FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2085967013> ?p ?o ?g. }

• W2085967013 endingPage "35578" @default.
• W2085967013 startingPage "35571" @default.
• W2085967013 abstract "Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14Δ cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane. Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14Δ cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane. Peroxisomal matrix proteins are imported post-translationally across the peroxisomal membrane as folded or even oligomeric proteins (1Brown L.A. Baker A. J. Cell. Mol. Med. 2003; 7: 388-400Crossref PubMed Scopus (55) Google Scholar, 2Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (81) Google Scholar). The identities of the proteins collectively called peroxins (which are required for this process) are known, but their precise roles are not well characterized (2Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (81) Google Scholar). Current evidence favors a cycling receptor model for this process (3Kunau W. Curr. Biol. 2001; 11: R659-R662Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 4Dammai V. Subramani S. Cell. 2001; 105: 187-196Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 5Nair D.M. Purdue P.E. Lazarow P.B. J. Cell Biol. 2004; 167: 599-604Crossref PubMed Scopus (100) Google Scholar). Two soluble import receptors, Pex5p and Pex7p, bind their cognate peroxisomal targeting signals (PTSs) 3The abbreviations used are: PTSperoxisomal targeting signalGSTglutathione S-transferaseNTAnitrilotriacetic acidMES4-morpholineethanesulfonic acidX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. 3The abbreviations used are: PTSperoxisomal targeting signalGSTglutathione S-transferaseNTAnitrilotriacetic acidMES4-morpholineethanesulfonic acidX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. in the cytosol and then shuttle to peroxisomes, where the PTS-containing proteins are imported. After releasing their cargo, the receptors recycle to the cytosol for additional rounds of import. Most peroxisomal matrix proteins possess one of two evolutionarily conserved PTSs, the C-terminal PTS1 or the N-terminal PTS2, which are specifically recognized by Pex5p and Pex7p, respectively (1Brown L.A. Baker A. J. Cell. Mol. Med. 2003; 7: 388-400Crossref PubMed Scopus (55) Google Scholar, 2Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (81) Google Scholar).A crucial step of the receptor cycle, which could provide directionality to the process, is the docking event at the peroxisomal membrane (1Brown L.A. Baker A. J. Cell. Mol. Med. 2003; 7: 388-400Crossref PubMed Scopus (55) Google Scholar, 2Eckert J.H. Erdmann R. Rev. Physiol. Biochem. Pharmacol. 2003; 147: 75-121Crossref PubMed Scopus (81) Google Scholar). Studies from several laboratories provide evidence that Pex14p is the central component of a docking complex, which also contains Pex17p and Pex13p. As Pex14p interacts with several other membrane-bound proteins in addition to both PTS receptors, it has been proposed to be the point of convergence of the two peroxisomal import pathways (6Albertini 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 (266) Google Scholar).There are conflicting data concerning the nature of the association of Pex14p with the peroxisomal membrane, and the topology of the protein is still not entirely solved. Although we originally report that Saccharomyces cerevisiae Pex14p is extractable with carbonate (6Albertini 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 (266) Google Scholar), others found that Pex14p from S. cerevisiae (7Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (107) Google Scholar) and other organisms (8Will G.K. Soukupova M. Hong X. Erdmann K.S. Kiel J.A.K.W. Dodt G. Kunau W.