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• W1976702924 abstract "Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence. Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence. The maintenance of peroxisome function depends on the formation of the peroxisomal membrane and the subsequent import of both membrane and matrix proteins. The matrix protein import occurs in a post-translational manner (1Lazarow P.B. Fujiki Y. Annu. Rev. Cell Biol. 1985; 1: 489-530Crossref PubMed Scopus (887) Google Scholar) and uses either one of the two well characterized peroxisomal targeting signals, PTS1 or PTS2, that are recognized and bound by specific receptor proteins Pex5p and Pex7p, respectively. Subsequently, the receptor-cargo complexes dock to distinct proteins accessible at the cis-side of the peroxisomal membrane. Pex13p and Pex14p physically bind both import receptors Pex5p and Pex7p, whereas Pex14p also interacts with Pex17p (2Albertini 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 (268) Google Scholar, 3Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (108) Google Scholar, 4Elgersma 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 (185) Google Scholar, 5Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (185) Google Scholar, 6Gould 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 (211) Google Scholar, 7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar, 8Shimizu 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 (144) Google Scholar, 9Snyder 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). These three peroxins are thought to form the receptor-docking complex (for review, see Refs. 10Lazarow P.B. Curr. Opin. Cell Biol. 2003; 15: 489-497Crossref PubMed Scopus (111) Google Scholar, 11Brown L.A. Baker A. J. Cell. Mol. Med. 2003; 7: 388-400Crossref PubMed Scopus (56) Google Scholar, 12Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 2000: 399-418Crossref Scopus (200) Google Scholar), being responsible for the initial binding of the receptor-cargo complex at the peroxisomal membrane. According to the widely accepted model of cycling receptors, the receptor-cargo complex is supposed to dissociate at the peroxisomal membrane in an entirely unknown manner, resulting in the sorting of the cargo protein to the peroxisomal matrix and the release of free receptors to the cytosol, where they are available for another round of import (13Dodt G. Gould S.J. J. Cell Biol. 1996; 135: 1763-1774Crossref PubMed Scopus (265) Google Scholar, 14Marzioch M. Erdmann R. Veenhuis M. Kunau W.-H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar). In contrast to the increasing knowledge on the peroxisomal matrix protein import, our understanding of the topogenesis of peroxisomal membrane proteins (PMPs) 2The abbreviations used are: PMP, peroxisomal membrane protein; MBP, maltose-binding protein; GFP, green fluorescent protein; ProtA, protein A; TEV, tobacco etch virus. is rather scarce. Most of the mutants lacking one of the known peroxins are characterized by a severe defect in matrix protein import, whereas the targeting of peroxisomal membrane proteins is not affected, indicated by the presence of peroxisomal remnant structures that contain a number of PMPs but no or only residual amounts of matrix proteins (15Santos M.J. Imanaka T. Shio H. Small G.M. Lazarow P.B. Science. 1988; 239: 1536-1538Crossref PubMed Scopus (242) Google Scholar). These and other findings led to the conclusion that PMP targeting occurs independently of the import of peroxisomal matrix proteins (5Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (185) Google Scholar, 6Gould 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 (211) Google Scholar). Among the 32 known proteins involved in peroxisome biogenesis, only Pex3p, Pex16p, and Pex19p are required for the biogenesis of the peroxisomal membrane. Cells lacking either a functional copy of PEX3, PEX16,or PEX19 contain neither peroxisomes nor obvious peroxisomal remnants (for review, see Ref. 16Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google Scholar), and the PMPs are rapidly degraded or mislocalized to other locations such as the endoplasmic reticulum and mitochondria (17Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (223) Google Scholar, 18Ghaedi K. Tamura S. Okumoto K. Matsuzono Y. Fujiki Y. Mol. Biol. Cell. 2000; 11: 2085-2103Crossref PubMed Scopus (97) Google Scholar, 19Baerends R.J.S. Rasmussen S.W. Hilbrands R.E. van der Heide M. Faber K.N. Reuvekamp P.T.W. Kiel J.A.K.W. Cregg J.M. van der Klei I.J. Veenhuis M. J. Biol. Chem. 1996; 271: 8887-8894Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Elgersma Y. Kwast L. van den Berg M. Snyder W.B. Distel B. Subramani S. Tabak H.F. EMBO J. 1997; 16: 7326-7341Crossref PubMed Scopus (189) Google Scholar, 21Pause B. Saffrich R. Hunziker A. Ansorge W. Just W.W. FEBS Lett. 2000; 471: 23-28Crossref PubMed Scopus (38) Google Scholar, 22Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (242) Google Scholar, 23Baerends R.J. Faber K.N. Kram A.M. Kiel J.A. van der Klei I.J. Veenhuis M. J. Biol. Chem. 2000; 275: 9986-9995Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 24Fransen M. Vastiau I. Brees C. Brys V. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2004; 279: 12615-12624Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 25Fang Y. Morrell J.C. Jones J.M. Gould S.J. J. Cell Biol. 2004; 164: 863-875Crossref PubMed Scopus (174) Google Scholar). The observation that most PMPs are recognized by Pex19p triggered the idea that this predominantly cytosolic protein might function as a chaperone and/or import receptor for peroxisomal membrane proteins (17Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (223) Google Scholar, 22Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (242) Google Scholar, 24Fransen M. Vastiau I. Brees C. Brys V. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2004; 279: 12615-12624Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 25Fang Y. Morrell J.C. Jones J.M. Gould S.J. J. Cell Biol. 2004; 164: 863-875Crossref PubMed Scopus (174) Google Scholar). This, however, is still a matter of debate (26Schliebs W. Kunau W.H. Curr. Biol. 2004; 14: R397-R399Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). PMPs use neither one of the two targeting signals, PTS1 and PTS2, that direct proteins to the peroxisomal matrix (for review, see Ref. 16Sparkes I.A. Baker A. Mol. Membr. Biol. 2002; 19: 171-185Crossref PubMed Scopus (34) Google Scholar). The targeting signal for peroxisomal membrane proteins (mPTS) has been identified for several proteins, including Pmp47p of Candida boidini, Pmp34p, Pmp22p from mammals, and Pex3p from various species (21Pause B. Saffrich R. Hunziker A. Ansorge W. Just W.W. FEBS Lett. 2000; 471: 23-28Crossref PubMed Scopus (38) Google Scholar, 27Dyer J.M. McNew J.A. Goodman J.M. J. Cell Biol. 1996; 133: 269-280Crossref PubMed Scopus (145) Google Scholar). In support of an import receptor function of Pex19p, most PMPs contain conserved binding sites for Pex19p that share a common motif that is essential and in conjunction with a transmembrane segment sufficient for peroxisomal targeting and membrane insertion (28Rottensteiner H. Kramer A. Lorenzen S. Stein K. Landgraf C. Volkmer-Engert R. Erdmann R. Mol. Biol. Cell. 2004; 7: 3406-3417Crossref Scopus (148) Google Scholar). Here we have analyzed the peroxisomal targeting and membrane association of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have defined the binding regions of Pex17p for other peroxins and demonstrated that the peroxisomal targeting and stability of Pex17p requires Pex19p as a chaperone or import receptor as well as Pex14p for a stable anchoring at the peroxisomal membrane. Strains and Culture Conditions—Saccharomyces cerevisiae strains used in this study are listed in Table 1. Strains in which the genomic copy of PEX17 contains epitope tags were generated as described by (29Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (813) Google Scholar) using primers KU1007 and KU1008 for PCR reactions. Transformants were selected for the appropriate marker and proper integration was confirmed by PCR. Localization of GFP-fusion proteins was analyzed in strain yHPR251, which expresses PTS2-DsRed from an integrated plasmid in strain UTL-7A (30Stein K. Schell-Steven A. Erdmann R. Rottensteiner H. Mol. Cell. Biol. 2002; 22: 6059-6069Crossref Scopus (96) Google Scholar). Yeast complete (YPD) and minimal media (S.D.) have been described previously (31Erdmann R. Veenhuis M. Mertens D. Kunau W.