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• W2164163003 abstract "Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Δpex13 or Δpex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Δpex4), and others (Δpex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Δpex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeastdoa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly. Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Δpex13 or Δpex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Δpex4), and others (Δpex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Δpex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeastdoa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly. peroxisomal targeting sequence polyacrylamide gel electrophoresis hemagglutinin ubiquitin ubiquitin carrier protein Peroxisome biogenesis proceeds via a complex, branched pathway, in which a cellular machinery consisting of more than 20 proteins (peroxins) effects the recognition, targeting, and import of proteins containing peroxisomal targeting sequences (PTSs)1 (reviewed in Refs.1Sacksteder K.A. Gould S.J. Annu. Rev. Genet. 2000; 34: 623-652Crossref PubMed Scopus (99) Google Scholar, 2Hettema E.H. Distel B. Tabak H.F. Biochim. Biophys. Acta Mol. Cell Res. 1999; 1451: 17-34Crossref PubMed Scopus (106) Google Scholar, 3Subramani S. Physiol. Rev. 1998; 78: 171-188Crossref PubMed Scopus (285) Google Scholar, 4Purdue P.E. Lazarow P.B. J. Biol. Chem. 1994; 269: 30065-30068Abstract Full Text PDF PubMed Google Scholar, 5Terlecky S.R. Fransen M. Traffic. 2000; 1: 465-473Crossref PubMed Scopus (44) Google Scholar). Multiple classes of PTS exist, of which one of the best characterized is the PTS2 family of NH2-terminal oligopeptides (6Osumi T. Tsukamoto T. Hata S. Yokota S. Miura S. Fujiki Y. Hijikata M. Miyazawa S. Hashimoto T. Biochem. Biophys. Res. Commun. 1991; 181: 947-954Crossref PubMed Scopus (232) Google Scholar, 7Swinkels B.W. Gould S.J. Bodnar A.G. Rachubinski R.A. Subramani S. EMBO J. 1991; 10: 3255-3262Crossref PubMed Scopus (518) Google Scholar, 8Erdmann R. Yeast. 1994; 10: 935-944Crossref PubMed Scopus (59) Google Scholar, 9Glover J.R. Andrews D.W. Subramani S. Rachubinski R.A. J. Biol. Chem. 1994; 269: 7558-7563Abstract Full Text PDF PubMed Google Scholar) utilized by thiolase and several other peroxisomal proteins, which are imported into peroxisomes via interaction with Pex7p, the PTS2 receptor (10Marzioch M. Erdmann R. Veenhuis M. Kunau W.-H. EMBO J. 1994; 13: 4908-4917Crossref PubMed Scopus (256) Google Scholar, 11Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (124) Google Scholar, 12Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (359) Google Scholar, 13Motley A.M. Hettema E.H. Hogenhout E.M. Brites P. Ten Asbroek A.L.M.A. Wijburg F.A. Baas F. Heijmans H.S. Tabak H.F. Wanders R.J.A. Distel B. Nat. Genet. 1997; 15: 377-380Crossref PubMed Scopus (223) Google Scholar, 14Purdue P.E. Zhang J.W. Skoneczny M. Lazarow P.B. Nat. Genet. 1997; 15: 381-384Crossref PubMed Scopus (224) Google Scholar, 15Elgersma Y. Elgersma-Hooisma M. Wenzel T. McCaffery J.M. Farquhar M.G. Subramani S. J. Cell Biol. 1998; 140: 807-820Crossref PubMed Scopus (74) Google Scholar). The importance of this pathway is highlighted by the observation that it is evolutionarily conserved between yeast and humans and by the fact that loss of the PTS2 branch of peroxisomal biogenesis through PEX7 mutation causes the lethal disorder rhizomelic chondrodysplasia punctata in humans (12Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (359) Google Scholar, 13Motley A.M. Hettema E.H. Hogenhout E.M. Brites P. Ten Asbroek A.L.M.A. Wijburg F.A. Baas F. Heijmans H.S. Tabak H.F. Wanders R.J.A. Distel B. Nat. Genet. 1997; 15: 377-380Crossref PubMed Scopus (223) Google Scholar, 14Purdue P.E. Zhang J.W. Skoneczny M. Lazarow P.B. Nat. Genet. 1997; 15: 381-384Crossref PubMed Scopus (224) Google Scholar), and inviability on oleic acid as carbon source in yeast. We recently identified a novel pair of yeast peroxins, Pex18p and Pex21p, which interact with Pex7p and are essential for PTS2 targeting (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). Pex18p and Pex21p are weakly homologous to each other and appear to demonstrate partial functional redundancy, in that full loss of PTS2 targeting requires the absence of them both. Thus, although thiolase is the only known Saccharomyces cerevisiae PTS2 protein, at least three peroxins are involved in its delivery to peroxisomes. Several reports concerning different species place Pex7p within peroxisomes and/or within the cytosol, raising the intriguing possibility that Pex7p acts as a mobile receptor, shuttling between the cytosol and peroxisomal surface and/or lumen (10Marzioch M. Erdmann R. Veenhuis M. Kunau W.