-H. Erdmann R. Mol. Cell. Biol. 1999; 19: 2265-2277Crossref PubMed Scopus (100) Google Scholar, 9Johnson 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, 10Shimizu 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, 11Komori 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, 12Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (40) Google Scholar, 13Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) behaves as an integral membrane protein. However, there is agreement that the C terminus of the protein is exposed to the cytosol (9Johnson 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, 10Shimizu 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, 12Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (40) Google Scholar, 13Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Rattus norvegicus Pex14p was found to be tightly associated with a fraction of Pex5p that behaves as an integral membrane protein (13Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). It was shown that the first 130 amino acids of Pex14p are highly protected from exogenously added protease by the peroxisomal membrane and that this domain is responsible for the strong interaction of Pex14p with the organelle membrane (12Oliveira M.E. Reguenga C. Gouveia A.M. Guimaraes C.P. Schliebs W. Kunau W.H. Silva M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (40) Google Scholar, 13Gouveia A.M. Reguenga C. Oliveira M.E. Sa-Miranda C. Azevedo J.E. J. Biol. Chem. 2000; 275: 32444-32451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).To explore the function of Pex14p in greater detail, we mapped its binding sites for the two PTS receptors. Unexpectedly, two separate and functionally different binding sites were found for the PTS receptor Pex5p, one in each of the two termini. The C-terminal binding site overlapped with a single binding site determined for Pex7p and was essential for matrix protein import. In contrast, deletion of the N-terminal binding site reduced the efficiency but did not abolish matrix protein import. We proposed a model in which the cytosolically exposed C terminus of Pex14p served as the actual docking site and in which the N terminus could be involved in a Pex14p-Pex5p association inside the peroxisomal membrane.EXPERIMENTAL PROCEDURESYeast Strains and Culture Conditions—The S. cerevisiae wild-type strain used in this study was UTL-7A (14Erdmann R. Wiebel F.F. Flessau A. Rytka J. Beyer A. Fröhlich K.U. Kunau W.-H. Cell. 1991; 64: 499-510Abstract Full Text PDF PubMed Scopus (269) Google Scholar). Yeast complete (yeast extract/peptone/dextrose) and minimal media (SD) have been described previously (15Erdmann R. Veenhuis M. Mertens D. Kunau W.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (261) Google Scholar). The yeast knock-out strain pex14Δ was a derivate of UTL-7A. To delete PEX14, the kanMX4 gene was used as a selective marker for insertion into the genomic locus (16Wach A. Brachat A. Pöhlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2225) Google Scholar). Deletion cassettes containing the kanMX4 gene and the 5′ and 3′ flanking regions of the Pex14p-ORF (open reading frame) were constructed by PCR using pFA6a-kanMX4 (16Wach A. Brachat A. Pöhlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2225) Google Scholar) as a template and Ku 289 (5′-gaaacctcaagtaaaacagagaagttgtaaggtgaataaggacgtacgctgcaggtcgac-3′) and Ku 290 (5′-aattacaatttccgttaaaaaactaattacttacatagaattgcgatcgatgaattcgagctcg-3′) as primers. For yeast two-hybrid experiments the yeast strain PCY2 was used (17Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (478) 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. YNDO medium contained the same components supplemented with 0.1% glucose. When necessary, auxotrophic requirements were added according to Ref. 18Ausubel 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 York1992: 13.1.1-13.113Google Scholar.Plasmids—For the expression of Myc-Pex7p, Pex14p and Pex14p fragments in a pex14 deletion strain of S. cerevisiae plasmids were cloned using the vectors pRS316, pRS416, and YEp351. For Pex14p and its derivatives, endogenous promoters and terminations were used. The resulting constructs are listed in TABLE ONE. Plasmids used for expressing