FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2136590099> ?p ?o ?g. }

• W2136590099 endingPage "16504" @default.
• W2136590099 startingPage "16498" @default.
• W2136590099 abstract "Of the ∼20 proteins required for peroxisome biogenesis, only four have been implicated in the process of peroxisomal membrane protein (PMP) import: Pex3p, Pex16p, Pex17p, and Pex19p. To improve our understanding of the role that Pex17p plays in PMP import, we examined the behavior of PMPs in a Pichia pastoris pex17 mutant. Relative to wild-type cells,pex17 cells appeared to have a mild reduction in PMP stability and slightly aberrant PMP behavior in subcellular fractionation experiments. However, we also found that the behavior of PMPs in the pex17 mutant was indistinguishable from PMP behavior in a pex5 mutant, which has no defect in PMP import, and was far different from PMP behavior in a pex3mutant, which has a bona fide defect in PMP import. Furthermore, we found that a pex14 mutant, which has no defect in PMP import, lacks detectable levels of Pex17p. Based on these and other results, we propose that Pex17p acts primarily in the matrix protein import pathway and does not play an important role in PMP import. Of the ∼20 proteins required for peroxisome biogenesis, only four have been implicated in the process of peroxisomal membrane protein (PMP) import: Pex3p, Pex16p, Pex17p, and Pex19p. To improve our understanding of the role that Pex17p plays in PMP import, we examined the behavior of PMPs in a Pichia pastoris pex17 mutant. Relative to wild-type cells,pex17 cells appeared to have a mild reduction in PMP stability and slightly aberrant PMP behavior in subcellular fractionation experiments. However, we also found that the behavior of PMPs in the pex17 mutant was indistinguishable from PMP behavior in a pex5 mutant, which has no defect in PMP import, and was far different from PMP behavior in a pex3mutant, which has a bona fide defect in PMP import. Furthermore, we found that a pex14 mutant, which has no defect in PMP import, lacks detectable levels of Pex17p. Based on these and other results, we propose that Pex17p acts primarily in the matrix protein import pathway and does not play an important role in PMP import. Eukaryotic cells contain specialized organelles that enhance their metabolic efficiency. Peroxisomes are a class of single-membrane bound organelle that play important roles in lipid metabolism in virtually all eukaryotes (1.Wanders R.J. Vreken P. Ferdinandusse S. Jansen G.A. Waterham H.R. van Roermund C.W. Van Grunsven E.G. Biochem. Soc. Trans. 2001; 29: 250-267Crossref PubMed Scopus (0) Google Scholar, 2.Wanders R.J. Tager J.M. Mol. Aspects Med. 1998; 19: 69-154Crossref PubMed Google Scholar, 3.Hettema E.H. Tabak H.F. Biochim. Biophys. Acta. 2000; 1486: 18-27Crossref PubMed Scopus (92) Google Scholar). Although eukaryotic cells can survive without peroxisomes, defects in peroxisome biogenesis have significant metabolic and developmental consequences. In humans, severe defects in peroxisome biogenesis cause Zellweger syndrome, a lethal neurological disorder (4.Gould S.J. Valle D. Trends Genet. 2000; 16: 340-344Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 5.Gould S.G. Valle D. Raymond G.V. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. 2. McGraw-Hill, New York2001: 3181-3217Google Scholar), and in yeast such defects eliminate growth on fatty acids, an important carbon and energy source in natural environments (3.Hettema E.H. Tabak H.F. Biochim. Biophys. Acta. 2000; 1486: 18-27Crossref PubMed Scopus (92) Google Scholar). Approximately 20 PEX genes are required for peroxisome biogenesis (6.Sacksteder K.A. Gould S.J. Annu. Rev. Genet. 