-H. EMBO J. 1994; 13: 4908-4917Crossref PubMed Scopus (256) Google Scholar, 11Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (124) Google Scholar, 12Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (359) Google Scholar, 15Elgersma Y. Elgersma-Hooisma M. Wenzel T. McCaffery J.M. Farquhar M.G. Subramani S. J. Cell Biol. 1998; 140: 807-820Crossref PubMed Scopus (74) Google Scholar). The initial characterization of Pex18p/Pex21p identified these proteins as potentially key players in this proposed “mobile receptor” mechanism. In addition to the PTS2-specific peroxins (Pex7p, Pex18p, and Pex21p), many other peroxins are required for import of peroxisomal proteins, including, but not limited to, those with a PTS2. These peroxins are believed to include components of a common translocation machinery acting downstream of the point at which the various branches of peroxisomal protein import converge. Examples include the peroxisomal membrane peroxins Pex13p and Pex14p, which have been reported to interact with both Pex7p and the PTS1 receptor, Pex5p, consistent with roles in a common docking site for the PTS1 and PTS2 branches (18Albertini M. Rehling P. Erdmann R. Girzalsky W. Kiel J.A.K.W. Veenhuis M. Kunau W.H. Cell. 1997; 89: 83-92Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 19Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (109) Google Scholar, 20Girzalsky W. Rehling P. Stein K. Kipper J. Blank L. Kunau W.H. Erdmann R. J. Cell Biol. 1999; 144: 1151-1162Crossref PubMed Scopus (151) Google Scholar, 21Komori M. Rasmussen S.W. Kiel J.A.K.W. Baerends R.J.S. Cregg J.M. van der Klei I.J. Veenhuis M. EMBO J. 1997; 16: 44-53Crossref PubMed Scopus (114) Google Scholar, 22Elgersma Y. Kwast L. Klein A. Voorn-Brouwer T. Van Den Berg M. Metzig B. America T. Tabak H.F. Distel B. J. Cell Biol. 1996; 135: 97-109Crossref PubMed Scopus (186) Google Scholar, 23Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (186) Google Scholar, 24Gould S.J. Kalish J.E. Morrell J.C. Bjorkman J. Urquhart A.J. Crane D.I. J. Cell Biol. 1996; 135: 85-95Crossref PubMed Scopus (212) Google Scholar). Still other peroxins, including Pex1p, Pex4p, and Pex6p, are required for both PTS1 and PTS2 packaging but not for peroxisomal membrane assembly; their molecular roles have yet to be established. Pex1p and Pex6p are AAA ATPases localized in various yeasts and mammals to either cytosol, peroxisomes, or vesicles, depending upon the species under investigation, but not yet firmly localized in S. cerevisiae(25Erdmann R. Wiebel F.F. Flessau A. Rytka J. Beyer A. Frohlich K.-U. Kunau W.-H. Cell. 1991; 64: 499-510Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 26Heyman J.A. Monosov E. Subramani S. J. Cell Biol. 1994; 127: 1259-1273Crossref PubMed Scopus (61) Google Scholar, 27Spong A.P. Subramani S. J. Cell Biol. 1993; 123: 535-548Crossref PubMed Scopus (82) Google Scholar, 28Voorn-Brouwer T. Van Der Leij I. Hemrika W. Distel B. Tabak H.F. Biochim. Biophys. Acta. 1993; 1216: 325-328Crossref PubMed Scopus (70) Google Scholar, 29Tamura S. Okumoto K. Toyama R. Shimozawa N. Tsukamoto T. Suzuki Y. Osumi T. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4350-4355Crossref PubMed Scopus (87) Google Scholar, 30Faber K.N. Heyman J.A. Subramani S. Mol. Cell. Biol. 1998; 18: 936-943Crossref PubMed Scopus (125) Google Scholar, 31Yahraus T. Braverman N. Dodt G. Kalish J.E. Morrell J.C. Moser H.W. Valle D. Gould S.J. EMBO J. 1996; 15: 2914-2923Crossref PubMed Scopus (161) Google Scholar, 32Tsukamoto T. Miura S. Nakai T. Yokota S. Shimozawa N. Suzuki Y. Orii T. Fujiki Y. Sakai F. Bogaki A. Yasumo H. Osumi T. Nat. Genet. 1995; 11: 395-401Crossref PubMed Scopus (101) Google Scholar, 33Titorenko V.I. Chan H. Rachubinski R.A. J. Cell Biol. 2000; 148: 29-43Crossref PubMed Scopus (123) Google Scholar). Pex4p is a ubiquitin-conjugating enzyme for which substrates have not been identified, localized to the peroxisome membrane (34Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar,35Wiebel F.F. Kunau W.