His6-Pex14-(1-58)-p, GST-Pex14-(234-341)-p, GST, and Pex5p in Escherichia coli are based on pET21d and pGEX-4T-2 vectors and are also listed in TABLE ONE. Detailed cloning strategies are available on request.TABLE ONEPlasmids used in this studyPlasmidVectorExpressed proteinReferencepRS316-PEX14pRS316Pex14plc6pKN14/21cpRS316Pex14-(59-341)-plcThis studypKN14/22cpRS416Pex14-(1-234)-plcThis studypUP14/9pPC86Pex14-(235-341)-plcThis studypUP14/18pPC86Pex14-(250-341)-plcThis studypUP14/10pPC86Pex14-(270-341)-plcThis studypUP14/20pPc86Pex14-(235-325)-plcThis studypUP14/19pPC86Pex14-(235-308)-plcThis studypPC86-PEX5pPC86Pex5plc36pPC86-PEX14pPC86Pex14plc6pPC97-PEX7pPC97Pex7plc31pPC97-PEX14pPC97Pex14plc6pKN14/11cpPC97Pex14-(1-58)-plcThis studypKN14/21bpPC97Pex14-(59-341)-plcThis studypKN14/34bpPC97Pex14-(59-234)-plcThis studypKN14/22bpPC97Pex14-(1-234)-plcThis studypKN14/12cpPC97Pex14-(1-249)-plcThis studypKN14/9bpPC97Pex14-(235-341)-plcThis studypKN14/18bpPC97Pex14-(250-341)-plcThis studypKN14/10bpPC97Pex14-(270-341)-plcThis studypKN14/20apPC97Pex14-(235-325)-plcThis studypKN14/19bpPC97Pex14-(235-308)-plcThis studypKN5/0-1pET21d(+)Pex5phcThis studypET21d-Pex14-(1-58)-His6pET21d(+)Pex14-(1-58)-p-His6hcThis studypGEX-4T-2-GST-Pex14-(235-341)pGEX-4T-2GST-Pex14-(235-341)-phcThis studypGEX-4T-3GSThcGE Healthcare, FreiburgpPR7/4YEp351Myc-Pex7phc31 Open table in a new tab Antibodies, Western Blotting—Anti-Pex13p, -Pex14p, -Pex3p, -Pex5p, -Fox1p, -catalase A, -Pcs60p, -Fox3p, -Myc, and anti-Fbp1p have been described previously (6Albertini 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 (266) Google Scholar, 19Girzalsky 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, 20Schäfer A. Kerssen D. Veenhuis M. Kunau W.H. Schliebs W. Mol. Cell. Biol. 2004; 24: 8895-8906Crossref PubMed Scopus (86) Google Scholar, 21Blobel F. Erdmann R. Eur. J. Biochem. 1996; 240: 468-476Crossref PubMed Scopus (58) Google Scholar, 22Erdmann R. Yeast. 1994; 10: 935-944Crossref PubMed Scopus (59) Google Scholar, 23Bigl M. Escherich K. Biol. Chem. 1994; 375: 153-160Crossref PubMed Scopus (10) Google Scholar). To produce anti-Pex14-(1-58)-p antibodies, Pex14-(1-58)-p-His6 was isolated using Ni2+-NTA-agarose. Pex14-(1-58)-p-His6 was expressed in BL21(DE3) E. coli cells. For induction, 0.1 mm isopropyl 1-thio-β-d-galactopyranoside was introduced. After lysis by French press in a 3× volume of lysis buffer (20 mm Tris/HCl, pH 7.9, 0.5 m NaCl, 5 mm imidazole), a centrifugation step of 25,000 × g for 30 min followed. The supernatant was filtered through a 0.45-μm membrane and supplied to a 1.2-ml Ni2+-NTA-agarose column. After washing using a 20× volume of washing buffer (20 mm Tris/HCl, pH 7.9, 0.5 m NaCl, 60 mm imidazole), bound proteins were eluted with elution buffer (20 mm Tris/HCl, pH 7.9, 0.5 m NaCl, 1 m imidazole) in 1-ml fractions. As protease inhibitors, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml chymostatin, 2 μg/ml leupeptin, and 2 μg/ml pepstatin were used. Fractions containing the fusion protein were used to immunize a rabbit.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. Proteins in polyacrylamide gels were visualized by Coomassie staining according to Ref. 24Lloyd M.D. Anal. Biochem. 1996; 241: 139-140Crossref PubMed Scopus (4) Google Scholar.In Vitro Binding Assays—Transformed E. coli BL21(DE3) cells were cultured and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Myc-Pex7p overexpression in pex14Δ cells was induced by the addition of 100 mg of CuSO4·5H2O/liter of culture containing 2% glucose. For the analysis of the interaction between Pex5p and Pex14-(1-58)-p-His6 as well as the binding of GST-Pex14-(235-341)-p to Myc-Pex7p, 0.2 g of E. coli BL21(DE3) cells expressing Pex14-(1-58)-p-His6, GST-Pex14-(235-341)-p, GST, 0.3 g of E. coli BL21(DE3) cells expressing Pex5p, and 1 g of pex14Δ yeast cells overexpressing Myc-Pex7p were resuspended in a 3× volume of lysis buffer (for Ni2+-NTA columns, 200 mm Tris/HCl, pH 8.0, 150 mm NaCl, 5 mm imidazole; for GSH-Sepharose columns, 50 