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (263) Google Scholar). YNO medium contained 0.1% oleic acid, 0.05% Tween 40, 0.1% yeast extract and 0.67% yeast nitrogen base without amino acids, adjusted to pH 6.0. When necessary, auxotrophic requirements were added according to (32Ausubel F.J. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1992Google Scholar).TABLE 1S. cerevisiae strains usedS. cerevisiae strainDescriptionSource or referenceUTL-7AMATα leu2-3, 112 ura3-52 trp131pex17ΔMATα, leu2-3, 112 ura3-52 trp1, pex17::LEU27UTL-7A-Pex17pProtAMATα leu2-3, 112 ura3-52 trp1, PEX17-TEV-ProtA-kanMX6This studypex12Δ-Pex17pProtAMATα, leu2-3, 112 ura3-52 trp1, pex12::LEU, PEX17-TEV-ProtA-kanMX6This studypex3Δ-Pex17pProtAMATα, leu2-3, 112 ura3-52 trp1, pex3::LEU, PEX17-TEV-ProtA-kanMX6This studypex19Δ-Pex17pProtAMATα, leu2-3, 112 ura3-52 trp1, pex19::LEU, PEX17-TEV-ProtA-kanMX6This studyPCY2MATα, gal4Δ, gal80Δ, URA3::GAL1-lacZ, lys2-801amber, his3-Δ200, trp1-Δ63, leu2 ade2-101ochre35 Open table in a new tab Oligonucleotides and Plasmids—The plasmids and oligonucleotides used are listed in Tables 2 and 3. To generate a maltose-binding protein-Pex17p fusion, the coding region of PEX17 was amplified by PCR using primers KU129 and KU130 as well as pRSPEX17 (7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar) as the template. The PCR product was digested with EcoRI/NotI and introduced into an EcoRI/NotI-digested pMal-C2 (New England Biolabs) resulting in plasmid pMal-PEX17. For the construction of Pex17pI80P, the mutation was introduced by PCR using gene splicing by overlap extension (33Yon J. Fried M. Nucleic Acids Res. 1989; 17: 4895Crossref PubMed Scopus (240) Google Scholar) with primers RE918/RE919 and pRSPEX17 as the template. For GFP fusion, mutated or wild-type PEX17 was amplified by PCR with primers RE1006/RE1007. The PEX17- and PEX17I80P-PCR products were digested with BamHI/EcoRI and subcloned into pUG36, resulting in plasmids pLH8 and pLH9, respectively.TABLE 2Plasmids usedPlasmidDescriptionSource or referenceOligonucleotidespPC97-PEX17GAL4-PEX17-(1-199)7pPC97-PEX17IIGAL4-PEX17-(1-167)This studyKU129/KU133pPC97-PEX17IIIGAL4-PEX17-(1-125)This studyKU129/KU132pPC97-PEX17IVGAL4-PEX17-(1-88)This studyKU129/KU131pPC97-PEX17VGAL4-PEX17-(1-51)This studyKU129/KU130pPC97-PEX17VIGAL4-PEX17-(52-199)This studyKU126/KU130pPC97-PEX17VIIGAL4-PEX17-(89-199)This studyKU127/KU130pPC97-PEX17VIIIGAL4-PEX17-(126-199)This studyKU128/KU133pPC97-PEX17IXGAL4-PEX17-(52-125)This studyKU126/KU132pPC97-PEX17XGAL4-PEX17-(52-88)This studyKU126/KU133pPC97-PEX17XIGAL4-PEX17-(89-125)This studyKU127/KU132pPC97-PEX17XIIGAL4-PEX17-(145-199)This studyKU183/KU130pPC97-PEX17XIIIGAL4-PEX17-(167-199)This studyKU184/KU130pPC86-PEX19GAL4-PEX1956pPC86-PEX14GAL4-PEX142pMal-PEX17MBP-PEX17This studyKU129/KU130pHL4GFP-PEX17-(52-88)This studyRE920/RE921pHL7GAL4-PEX17180PThis studyKU129/KU130pLH8GFP-PEX17This studyRE1006/RE1007pLH9GFP-PEX17I80PThis studyRE1006/RE1007pLH10GFP-PEX17-(52-88/167-199)This studyRE920/1007/1084/1085pLH12GFP-PEX17-(167-199)This studyRE920/RE1007 Open table in a new tab TABLE 3Oligonucleotides usedOligonucleotideKU1265′-CCGGAATTCAGATGTTTGTTGAACCAACG-3′KU1275′-CCGGAATTCAGATGACCCCAGTATCGTCG-3′KU1285′-CCGGAATTCAGATGATTTATCAACTGCAA-3′KU1295′-CCGGAATTCAGATGACATCGATTAACAGT-3′KU1305′-CGGGATCCGAGCTCTTACCTTGGCACTTGGCCATT-3′KU1315′-CGGGATCCGAGCTCTTAAGCAATAACAAAATATAG-3′KU1325′-CGGGATCCGAGCTCTTACACCAGACGTTTTTGCAA-3′KU1335′-CGGGATCCGAGCTCTTATTCAGCCCAATGGCTATT-3′KU1835′-CCGGAATTCAGATGGGGCCAACCATCTGAAAGT-3′KU1845′-CCGGAATTCAGATGTTAACAGATAGGTCCCGA-3′KU10075′-AGAAATCAAAGGTTGGTTTGTGAATGGCCAAGTGCCA AGGCGTACGCTGCAGGTCGAC-3′KU10085′-CACTAGAGCGTTTTAAATTCAATGCTATTATTTTTGA TTGATCGATGAATTCGAGCTCG-3′RE9185′-AGAAGAATCCCAGCGCAATTG-3′RE9195′-CAATTGCGCTGGGATTCTTCT-3′RE10065′-GCACTAGTGGATCCATGACATCGATTAACAGT-3′RE10075′-GCGAATTCCTGCAGTTACCTTGGCACTTGGCC-3′RE10845′-AAACGACTGGTGTTAACAGATAGG-3′RE10855′-CCTATCTGTTAACACCAGTCGTTT-3′ Open table in a new tab Yeast Cell Extracts—Yeast cells were grown on 