-H. Nature. 1992; 359: 73-78Crossref PubMed Scopus (163) Google Scholar). In this paper, we report that the functioning of Pex18p and Pex21p is accompanied by their rapid proteolytic turnover and that this pathway of peroxin degradation is obligatorily connected to ongoing peroxisome assembly. This represents, to the best of our knowledge, the first instance of an organelle biogenesis factor that is constitutively degraded during its normal function. Polyclonal rabbit antisera against S. cerevisiae Pex7p and Pex18p were raised at Covance Research Products, Inc. (Denver, PA) using standard procedures. Antigens (prepared as slices excised from SDS-PAGE gels) corresponding to the NH2-terminal 156 residues of Pex7p and the COOH-terminal 200 residues of Pex18p were purified as His6-tagged proteins from overexpressing bacterial clones as described previously (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). The Pex7p antigen was prepared in the presence of 6 m urea to overcome its low solubility. The anti-Pex18p serum was affinity-purified on amino-link columns (Pierce) coupled with His6-tagged antigen, eluted with 0.1m glycine, pH 2.5, and adjusted to pH 8.0 with Tris base. 10 ml of sera yielded 0.8 ml of purified antibody. All of thepex mutants described in this paper were generated by targeted disruption from wild-type haploid W303 of the correspondingPEX genes. W303Δpex5, W303Δpex7, and W303Δpex18 and have been described previously (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). W303Δpex1, in which the entire PEX1 coding region is replaced with the LEU2 marker gene, was a kind gift of Dr. Marek Skoneczny. W303Δpex4 was generated by transformation of W303 with the HindIII-NotI insert of plasmid pPEX4::TRP1 (see below) and selection of tryptophan auxotrophs. All double knockout strains were generated by successive transformations with the appropriate disruption constructs. W303Δpep4 was generated by transformation of W303 with theEcoRI-XbaI insert of plasmid pBJ3287 (a kind gift of Dr. Beth Jones, Carnegie Mellon University) and selection of histidine auxotrophs. All new disruption strains were checked by polymerase chain reaction and/or Western blotting. MHY623, in whichDOA4 has been disrupted, and the congenic wild-type strain MHY501 (36Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (341) Google Scholar) were kind gifts of Dr. Marc Hochstrasser (University of Chicago). Y0238, in which UBC4 and UBC5 have been disrupted, and the congenic wild-type strain Y0002 were kind gifts of Dr. Stefan Jentsch (University of Heidelberg). For induction of peroxisomes, cells were precultured to a density of 5 × 107/ml in YPD (1% yeast extract, 2% peptone, 2% dextrose) and then inoculated at a density of 5 × 106/ml into YPEO (1% yeast extract, 2% peptone, 2% ethanol, 0.1% oleic acid, 0.25% Tween 40) and grown for a further 18 h. Where appropriate, cycloheximide was then added to a concentration of 10 μg/ml (0.2 μl/ml of a 50 mg/ml stock solution in ethanol), and growth was continued for up to 30 min, prior to protein extraction. For expression of hemagglutinin epitope-tagged ubiquitin (HA-Ub) from plasmid pKR81, galactose was added to the YPEO at a concentration of 0.01%. For strains harboring plasmids, preculturing was in SCD (0.67% yeast nitrogen base, 2% dextrose supplemented with all amino acids, adenine, and uracil, except those omitted to maintain plasmid selection). For some experiments, YPEO was replaced with YPE (1% yeast extract, 2% peptone, 2% ethanol). For assessment of Pex18p levels in YPD (see Fig. 1 B), aliquots of YPD precultures (2 × 107 cells/ml) were removed and processed for protein extraction (see below). For the pulse labeling shown in Fig. 2 B, peroxisome induction was in SCEO lacking methionine (0.67% yeast nitrogen base, 2% ethanol, 0.1% oleic acid, 0.25% Tween 40 supplemented with adenine, uracil, and all amino acids except methionine) for 18 h, after which cells were diluted into fresh SCEO lacking methionine, supplemented with 100 μCi/ml [35S]methionine, to a density of 5 × 107 cells/ml, and incubated for a further 5 min prior to protein extraction.Figure 2Strains with vastly different levels of Pex18p protein show little variation in PEX18 mRNA abundance or synthetic rate of Pex18p. A, total RNA was prepared from yeast grown in YPEO or YPE, and 20-μg aliquots were analyzed by Northern blotting with probes against PEX18 and actin (as a loading control). B, yeast cells grown in SCEO without methionine were pulse-labeled with radiolabeled methionine. Pex18p was immunoprecipitated from total cellular protein extracts (prepared by glass bead homogenization); its radioactivity was visualized by SDS-PAGE and fluorography.