are observed in pex19 mutants of S. cerevisiae (Götte et al., 1998), and only small vesicular and tubular structures are found in P. pastoris pex19 mutants (Snyder et al., 1999a; Hettema et al., 2000). Isopycnic density gradient centrifugation also revealed the presence of vesicular structures in Y. lipolytica pex19 mutants, some of which were found to be of the same density as wild-type peroxisomes. These vesicular structures were shown to contain all peroxisomal matrix proteins tested. Therefore, it appears that Y. lipolytica pex19 mutants are almost able to form mature, functional peroxisomes, but that some step or steps in this process are missing.

Pex19p has been suggested to act as a membrane protein receptor, recognizing peroxisomal membrane proteins (PMP) after their synthesis in the cytosol on polyribosomes and recruiting them to the peroxisomal membrane, most likely with the help of other proteins (Sacksteder et al., 2000). Pex19p has been shown to interact with the domains of PMPs that are required for targeting to the peroxisomal membrane. However, these domains encompass more than the minimal sequence required for targeting the protein to the peroxisomal membrane, and therefore the amino acids required for targeting and for binding of Pex19p may be different. In fact, it has been demonstrated that the targeting sequences and the Pex19p-binding domains for several PMPs of P. pastoris and of human cells are separable (Snyder et al., 2000). This has led to the proposal that Pex19p may perform a chaperone-like function (Snyder et al., 2000). In this way, Pex19p would perform an important role in stabilizing the interaction between a putative membrane protein receptor and a PMP or the interactions of various proteins at the translocation/insertion step of a membrane protein at the peroxisomal membrane. Indeed, S. cerevisiae Pex19p has been shown to influence PMP stability (Hettema et al., 2000). A distribution for Pex19p that is partly cytosolic and partly peroxisomal has lent support to this idea. However, it has also been shown that Pex19p does not interact with newly synthesized PMPs in the cytosol and so is probably not involved in a receptor protein/PMP complex in the cytosol (Snyder et al., 2000), but rather is probably involved in stabilizing PMP interactions within the peroxisomal membrane itself.

Our findings on Y. lipolytica Pex19p are consistent with this last hypothesis. pex19 mutants of Y. lipolytica are capable of forming structures that are morphologically similar to wild-type peroxisomes and that are surrounded by a membrane whose protein composition is similar to that of wild-type peroxisomes. Therefore, in Y. lipolytica, Pex19p appears not to function as a PMP receptor. A significant reduction in the levels of the peroxisomal integral membrane protein Pex2p in pex19 mutants lends support to the proposal that Pex19p is important to the stability of PMPs in Y. lipolytica. It is interesting to note that Pex2p has recently been shown to interact with Pex19p (Snyder et al., 2000). Although Y. lipolytica pex19 mutants do contain peroxisome-like structures, these structures do not function fully as peroxisomes, because pex19 mutants cannot use oleic acid as a sole carbon source. This inability to use oleic acid may be the result of the lack of coordination of PMP activity by Pex19p, which leads to secondary effects such as reduced import of matrix proteins due to improper functioning and/or assembly of the matrix protein translocation machinery controlled by the action of Pex19p.

In closing, we have isolated and characterized the peroxin Pex19p of the yeast Y. lipolytica. Pex19p is not required for the assembly of the peroxisomal membrane in Y. lipolytica, because pex19 mutants of this yeast are capable of assembling subcellular structures that resemble wild-type peroxisomes morphologically and biochemically. From studies of peroxisome biogenesis in different organisms, it is becoming apparent that although the overall mechanism of peroxisome assembly has been conserved during evolution, the roles played by individual peroxins may not necessarily be the same in different organisms and that differences in the overall molecular activities of peroxins may modulate the assembly of peroxisomes to suit the specialized requirements of a particular organism.