0.3% SD medium to late log phase and subsequently for 15 h in YNOD (0.1% dextrose, 0.1% oleic acid, 0.05% Tween 40, 0.1% yeast extract, and 0.67% yeast nitrogen base). Cells were harvested and aliquots of 30 mg of cells were resuspended in 300 μlof potassium-phosphate buffer, pH 7.4, containing 20% trichloroacetic acid. The samples were frozen at -80 °C for at least 30 min. Samples were sedimented, washed twice with ice-cold 50% acetone, and resuspended in 80 μl of 10% SDS/0.1 m NaOH and 20 μl of SDS loading buffer (5% β-mercaptoethanol, 15% glycerol, 0.01% bromphenol blue). Two-hybrid Analysis—The two-hybrid assay was based on the method of Fields and Song (34Fields S. Song O.K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar). Open reading frames were fused to the DNA-binding domain or trans-activating domain of GAL4 in the vectors pPC86 and pPC97 (35Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (480) Google Scholar). To generate PEX17 and PEX17I80P constructs in pPC97, PEX17 fragments were amplified by PCR using pPC97-PEX17 (7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar) or pLH9 as a template. The PCR fragments were first cloned into EcoRI/SacI-prepared pPC86 and subsequently into SalI/SacI-digested pPC97. Co-transformation of two-hybrid vectors into strain PCY2 was performed according to Gietz and Woods (36Gietz R.D. Woods R.A. Johnston J.A. Molecular Genetics of Yeast: Practical Approaches. Oxford University Press, Oxford1994: 121-134Google Scholar). Transformed yeast cells were plated onto SD medium without tryptophan and leucine. β-Galactosidase filter assays were performed according to Rehling et al. (37Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W.-H. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar). In Vitro Binding Assays—For in vitro binding of Pex17p to Pex14p, the maltose-binding protein (MBP) and MBP-Pex17p of soluble fractions obtained from pMal-C2- and pMal-PEX17-transformed Escherichia coli Bl21, respectively, were bound for 2 h at 4°C to amylose resin (New England Biolabs). After washing of the matrix with buffer A (50 mm NaCl, 50 mm HEPES, pH 7.4), the soluble fractions of bacteria-expressed Pex14pHis6 were added and incubated with the matrix for 2 h at 4°C with gentle rotation. After washing the matrix with buffer A, bound proteins were eluted with 10 mm maltose in 50 mm Tris-HCl, pH 7.4. The eluted samples were analyzed by immunoblot analysis. Antibodies and Western Blotting—Anti-Pex3p, -Pex5p, -Pex10p, -Pex12p, -Pex13p, -Pex14p, -Pex17p, and -Fbp1p (fructose-1,6-bisphosphatase) have been described previously (2Albertini 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 (268) Google Scholar, 7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar, 38Albertini M. Girzalsky W. Veenhuis M. Kunau W.-H. Eur. J. Cell Biol. 2001; 80: 257-270Crossref PubMed Scopus (52) Google Scholar, 39Girzalsky W. Rehling P. Stein K. Kipper J. Blank L. Kunau W.-H. Erdmann R. J. Cell Biol. 1999; 144: 1151-1162Crossref PubMed Scopus (149) Google Scholar, 40Höhfeld J. Veenhuis M. Kunau W.-H. J. Cell Biol. 1991; 114: 1167-1178Crossref PubMed Scopus (175) Google Scholar, 41Entian K.D. Vogel R.F. Rose M. Hofmann L. Mecke D. FEBS Lett. 1988; 236: 195-200Crossref PubMed Scopus (28) Google Scholar). Anti-rabbit IgG-coupled horseradish peroxidase (Amersham Biosciences) was used as the secondary antibody. Immunoreactive complexes were visualized using anti-rabbit IgG-coupled horseradish peroxidase in combination with the ECL™ system from Amersham Biosciences. Miscellaneous Methods—Analysis of live cells for DsRed and GFP fluorescence was performed with a Zeiss Axioplan microscope and Axio-Vision version 4.1 software (Zeiss, Jena, Germany). Immunopurification of native complexes using hsIgG-coupled Sepharose was performed as described previously (42Agne B. Meindl N.M. Niederhoff K. Einwächter H. Rehling P. Sickmann A. Meyer H.E. Girzalsky W. Kunau W.H. Mol. Cell. 2003; 11: 635-646Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Genomically Tagged Pex17p-Protein A (ProtA) Is Functional—To isolate and characterize Pex17p, we constructed strains expressing this peroxin fused to two IgG-binding domains