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For FLAG tagging, the complete coding sequence of the PEX18 gene was excised as a BamHI fragment from clone pYcp-PEX18 (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar) and cloned into the BglII site of pESC-LEU (Stratagene, La Jolla, CA). This produced a sequence encoding an in-frame fusion between the FLAG tag and the amino terminus ofPEX18, which was then excised as an SpeI fragment and cloned into the XbaI site of Yep351-POT1, which contains the POT1 (thiolase) promoter in the episomal vector Yep351. This produced a clone encoding FLAG-Pex18p under the regulation of the oleate-responsive POT1 promoter. To produce a similar clone lacking the FLAG tag, the PEX18 gene was excised as anSpeI fragment from clone pYcp-PEX18 (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar) and cloned directly into the into the XbaI site of Yep351-POT1. Plasmid pKR81, encoding HA-Ub (37Robzyk K. Recht J. Osley M.A. Science. 2000; 287: 501-504Crossref PubMed Scopus (529) Google Scholar) under the regulation of the GAL1 promoter, was a kind gift of Dr. K. Robzyk (Memorial Sloan Kettering Cancer Center, New York). For construction of plasmid pPEX4::TRP1, which was used for targeted disruption of PEX4 from W303 wild-type yeast, PEX4 was polymerase chain reaction-amplified from W303 genomic DNA using primers 5′-CCTGTTGCTTTACACCATTGA-3′ (maps to 593 bases upstream of PEX4 coding region) and 5′-GATACTGCAGTCAATGGTTGTTGATCCGCT-3′ (maps to the 3′ end ofPEX4 coding sequence). This polymerase chain reaction product was cloned into pCR-TOPO2.1 (Invitrogen, Carlsbad, CA), linearized with StuI, and ligated to aSmaI-StuI fragment containing the TRP1 gene. A fragment containing the TRP1 gene flanked byPEX4 sequences was excised from the resultant construct and used for transformation of W303. PEX4 knockouts were verified by polymerase chain reaction. Preparation of total cellular RNA by the hot acidic phenol method and Northern blotting were as described (38Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York, NY1987Google Scholar). 32P-Labeled probes were prepared by random priming from the coding regions of yeast PEX18 and actin genes, the latter of which were a kind gift of Drs. Igor Karpichev and Gillian Small (Mount Sinai School of Medicine, New York). Radioactive bands on the washed blots were visualized by autoradiography or with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Preparation by glass bead homogenization of total cellular protein extracts for immunoblotting was as described previously (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). Preparation by glass bead homogenization of total cellular protein extracts for immunoprecipitation with anti-Pex18p (Figs. 2 Band 4 B) was by this same method but using IP buffer (50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 150 mm NaCl, 0.1% Triton X-100, supplemented as usual with protease inhibitor mixture (39Thieringer R. Shio H. Han Y. Cohen G. Lazarow P.B. Mol. Cell Biol. 1991; 11: 510-522Crossref PubMed Scopus (62) Google Scholar)) in place of our normal protein extraction buffer. Immunoprecipitations, with affinity-purified anti-Pex18p, were as described previously (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). For preparation of total protein extracts prior to immunoprecipitation of Pex18p-ubiquitin conjugates with anti-FLAG, a modified extraction procedure was employed in order to minimize the risk of proteolytic degradation of these conjugates. Cells were grown in 25 ml of YPEO and washed and collected as usual. All subsequent steps were performed at room temperature. Pellets were resuspended in 0.5 ml of 40% trichloroacetic acid and centrifuged at 14,000 rpm. The supernatant was discarded, and the pellets were briefly frozen in liquid nitrogen and then thawed and