2000; 34: 623-652Crossref PubMed Scopus (98) Google Scholar). Two major classes of peroxins have been identified. Most PEX genes are required for import of peroxisomal matrix enzymes but are not required for peroxisome membrane biogenesis or peroxisomal membrane protein (PMP) 1The abbreviations used are: PMPperoxisomal membrane proteinWTwild typePNSpostnuclear supernatant1The abbreviations used are: PMPperoxisomal membrane proteinWTwild typePNSpostnuclear supernatant import.PEX5, which encodes the import receptor for most newly synthesized peroxisomal matrix enzymes, is the exemplar of this class (7.McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 9.Chang C.C. South S. Warren D. Jones J. Moser A.B. Moser H.W. Gould S.J. J. Cell Sci. 1999; 112: 1579-1590Crossref PubMed Google Scholar, 10.Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-124Crossref PubMed Scopus (379) Google Scholar, 11.van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (201) Google Scholar, 12.van der Klei I.J. Hibrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 13.Baes M. Gressens P. Baumgart E. Carmeliet P. Casteels M. Fransen M. Evrard P. Fahimi D. Declercq P.E. Collen D. van Veldhoven P.P. Mannaerts G.P. Nat. Genet. 1997; 17: 49-57Crossref PubMed Scopus (217) Google Scholar). The second major class of PEX genes is required for the synthesis of peroxisomal membranes and/or the import of PMPs; this group includes PEX3, PEX16,PEX17, and PEX19 (4.Gould S.J. Valle D. Trends Genet. 2000; 16: 340-344Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 14.Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (199) Google Scholar). Pex3p is an integral PMP that is necessary for the formation of peroxisomes. Yeast and humanpex3 mutants lack detectable peroxisomal structures, and PMPs are either rapidly degraded or mislocalized to the mitochondrion in these cells (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 15.South S.T. Sacksteder K.A. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 149: 1345-1360Crossref PubMed Scopus (119) Google Scholar). Similar phenotypes are observed in cells lacking Pex19p (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 16.Matsuzono Y. Kinoshita N. Tamura S. Shimozawa N. Hamasaki M. Ghaedi K. Wanders R.J. Suzuki Y. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2116-2121Crossref PubMed Scopus (186) Google Scholar, 17.Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (234) Google Scholar), a putative import receptor/chaperone for newly synthesized PMPs (17.Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (234) Google Scholar, 18.Jones J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2001; 153: 1141-1150Crossref PubMed Scopus (90) Google Scholar) that interacts with PEX3(19.Gotte K. Girzalsky W. Linkert M. Baumgart E. Kammerer S. Kunau W.-H. Erdmann R. Mol. Cell. Biol. 1998; 18: 616-628Crossref PubMed Scopus (161) Google Scholar, 20.Snyder W.B. Faber K.N. Wenzel T.J. Koller A. Luers G.H. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 1745-1761Crossref PubMed Scopus (90) Google Scholar, 21.Fransen M. Wylin T. Brees C. Mannaerts G.P. Van Veldhoven P.P. Mol. Cell. Biol. 2001; 21: 4413-4424Crossref PubMed Scopus (109) Google Scholar). Human PEX16, like PEX3 andPEX19, is also required for peroxisome membrane biogenesis and PMP import (22.Honsho M. Tamura S. Shimozawa N. Suzuki Y. Kondo N. Fujiki Y. Am. J. Hum. Genet. 1998; 63: 1622-1630Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23.South S.T. Gould S.J. J. Cell Biol. 1999; 144: 255-266Crossref PubMed Scopus (188) Google Scholar), although it seems to function differently in the yeast Yarrowia lipolyitca (24.Eitzen G.A. Szilard R.K. Rachubinski R.A. J. Cell Biol. 1997; 137: 1265-1278Crossref PubMed Scopus (108) Google Scholar) and appears to be absent from the yeast Saccharomyces cerevisiae (25.Tabak H.F. Braakman I. Distel B. Trends Cell Biol. 1999; 9: 447-453Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The fourth peroxin implicated in PMP biogenesis is Pichia pastorisPex17p (26.Snyder W.B. Koller A. Choy A.J. Johnson M.A. Cregg J.M. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 4005-4019Crossref PubMed Scopus (51) Google Scholar). This peroxin was identified in the yeast P. pastoris in a screen for mutants that are defective in the import of a PMP-GFP fusion protein (26.Snyder W.B. Koller A. Choy A.J. Johnson M.A. Cregg J.M. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 4005-4019Crossref PubMed Scopus (51) Google Scholar). Furthermore, the pex17Δ mutant was thought to import PMPs less efficiently than wild-type cells. Based on these results, Snyder et al. (26.Snyder W.B. Koller A. Choy A.J. Johnson M.A. Cregg J.M. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 4005-4019Crossref PubMed Scopus (51) Google Scholar) and Subramani et al. (14.Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (199) Google Scholar) have proposed that Pex17p plays a specific role in PMP import, with the matrix protein import defect ofpex17Δ cells resulting indirectly from this defect in PMP biogenesis. These results are somewhat different from those reported for S. cerevisiae PEX17, which appears to participate only in matrix protein import (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 27.Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (124) Google Scholar). peroxisomal membrane protein wild type postnuclear supernatant peroxisomal membrane protein wild type postnuclear supernatant To improve our understanding of PMP import we began an investigation into the role of PEX17 in this process. An analysis ofpex17Δ cells revealed that they do have slightly reduced levels of PMPs and that PMPs displayed an aberrant fractionation behavior, at least as compared with wild-type (WT) cells. However, we also observed these changes in pex5Δ mutants, which are impaired in matrix protein import but unaffected in PMP import or peroxisome membrane biogenesis. Furthermore, pex17Δ cells show a phenotype that is very different from that of pex3Δ cells, which have a clear role in PMP biogenesis. Based on these and other results, we conclude that P. pastoris Pex17p plays an important role in peroxisomal matrix protein import but has no detectable role in PMP import or any other aspect of PMP biogenesis. The yeast strains used in this study are listed in Table I. The pex3Δ mutant was generated by PCR-mediated gene disruption (28.Baker-Brachmann C. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2548) Google Scholar). A DNA fragment containing the KanMX cassette (28.Baker-Brachmann C. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2548) Google Scholar), 90 base pairs 5′ of the PEX3 open reading frame and 257 base pairs 3′ of the PEX3 open reading frame was generated by PCR and introduced into SGY55 by electroporation (29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). Transformants were selected on YPD plates containing 300 μg/ml G418. Replacement of thePEX3 open reading frame with the KanMX cassette was confirmed by PCR. Cells were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose), SYOLT (0.17% yeast nitrogen base without amino acids or ammonium sulfate, 0.5% ammonium sulfate, 0.05%l-histidine, 0.05% yeast extract, 0.18% oleic acid, 0.02% Tween 40), or SM (0.17% yeast nitrogen base without amino acids or ammonium sulfate, 0.5% ammonium sulfate, 0.05% l-histidine, 0.5% methanol) as described (29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar).Table IYeast strainsStrainGenotypeSource or referenceSGY551-aSGY55 was used as wild type in all experiments described here.arg4-1 his4Δ∷ARG4Crane et al.(29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar)CHPP01arg4-1 his4Δ∷ARG4 pex3Δ∷KAN rThis studySGY29arg4-1 his4Δ∷ARG4 leu2∷ARG4 pex5Δ∷LEU2Laboratory stockCYPP21arg4-1 his4Δ∷ARG4 pex17Δ∷ARG4Laboratory stock1-a SGY55 was used as wild type in all experiments described here. Open table in a new tab Antibodies to P. pastoris Pex10p and Pex12p have been described previously (30.Kalish J.E. Theda C. Morrell J.C. Berg J.M. Gould S.J. Mol. Cell. Biol. 1995; 15: 6406-6419Crossref PubMed Scopus (62) Google Scholar, 31.Kalish J.E. Keller G.A. Morrell J.C. Mihalik S.J. Smith B. Cregg J.M. Gould S.J. EMBO J. 1996; 15: 3275-3285Crossref PubMed Scopus (67) Google Scholar). Anti-Pex13p antibodies were a generous gift from D. Crane (Griffith