derived from Staphylococcus aureus ProtA, with a cleavage site for the tobacco etch virus (TEV) protease inserted between Pex17p and the ProtA tag (29Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (813) Google Scholar). The corresponding wild-type strain was initially tested for growth on plates containing oleic acid as the sole carbon source, which will support cell growth only if peroxisomal β-oxidation is functional. The pex17Δ strain was unable to grow on this medium in line with previous findings (7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar) and typical for peroxisomal mutant strains of S. cerevisiae (31Erdmann R. Veenhuis M. Mertens D. Kunau W.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (263) Google Scholar). The genomically tagged wild-type strain expressing Pex17p-ProtA grew at a rate indistinguishable from the corresponding non-tagged wild type (Fig. 1A). These data are corroborated by electron microscopic analysis of the ultrastructure of the cells. pex17Δ cells are characterized by the mislocalization of peroxisomal matrix proteins to the cytosol, which leads to the absence of morphologically detectable peroxisomes, whereas the strain harboring the ProtA-tagged Pex17p was indistinguishable from the wild-type cells (Fig. 1B). Thus, the genomic tagging did neither interfere with the growth behavior of the cells on oleic acid medium nor with the morphological appearance of peroxisomes, indicating that the TEV-ProtA-tagged Pex17p is functional in vivo. Steady State Level of Pex17p in pex Mutants—Previous studies have demonstrated that, in cells lacking either Pex3p or Pex19p, the levels of integral peroxisomal membrane proteins that rely on these PMPs are strongly reduced (17Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (223) Google Scholar, 43Otzen M. Perband U. Wang D. Baerends R.J. Kunau W.H. Veenhuis M. van der Klei I.J. J. Biol. Chem. 2004; 279: 19181-19190Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 44Hazra P.P. Suriapranata I. Snyder W.B. Subramani S. Traffic. 2002; 3: 560-574Crossref PubMed Scopus (102) Google Scholar). To investigate whether the membrane-associated Pex17p is also subjected to a rapid turnover in these mutant cells, we determined the steady-state level of Pex17p-ProtA in pex3Δ and pex19Δ as well as pex12Δ cells, a mutant strain which is defective only in matrix protein import, whereas the membrane protein transport remains unaffected (38Albertini M. Girzalsky W. Veenhuis M. Kunau W.-H. Eur. J. Cell Biol. 2001; 80: 257-270Crossref PubMed Scopus (52) Google Scholar). Yeast strains were grown on oleic acid-containing medium, and cell lysates were subjected to SDS-PAGE and immunoblot analysis. Samples were probed for the presence of Pex17p, its binding partner Pex14p (2Albertini 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 (268) Google Scholar, 7Huhse B. Rehling P. Albertini M. Blank L. Meller K. Kunau W.-H. J. Cell Biol. 1998; 140: 49-60Crossref PubMed Scopus (126) Google Scholar, 9Snyder 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, 45Johnson M.A. Snyder W.B. Lin Cereghino J. Veenhuis M. Subramani S. Cregg J.M. Yeast. 2001; 18: 621-641Crossref PubMed Scopus (44) Google Scholar), as well as for cytosolic Fbp1p (41Entian K.D. Vogel R.F. Rose M. Hofmann L. Mecke D. FEBS Lett. 1988; 236: 195-200Crossref PubMed Scopus (28) Google Scholar) as an internal control (Fig. 2A). In pex12Δ cells, the Pex17p-ProtA steady-state level was the same as in the wild-type cells. Remarkably, the protein abundance of Pex17p was drastically decreased in strains deficient in either PEX3 or PEX19, whereas the steady-state level of Pex14p, a membrane-associated protein (2Albertini 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 (268) Google Scholar, 3Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (108) Google Scholar), was not affected (Fig. 2A). Thus, the lower Pex17p concentration is not due to a lack of Pex14p, the deficiency of which has been demonstrated to cause Pex17p instability in Pichia pastoris (46Harper C.C. South S. McCaffery J.M. Gould S.J. J. Biol. Chem. 2002; 277: 16498-16504Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). However, the