resuspended in 0.5 ml of 20% trichloroacetic acid. Glass beads were added, and the samples were then vortexed vigorously for 2 min, after which the extracts were decanted from the beads and centrifuged at 3000 rpm for 10 min. The resultant pellets were resuspended in 200 μl of SDS-PAGE sample buffer, and 50 μl of unbuffered 2 m Tris base was added (to adjust the pH). Samples were boiled for 3 min and centrifuged at 3000 rpm for 10 min. 200-μl aliquots of the blue supernatant were then recovered and added to 770 μl of fresh IP buffer (supplemented with 0.5 μg/ml bovine serum albumin) and 30 μl of anti-FLAG M2-agarose affinity gel (Sigma). Samples were rotated at 4 °C for 1 h, after which the anti-FLAG M2-agarose affinity gel was recovered by centrifugation at 1000 rpm for 1 min and washed twice with 1 ml of IP buffer. Bound proteins were eluted with 40 μl of 0.2 mg/ml FLAG peptide (Sigma) in IP buffer. The final eluate was processed as usual for SDS-PAGE. SDS-PAGE and immunoblotting were as described previously (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar), except that prior to blotting with anti-ubiquitin, the nitrocellulose membrane was autoclaved for 20 min. This greatly enhances the signal from both free ubiquitin and ubiquitin conjugates (data not shown). Antibody dilutions used were as follows: 1:100 affinity-purified anti-Pex18p, 1:500 anti-Pex7p serum, and 1:100 anti-ubiquitin (Sigma). For the pulse-labeling experiment shown in Fig. 2 B, the SDS-PAGE gel was incubated in 1 m sodium salicylate for 1 h prior to drying and fluorography. A polyclonal antiserum raised against bacterially expressed Pex18p (see “Experimental Procedures”) was used to monitor Pex18p expression levels in wild-type and variouspex mutant yeast strains. Pex18p could be detected in extracts from oleate-induced wild-type yeast as a doublet with an estimated size of about 36 kDa (see Fig. 1), compared with the predicted size, based on the sequence of the PEX18 gene, of ∼32 kDa (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). The reason for this apparent size difference is unclear, but it was also observed for bacterially expressed Pex18p (which likewise runs as a doublet), indicating that its mobility in SDS-PAGE may be an intrinsic feature of the protein rather than due to covalent modifications. Treatment of extracts with iodoacetamide, phosphatases, or hydroxylamine did not alter the Pex18p gel migration pattern (data not shown). Both bands of the doublet were absent in extracts from W303Δpex18 yeast, confirming that they correspond to Pex18p (Fig. 1 A). Remarkably, the abundance of Pex18p was greatly increased in strains with generalized defects in the import of peroxisomal matrix proteins (W303Δpex1, W303Δpex4, W303Δpex13, and W303Δpex14) (Figs. 1 and3 B). On the other hand, Pex18p abundance was close to normal in W303Δpex7 and W303Δpex5, which display partial defects in this process. In common with many peroxisomal proteins and peroxins, Pex18p expression was oleate-inducible, as shown by the apparent absence or near absence of immunoreactive protein in strains grown in YPD or YPE (Fig. 1 B). To investigate the mechanism of the variation in Pex18p levels, RNA was extracted from oleate-induced strains and analyzed by Northern blotting. PEX18 mRNA levels were similar to the wild-type level in several pex mutants, including W303Δpex5, W303Δpex7, W303Δpex13, and W303Δpex14 (shown for W303Δpex14 in Fig. 2 A), suggesting that the variation in Pex18p levels is not primarily due to differences in abundance of PEX18 message. In agreement with the immunoblot data shown in Fig. 1 B, PEX18 mRNA levels were much lower in the absence of oleate. The rate of Pex18p synthesis was tested directly by pulse-labeling oleate-grown cells with [35S]methionine and then analyzing incorporation into Pex18p by immunoprecipitation with anti-Pex18p, followed by SDS-PAGE and fluorography (Fig. 2 B). Little difference was observed between wild type and W303Δpex14, suggesting that Pex18p is made at similar rates in these strains. Since neither the levels of PEX18 mRNA nor the rates of Pex18p synthesis vary substantially between strains with grossly different levels of Pex18p, we next addressed the question of Pex18p turnover. Cells were grown and induced as usual, and then growth was continued in the presence or absence