University, Brisbane, Australia). We raised antibodies to a 19-amino acid peptide containing the COOH-terminal 15 amino acids of P. pastoris Pex3p (NH2-CKKKELNDLSASVYSNFDP-COOH). Anti-Pex17p antibodies were raised to an MBP-Pex17p fusion protein expressed in and purified fromEscherichia coli (amino acids 1–267). Whole-cell lysates were prepared as described (29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar) and detected by immunoblotting (29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). All differential centrifugation and density gradient centrifugation experiments were performed as described previously (29.Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). Assays were performed on supernatant and pellet fractions of postnuclear supernatants generated from oleic acid-induced pex17Δ, pex5Δ, and WT strains spun for 1 h at 250,000 × g.Glyceraldehyde-3-phosphate dehydrogenase activity was determined at 25 °C by increasing A 340 over 160 s. Each fraction was diluted 30-fold into the reaction buffer (13 mm sodium pyrophosphate, pH 8.5, 26 mm sodium arsenate, 25 μm NAD+, 3 mmdithiothreitol), and the reaction was started with the addition ofdl-glyceraldehyde-3-phosphate to a concentration of 500 μm. Succinate dehydrogenase activity was determined by incubating 25 μl of each lysate at 37 °C in the presence of 50 mm potassium phosphate, pH 6.8, 0.1%p-iodonitrotetrazolene, and 50 mmNa2 succinic acid for 10 min. The reactions were stopped by the addition of trichloroacetic acid to 5%. Color was developed by the addition of 1 ml of ethylene glycol monomethyl ether, and theA 440 was read. Strains were induced in SM media as described above. Yeast cells (at 24 °C) were pelleted, washed in ddH20, 3% glutaraldehyde, 100 mmcacodylate buffer, 5 mm CaCl2, and 5 mm MgCl2 for 1 h at room temperature, pH 7.4. Cells were then washed briefly in 100 mm cacodylate buffer, pH 7.4, dispersed/embedded in 2% low temperature agarose (∼1:1), cooled, and subsequently cut into small blocks (∼1 mm3). Blocks were then post-fixed in 4% KMnO4, prepared in ddH2O for 1 h at room temperature, washed thoroughly (4 times, 10 min total) in ddH2O, treated with 0.5% sodium meta-periodate for 15 min at room temperature, washed twice in ddH2O, and placed into filtered 2% uranyl acetate overnight at room temperature (in the dark). Blocks were then rapidly dehydrated through a graded series of EtOH (4 °C), followed by three washes in 100% EtOH (15 min each) and then two washes with propylene oxide, and placed into a 50:50 mixture of propylene oxide and Spurr resin. Samples were incubated overnight under vacuum and were subsequently given two changes of Spurr resin over 6–8 h and left overnight in a third change under vacuum throughout the next day. The samples were placed in beam capsules containing fresh Spurr resin, which were placed in a polymerizing oven at 80 °C for 24–48 h. 60-nm sections were cut on a Leica UCT ultramicrotome, collected onto 400-mesh nickel grids, post-stained with lead citrate (2–5 min), and observed in a Philips EM 410 at 80 kV. Previous studies have shown that defects in PMP import result in rapid turnover and low steady state levels of most PMPs (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar,15.South S.T. Sacksteder K.A. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 149: 1345-1360Crossref PubMed Scopus (119) Google Scholar, 16.Matsuzono Y. Kinoshita N. Tamura S. Shimozawa N. Hamasaki M. Ghaedi K. Wanders R.J. Suzuki Y. Kondo N. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2116-2121Crossref PubMed Scopus (186) Google Scholar, 17.Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (234) Google Scholar, 23.South S.T. Gould S.J. J. Cell Biol. 1999; 144: 255-266Crossref PubMed Scopus (188) Google Scholar, 32.Kinoshita N. Ghaedi K. Shimozawa N. Wanders R.J.A. Matsuzono Y. Imanaka T. Okumoto K. Suzuki Y. Kondo N. Fujiki Y. J. Biol. Chem. 1998; 273: 24122-24130Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To determine whether this was also true for cells lacking Pex17p, we examined the abundance of three integral PMPs in aP. pastoris pex17Δ strain: Pex3p (33.Wiemer E.A.C. Luers G.H. Faber K.N. Wenzel T. Veenhuis M. Subramani S. J. Biol. Chem. 1996; 271: 18973-18980Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), Pex10p (30.Kalish J.E. Theda C. Morrell J.C. Berg J.M. Gould S.J. Mol. Cell. Biol. 1995; 15: 6406-6419Crossref PubMed Scopus (62) Google Scholar), and Pex12p (31.Kalish J.E. Keller G.A. Morrell J.C. Mihalik S.J. Smith B. Cregg J.M. Gould S.J. EMBO J. 1996; 15: 3275-3285Crossref PubMed Scopus (67) Google Scholar). To control for the specificity of any phenotypes we might detect, we also examined the phenotypes of pex3Δ cells, which have a well established defect in PMP import (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 15.South S.T. Sacksteder K.A. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 149: 1345-1360Crossref PubMed Scopus (119) Google Scholar, 33.Wiemer E.A.C. Luers G.H. Faber K.N. Wenzel T. Veenhuis M. Subramani S. J. Biol. Chem. 1996; 271: 18973-18980Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), andpex5Δ cells, which are defective in matrix protein import but are not defective in PMP import (7.McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 9.Chang C.C. South S. Warren D. Jones J. Moser A.B. Moser H.W. Gould S.J. J. Cell Sci. 1999; 112: 1579-1590Crossref PubMed Google Scholar). These strains were grown in YPD to mid-log phase, washed, and incubated in oleate-containing medium for 18 h. Cells were then harvested and lysed, and total cellular protein was collected by trichloroacetic acid precipitation. Equal amounts of protein from each strain were separated by SDS-PAGE and blotted with anti-PMP antibodies. As previously reported for pex3Δ mutants of humans and S. cerevisiae, P. pastoris pex3Δ mutants had low steady state levels of PMPs (Fig. 1). Steady state levels of Pex3p, Pex10p, and Pex12p were also somewhat lower inpex17Δ cells than in WT cells, although they were clearly higher than in the pex3Δ strain. The levels of these three PMPs in the pex5Δ cells were the same as inpex17Δ cells. This was somewhat surprising given that all prior studies of PMP biogenesis in pex5Δ mutants have supported the hypothesis that Pex5p has no role in PMP import (7.McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 9.Chang C.C. South S. Warren D. Jones J. Moser A.B. Moser H.W. Gould S.J. J. Cell Sci. 1999; 112: 1579-1590Crossref PubMed Google Scholar, 10.Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-124Crossref PubMed Scopus (379) Google Scholar, 11.van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (201) Google Scholar, 12.van der Klei I.J. Hibrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 13.Baes M. Gressens P. Baumgart E. Carmeliet P. Casteels M. Fransen M. Evrard P. Fahimi D. Declercq P.E. Collen D. van Veldhoven P.P. Mannaerts G.P. Nat. Genet. 1997; 17: 49-57Crossref PubMed Scopus (217) Google Scholar,34.Gatto Jr., G.J. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Struct. Biol. 2000; 7: 1091-1095Crossref PubMed Scopus (290) Google Scholar). To better understand the role of Pex17p in PMP import, we next used subcellular fractionation experiments in an attempt to assess the import of PMPs in pex17Δ cells, as well as inpex3Δ, pex5Δ, and WT controls. Each strain was grown in YPD, incubated in oleate-containing medium for 18 h, and converted to spheroplasts. Cells were lysed in a Dounce homogenizer and cleared of nuclei and cell debris by low speed centrifugation. The resulting postnuclear supernatants (PNSs) were separated by centrifugation at 25,000 × g for 30 min. Equal proportions of each fraction were then separated by SDS-PAGE and blotted with antibodies to Pex3p, Pex10p, and Pex12p (Fig. 2). The levels of these PMPs in extracts prepared from pex3Δ cells were below the level of detection. In cells lacking Pex17p, ∼20–30% of the Pex10p and Pex12p and ∼50% of the Pex3p were detected in the 25kgsupernatant, which could be interpreted as evidence thatpex17Δ cells have a partial PMP import defect. However,pex5Δ cells have a similar phenotype, raising questions about whether this assay is an accurate measure of PMP import. Snyder et al. (26.Snyder W.B. Koller A. Choy A.J. Johnson M.A. Cregg J.M. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 4005-4019Crossref PubMed Scopus (51) Google Scholar) previously reported that the PMPs present in the 25,000 × gsupernatant fraction of pex17Δ mutant extracts were largely resistant to sedimentation at higher speeds (100,000 ×g). Based in part on this result, they concluded thatpex17Δ cells have a significant pool of soluble, cytosolic PMPs (26.Snyder W.B. Koller A. Choy A.J. Johnson M.A. Cregg J.M. Rangell L. Keller G.A. Subramani S. Mol. Biol. Cell. 1999; 10: 4005-4019Crossref PubMed Scopus (51) Google Scholar). However, other studies have suggested that cell homogenates contain significant levels of peroxisome-derived “microsomes” that may pellet only at higher speeds (35.Heinemann P. Just W.W. FEBS Lett. 1992; 300: 179-182Crossref PubMed Scopus (49) Google Scholar, 36.van Roermund C.W.T. van den Berg M. Wanders R.J.A. Biochim. Biophys. Acta. 1995; 1245: 348-358Crossref PubMed Scopus (24) Google Scholar, 37.Titorenko V.I. Chan H. Rachubinski R.A. J. Cell Biol. 2000; 148: 29-44Crossref PubMed Scopus (120) Google Scholar, 38.Titorenko V.I. Rachubinski R.A. J. Cell Biol. 2000; 150: 881-886Crossref PubMed Scopus (94) Google Scholar). Postnuclear supernatants were again generated from oleic acid-induced pex17Δ,pex5Δ, and WT strains and spun for 1 h at 250,000 × g. Enzymatic assays of glyceraldehyde-3-phosphate dehydrogenase and succinate dehydrogenase, which are cytosolic and mitochondrial enzymes, respectively, demonstrate that these conditions are sufficient to pellet organelles but not soluble proteins. Equal proportions of each fraction were then separated by SDS-PAGE, transferred to membranes, and probed with antibodies to Pex3p, Pex10p, and Pex12p (Fig. 3 A). Under these conditions, all three PMPs were found primarily in the pellet fraction ofpex17Δ cells. Similar results were observed for thepex5Δ mutant. Thus, it appears that PMPs present in the 25,000 × g supernatant of pex17Δ andpex5Δ mutants do not represent pools of soluble, cytosolic PMPs. The sedimentation of Pex3p, Pex10p, and Pex12p from pex17Δ and pex5Δ lysates could reflect the insertion of these proteins into peroxisomal membranes. However, their sedimentation behavior could also reflect a more complicated situation. For example, a portion of each PMP may be properly inserted into peroxisome membranes, whereas other subsets of these PMPs may be only peripherally associated with membranes or may exist in large protein aggregates. To determine the proportion of each PMP that was inserted into the peroxisome membrane in each cell type, we incubated the PNSs frompex17Δ, pex5Δ, and WT strains with 0.1m Na2CO3, pH 11.5 (39.Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1374) Google Scholar), to extract non-integral proteins from cellular membranes. Integral membrane proteins were then collected in the pellet fraction by centrifugation at 250,000 × g for 1 h. Equal proportions of each fraction were then processed for immunoblot using antibodies to Pex3p, Pex10p, and Pex12p (Fig. 3 B). In both thepex17Δ and pex5Δ mutants, ∼50% of their PMPs were released to the supernatant by carbonate extraction. The amounts of PMPs released from WT peroxisome membranes were similar to the amounts of PMPs released from pex17Δ andpex5Δ mutants, although they corresponded to only 10% of the total PMPs in WT cells as opposed to 50% of the PMPs in these two mutants. As part of our basic characterization of Pex17p, we examined its abundance in an array of P. pastoris pex mutants. We generated antibodies to recombinant Pex17p. These antibodies recognize a protein of ∼25 kDa (the predicted molecular mass of Pex17p is 30,497 Da) inP. pastoris cell extracts. The protein recognized by these antibodies is absent