mm Tris/HCl, pH 7.5, 100 mm NaCl, 0.2% (w/v) Triton X-100). Cells were lysed by the addition of a 4× volume of glass beads (℘ 0.5 mm) and vortexing 10 times for 1 min each. Glass beads, cell debris, and unlysed cells were sedimented by a centrifugation step at 1500 × g for 5 min. Thereafter, E. coli lysates were centrifuged for 10 min at 88,000 × g and yeast lysates for 30 min at 200,000 × g to pellet insoluble material. Supernatants were mixed and incubated with 100 μl of Ni2+-NTA-agarose (Qiagen) or 100 μl of GSH-Sepharose (GE Healthcare) as needed for 2 h at 4°C. The resin columns were collected in minispin columns (MobiTec). The Ni2+-NTA-agarose was washed 10 times using 200 μl of lysis buffer, followed by 10 times using 100 μl of 200 mm Tris/HCl, pH 8.0, 150 mm NaCl, and 20 mm imidazole, 10 times using 200 mm Tris/HCl, pH 8.0, 150 mm NaCl, and 40 mm imidazole, and 10 times using 200 mm Tris/HCl, pH 8.0, 150 mm NaCl, and 60 mm imidazole. Elution was carried out using 200 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 m imidazole. Every two fractions were pooled before analysis. The GSH-Sepharose was washed 10 times using 200 μl of lysis buffer. Proteins were eluted with lysis buffer enriched with 10 mm reduced glutathione. Every two fractions were pooled before analysis. As protease inhibitors 1 mm phenylmethylsulfonyl fluoride, 5 mm NaF, 2 μg/ml leupeptin, and 2 μg/ml pepstatin were used.For the analysis of the binding between GST-Pex14-(235-341)-p and Pex5p, 2 g of cells expressing one or the other fusion protein or GST were used. Lysis buffer did not contain Triton X-100 but 1 mm dithiothreitol. As protease inhibitors, 2 μm leupeptin, 2 μm pepstatin, 200 μm Pefabloc SC, 5 mm benzamidine, and 5 mm NaF were used. Cells were lysed using a French pressure cell press. Soluble fractions were obtained by centrifugation as described above, mixed, and incubated at 4 °C for 1 h before they were subjected to columns with 0.8 ml of GSH-Sepharose. Washing was performed using 32 ml of lysis buffer. Recovered proteins were eluted with 16 ml of lysis buffer enriched with 10 mm reduced glutathione in 0.5-ml fractions.Cell Fractionation—Spheroplasting of yeast cells, homogenization, and differential centrifugation at 25,000 × g of homogenates were performed as described previously (15Erdmann R. Veenhuis M. Mertens D. Kunau W.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (261) Google Scholar).For separation of cell organelles by density gradient centrifugation, post-nuclear supernatants of wild-type and mutant strains were loaded onto continuous 20-53% (w/v) sucrose density gradients (25 ml). The gradient buffer contained 5 mm MES, 1 mm EDTA, 1 mm KCl, 0.1% (v/v) ethanol/KOH, pH 6.0, at 4 °C. After a centrifugation step of 1.5 h (Sorvall-SV288, 19,500 rpm), ∼30 fractions of 1 ml each were collected. 500 μl of each fraction of any gradient were processed for Western blotting using trichloroacetic acid, whereas the other part was used for enzyme measurements.The suborganellar localization of proteins was determined by extraction of 25,000 × g organelle pellets by either high salt treatment with buffer containing 10 mm Tris/HCl, pH 8.0, 500 mm KCl, and 1 mm phenylmethylsulfonyl fluoride or using carbonate buffer containing 100 mm Na2CO3, pH 11.5, and 1 mm phenylmethylsulfonyl fluoride. After incubation for 30 min at 4 °C, the samples were spun for 1 h at 200,000 × g through a cushion of 10 mm Tris/HCl, pH 8.0, and 250 mm sucrose.Enzyme Assays—Catalase (EC 1.11.1.6) and cytochrome c oxidase (EC 1.9.3.1) were assayed according to published procedures (25Baudhuin P. Methods Enzymol. 1974; 31: 356-368Crossref PubMed Scopus (64) Google Scholar, 26Graham J.M. Methods Mol. Biol. 1993; 19: 29-40PubMed Google Scholar, 27Bergmeyer H.U. Gawehn K. Grassel M. Bergmeyer H.U. Methoden der Enzymatischen Analyse I. Verlag Chemie, Weinheim, Germany1974: 479-480Google Scholar, 28Douma A.C. Veenhuis M. de Koning W. Evers M. Harder W. Arch. Microbiol. 