of the protein synthesis inhibitor, cycloheximide. As shown in Fig. 3 A, Pex18p rapidly disappeared from wild-type cells treated with cycloheximide, being barely detectable within 20 min. In contrast, the high level of Pex18p in W303Δpex14 cells was maintained after 20 min in cycloheximide (Fig. 3 A) and showed no discernible decline even 5 h after administration of cycloheximide (not shown). The turnover of Pex18p in W303Δpex7 cells, which maintain Pex18p at a level similar to that seen in wild-type cells, is similar to the turnover rate in wild-type cells (Fig. 3 A). Analysis of additional pex mutants revealed a consistent pattern. Mutants that have a generalized defect in peroxisome biogenesis accumulated high levels of Pex18p, which turned over slowly, if at all, whereas W303Δpex7 and W303Δpex5, which are defective only for PTS2 and PTS1 targeting, respectively, behaved similarly to wild type (Fig. 3 B). Thus, in mutants W303Δpex13 and W303Δpex14, where Pex18p is unable to fulfill its role of PTS2 protein delivery to peroxisomes, Pex18p is stabilized. Notably in W303Δpex4, which lacks the peroxisomal member of the ubiquitin-conjugating enzyme family, Pex18p is likewise stabilized. Stabilization also occurs in W303Δpex1. In W303Δpex5, where PTS2 packaging is functional, Pex18p is turned over as in wild-type cells. Pex18p is known to interact with Pex7p (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar). The instability of Pex18p in W303Δpex7 suggests that Pex18p may be stabilized by its interaction with Pex7p. Pex21p, whose function can partially compensate for an absence of Pex18p (16Purdue P.E. Yang X. Lazarow P.B. J. Cell Biol. 1998; 143: 1859-1869Crossref PubMed Scopus (136) Google Scholar), also turns over rapidly in wild-type yeast (data not shown). In cells defective in peroxisome biogenesis (W303Δpex13 or W303Δpex14), Pex21p is stabilized little, if at all, unless Pex18p is missing, in which case there is a striking stabilization and accumulation of Pex21p. As for Pex18p, this stabilization requires Pex7p to be present. In order to investigate further the possible involvement of Pex7p in Pex18p stabilization, we disrupted thePEX7 gene from several pex mutants and assessed the consequences for Pex18p accumulation and turnover. As shown in Fig. 4 A, the absence of Pex7p completely abolishes Pex18p stabilization in the W303Δpex13 background. W303Δpex13Δpex7appeared identical to W303Δpex7 with respect to the abundance of Pex18p and its turnover. Similar Pex18p instability was observed in W303Δpex14Δpex7 (Fig. 4 A) and W303Δpex4Δpex7 (not shown). These data support the idea that Pex18p becomes stabilized in pex mutants through persistence of its interaction with Pex7p. To test directly whether the elevated Pex18p is associated with Pex7p, anti-Pex18p immunoprecipitates were immunoblotted with anti-Pex7p antisera (Fig. 4 B). Pex7p was co-immunoprecipitated with Pex18p from wild-type cells; the amount of immunoprecipitated Pex7p was considerably greater with W303Δpex14 cells. In neither case was immunoprecipitation of Pex7p complete, suggesting that the cellular Pex7p is not saturated with Pex18p, even when the latter is accumulated to the levels seen in W303Δpex14. To investigate the mechanism of the rapid turnover of Pex18p in wild-type cells, we first analyzed Pex18p turnover in strains defective in various aspects of degradation of cellular proteins. W303Δpep4, which lacks the vacuolar proteinase A, had wild-type levels of Pex18p abundance and turnover (Fig. 5). On the other hand, Y0238, which lacks two functionally related ubiquitin-conjugating enzymes, Ubc4p and Ubc5p, showed accumulation of high levels of Pex18p and impaired Pex18p turnover. A congenic wild type strain (Y0002; Fig. 5) and various otherubc mutants, including a strain lacking Ubc6p and Ubc7p (Y0241; not shown), demonstrated normal Pex18p" @default.
• W2164163003 created "2016-06-24" @default.
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• W2164163003 date "2001-12-01" @default.
• W2164163003 modified "2023-09-27" @default.
• W2164163003 title "Pex18p Is Constitutively Degraded during Peroxisome Biogenesis" @default.
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• W2164163003 doi "https://doi.org/10.1074/jbc.m106823200" @default.
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