from pex17Δ cells, colocalizes with peroxisomes in density gradient centrifugation experiments, and behaves as an integral PMP, indicating that these antibodies are specific for Pex17p (Fig. 4). These antibodies were then used to assess the abundance of Pex17p in the pex1,pex2, pex3, pex4, pex5,pex6, pex8, pex10, pex12,pex14, pex17, and pex22 mutants (Fig. 5). Whole cell lysates were generated by alkaline lysis, and equal amounts of protein from each strain were separated by SDS-PAGE, transferred to membranes, and probed with anti-Pex17p antibodies. As a control, we determined the levels of Pex13p in these same samples. As expected, Pex17p was absent from thepex17Δ mutant. Levels were also reduced in thepex3Δ mutant, consistent with the rapid degradation of all known PMPs in these cells (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 15.South S.T. Sacksteder K.A. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 149: 1345-1360Crossref PubMed Scopus (119) Google Scholar, 32.Kinoshita N. Ghaedi K. Shimozawa N. Wanders R.J.A. Matsuzono Y. Imanaka T. Okumoto K. Suzuki Y. Kondo N. Fujiki Y. J. Biol. Chem. 1998; 273: 24122-24130Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) (see Fig. 1). However, we observed that Pex17p was also undetectable in the P. pastoris pex14 mutant, and it is interesting to note that thepex14 mutants of P. pastoris (40.Johnson M.A. Snyder W.B. Cereghino J.L. Veenhuis M. Subramani S. Cregg J.M. Yeast. 2001; 18: 621-641Crossref PubMed Scopus (43) Google Scholar), S. cerevisiae (8.Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (222) Google Scholar, 41.Albertini 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 (265) Google Scholar), and mammalian cells (42.Shimizu N. Itoh R. Hirono Y. Otera H. Ghaedi K. Tateishi K. Tamura S. Okumoto K. Harano T. Mukai S. Fujiki Y. J. Biol. Chem. 1999; 274: 12593-12604Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) have all been reported to import PMPs normally. Pex17p abundance was slightly reduced in cells lacking Pex13p, another Pex14p-binding protein (40.Johnson M.A. Snyder W.B. Cereghino J.L. Veenhuis M. Subramani S. Cregg J.M. Yeast. 2001; 18: 621-641Crossref PubMed Scopus (43) Google Scholar, 41.Albertini 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 (265) Google Scholar, 43.Girzalsky W. Rehling P. Stein K. Kipper J. Blank L. Kunau W.H. Erdmann R. J. Cell Biol. 1999; 144: 1151-1162Crossref PubMed Scopus (147) Google Scholar). Equal loading and transfer of the pex14 sample can be deduced from the fact that Pex13p levels were normal in this strain.Figure 5Pex17p is undetectable inpex14 − cells. Whole-cell protein extracts were generated from P. pastoris pexmutants. Equal amounts of protein from each sample were separated by SDS-PAGE and analyzed by Western blot. Pex17p is absent frompex17Δ cells, is found at low levels in pex3Δ cells, and is absent from pex14 − cells. Antibodies against Pex13p were used as a lo" @default.
• W2136590099 created "2016-06-24" @default.
• W2136590099 creator A5003907612 @default.
• W2136590099 creator A5005551232 @default.
• W2136590099 creator A5020715530 @default.
• W2136590099 creator A5042584066 @default.
• W2136590099 date "2002-05-01" @default.
• W2136590099 modified "2023-10-10" @default.
• W2136590099 title "Peroxisomal Membrane Protein Import Does Not Require Pex17p" @default.
• W2136590099 cites W1570204140 @default.
• W2136590099 cites W171263212 @default.
• W2136590099 cites W1851584592 @default.
• W2136590099 cites W1877409741 @default.
• W2136590099 cites W1920423370 @default.
• W2136590099 cites W1964697500 @default.
• W2136590099 cites W1967503648 @default.
• W2136590099 cites W1974662072 @default.
• W2136590099 cites W2006120283 @default.
• W2136590099 cites W2011100117 @default.
• W2136590099 cites W2014664955 @default.
• W2136590099 cites W2064948539 @default.
• W2136590099 cites W2069547574 @default.
• W2136590099 cites W2071925175 @default.
• W2136590099 cites W2082926317 @default.
• W2136590099 cites W2094732318 @default.