instability of Pex17p in these mutants reflects the behavior of an integral membrane protein. As there is no doubt that Pex17p is a peripheral membrane protein that associates with the peroxisomal membrane by binding to Pex14p, these data establish that not only the stability of integral PMPs but also of the membrane-associated Pex17p depends on proteins, which have been shown to be essential for proper targeting of integral peroxisomal membrane proteins. Both Pex14p and Pex19p Are Part of Pex17p Complexes—The reduced level of Pex17p in cells lacking Pex19p tempted us to analyze whether both proteins are associated in vivo, as previously reported for P. pastoris (9Snyder 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). We performed IgG affinity chromatography with the ProtA-tagged Pex17p as bait to identify associated protein components. Total cell membranes were solubilized with digitonin and subjected to affinity purification. Pex17p and bound proteins were eluted by cleavage with the TEV protease. Wild-type cells expressing no ProtA fusion served as the control. Aliquots of the TEV protease eluates, representing equal amounts of total membranes from each strain, were separated by SDS-PAGE and subjected to Western blot analysis (Fig. 2B). Pex13p and Pex14p, two docking complex constituents (12Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 2000: 399-418Crossref Scopus (200) Google Scholar, 47Holroyd C. Erdmann R. FEBS Lett. 2001; 501: 6-10Crossref PubMed Scopus (66) Google Scholar, 48Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (82) Google Scholar, 49Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar), the PTS1 receptor Pex5p as well as minor amounts of the proposed PMP receptor Pex19p were present in the eluate from cells harboring the tagged Pex17p. The specificity of the isolation procedure is documented by the absence of the prominent peroxisomal membrane protein Pex11p (50Erdmann R. Blobel G. J. Cell Biol. 1995; 128: 509-523Crossref PubMed Scopus (233) Google Scholar, 51Marshall P.A. Krimkevich Y.I. Lark R.H. Deyer J.M. Veenhuis M. Goodman J.M. J. Cell Biol. 1995; 129: 345-355Crossref PubMed Scopus (174) Google Scholar) and the mitochondrial proteins Tom40p and Tim22p (52Rehling P. Wiedemann N. Pfanner N. Truscott K.N. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 291-336Crossref PubMed Scopus (64) Google Scholar) in the eluates and the complete depletion of Pex17p-ProtA from the detergent extract upon incubation with IgG-Sepharose. These data corroborate that Pex17p belongs to the docking complex of the peroxisomal protein import machinery. To determine whether the observed Pex17p interactions with Pex14p are mediated by one of the components identified in the in vivo complex or whether they represent direct protein/protein interactions, in vitro binding assays were performed. MBP and chimeric MBP-Pex17p were immobilized on amylose resin and incubated with lysates containing Pex14p fused to a hexahistidyl tag. As shown in Fig. 3, Pex14pHis6 only bound to the column when the matrices were loaded with MBP-Pex17p, whereas binding to the MBP control was not observed. Interestingly, the same was true for recombinant GST-Pex19p, which was exclusively recovered from the column loaded with MBP-Pex17p, although the amount of Pex19p bound to Pex17p was only just above the detection level (data not shown). Nevertheless, the data support the idea of a specific interaction of Pex17p and Pex19p. In line with this assumption, a small but significant amount of Pex19p was pulled down by Pex17p in vivo. From these findings, we conclude that Pex17p interacts with both Pex14p and Pex19p in a direct manner. Distinct Binding Sites of Pex17p for Pex14p and Pex19p—We used the two-hybrid system to identify the regions within Pex17p that contribute to the association with Pex14p and Pex19p. Sets of full-length Pex14p or Pex19p fusions with the Gal4p activation doma" @default.
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• W1976702924 title "Pex19p-dependent Targeting of Pex17p, a Peripheral Component of the Peroxisomal Protein Import Machinery" @default.
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