• W2136590099 cites W2101378834 @default.
• W2136590099 cites W2103186456 @default.
• W2136590099 cites W2106462570 @default.
• W2136590099 cites W2111781163 @default.
• W2136590099 cites W2111864062 @default.
• W2136590099 cites W2112798765 @default.
• W2136590099 cites W2114625295 @default.
• W2136590099 cites W2116488364 @default.
• W2136590099 cites W2116652387 @default.
• W2136590099 cites W2118176667 @default.
• W2136590099 cites W2124967428 @default.
• W2136590099 cites W2127006597 @default.
• W2136590099 cites W2130350654 @default.
• W2136590099 cites W2131374243 @default.
• W2136590099 cites W2131521418 @default.
• W2136590099 cites W2131709970 @default.
• W2136590099 cites W2132262455 @default.
• W2136590099 cites W2132778321 @default.
• W2136590099 cites W2136612214 @default.
• W2136590099 cites W2144973878 @default.
• W2136590099 cites W2146503498 @default.
• W2136590099 cites W2152015543 @default.
• W2136590099 cites W2152105168 @default.
• W2136590099 cites W2160107586 @default.
• W2136590099 cites W2168101937 @default.
• W2136590099 cites W2170727893 @default.
• W2136590099 cites W2290606973 @default.
• W2136590099 cites W4231809167 @default.
• W2136590099 doi "https://doi.org/10.1074/jbc.m111728200" @default.
• W2136590099 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11859077" @default.
• W2136590099 hasPublicationYear "2002" @default.
• W2136590099 type Work @default.
• W2136590099 sameAs 2136590099 @default.
• W2136590099 citedByCount "18" @default.
• W2136590099 countsByYear W21365900992013 @default.
• W2136590099 crossrefType "journal-article" @default.
• W2136590099 hasAuthorship W2136590099A5003907612 @default.
• W2136590099 hasAuthorship W2136590099A5005551232 @default.
• W2136590099 hasAuthorship W2136590099A5020715530 @default.
• W2136590099 hasAuthorship W2136590099A5042584066 @default.
• W2136590099 hasBestOaLocation W21365900991 @default.
• W2136590099 hasConcept C12554922 @default.
• W2136590099 hasConcept C127078168 @default.
• W2136590099 hasConcept C170493617 @default.
• W2136590099 hasConcept C185592680 @default.
• W2136590099 hasConcept C41625074 @default.
• W2136590099 hasConcept C55493867 @default.
• W2136590099 hasConcept C86803240 @default.
• W2136590099 hasConcept C95444343 @default.
• W2136590099 hasConceptScore W2136590099C12554922 @default.
• W2136590099 hasConceptScore W2136590099C127078168 @default.
• W2136590099 hasConceptScore W2136590099C170493617 @default.
• W2136590099 hasConceptScore W2136590099C185592680 @default.
• W2136590099 hasConceptScore W2136590099C41625074 @default.
• W2136590099 hasConceptScore W2136590099C55493867 @default.
• W2136590099 hasConceptScore W2136590099C86803240 @default.
• W2136590099 hasConceptScore W2136590099C95444343 @default.
• W2136590099 hasIssue "19" @default.
• W2136590099 hasLocation W21365900991 @default.
• W2136590099 hasOpenAccess W2136590099 @default.
• W2136590099 hasPrimaryLocation W21365900991 @default.
• W2136590099 hasRelatedWork W1964807379 @default.
• W2136590099 hasRelatedWork W1987794894 @default.
• W2136590099 hasRelatedWork W2028253379 @default.
• W2136590099 hasRelatedWork W2104937747 @default.
• W2136590099 hasRelatedWork W2157864057 @default.
• W2136590099 hasRelatedWork W2399030784 @default.
• W2136590099 hasRelatedWork W2893110889 @default.
• W2136590099 hasRelatedWork W2898957604 @default.
• W2136590099 hasRelatedWork W3000690042 @default.
• W2136590099 hasRelatedWork W3010358308 @default.
• W2136590099 hasVolume "277" @default.
• W2136590099 isParatext "false" @default.

• next