1985; 143: 237-243Crossref Scopus (121) Google Scholar).Two-Hybrid Assay—The two-hybrid assay was based on the method of Fields and Song (29Fields S. Song O.K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar). The open reading frames or coding regions of specific fragments of PEX genes were fused to the DNA binding domain or transcription-activating domain of GAL4 into the vectors pPC86 and pPC97 (17Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (478) Google Scholar) (see TABLE ONE). Co-transformation of two-hybrid vectors into PCY2 yeast cells was performed according to Gietz and Sugino (30Gietz R.D. Sugino A. Gene. 1988; 74: 527-534Crossref PubMed Scopus (2506) Google Scholar). Double transformants were selected on SD synthetic medium without tryptophan and leucine. β-galactosidase activity of transformed cells was determined by a filter assay described by Rehling et al. (31Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W.-H. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar), using X-gal as substrate.Electron Microscopy—Potassium permanganate fixation and preparation of intact yeast cells were performed according to Ref. 15Erdmann R. Veenhuis M. Mertens D. Kunau W.-H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (261) Google Scholar.Indirect Immunofluorescence Microscopy—Immunolabeling of yeast cells and fluorescence microscopy were performed as described by Rehling et al. (31Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W.-H. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar).RESULTSPex5p Binds the N and C Termini of ScPex14p—To understand how ScPex14p facilitates the import of peroxisomal matrix proteins, we first mapped the binding sites of the two PTS receptors within this protein. We used the yeast two-hybrid system and first tested N- and C-terminal deletion mutants of Pex14p for the binding of Pex5p. The short N-terminal fragment Pex14-(1-58)-p was chosen because it comprises the most conserved region of Pex14 proteins (data not shown), and Schliebs et al. reports that the analogous region of human Pex14p directly binds HsPex5p (32Schliebs W. Saidowsky J. Agianian B. Dodt G. Herberg F.W. Kunau W.H. J. Biol. Chem. 1999; 274: 5666-5673Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). As expected, Pex14-(1-58)-p did interact with the PTS1 receptor (Fig. 1A). Surprisingly, the complementary fragment Pex14-(59-341)-p, which was actually meant as a control, also interacted with the PTS1 receptor. To test whether ScPex14p contains two separate binding sites of Pex5p, we used a series of N-terminal deletions and found that the C-terminal fragment Pex14-(235-341)-p interacts with Pex5p, whereas the core fragment Pex14-(59-234)-p missing both termini does not (Fig. 1A). In addition, we verified the interaction of both termini of Pex14p with the PTS1 receptor using an in vitro assay. For this purpose, Pex14-(1-58)-p and Pex14-(235-341)-p (fused to a His6 or GST tag, respectively) and Pex5p (without any tag) were expressed in E. coli. Both recombinant Pex14p fragments clearly bound Pex5p, whereas this was not the case in the controls (Fig. 2, A and B). These data strongly indicated the existence of two separate direct Pex5p binding sites on ScPex14p, one in the N-terminal 58 amino acid residues and one in the last 106 amino acid residues.FIGURE 2In vitro binding assay. The binding of Pex14-(1-58)-p-His6 and GST-Pex14-(235-341)-p to Pex5p or Pex7p was studied in vitro by pull-down experiments. Soluble fractions of BL21(DE3) cells heterologously expressing Pex5p, Pex14-(1-58)-p-His6, GST-Pex14-(235-341)-p, GST, or no host protein as well as soluble fractions of yeast cells overexpressing Myc-Pex7p were mixed as indicated and incubated with Ni2+-NTA-agarose (A) or GSH-Sepharose (B and C). Recovered proteins of the elution fractions were analyzed by SDS-PAGE and Western blotting using anti-Pex5p and anti-Pex14p antibodies (A), antibodies against Pex5p and GST (B), or anti-Pex14p antibodies and antibodies against the Myc epitope (C). Direct binding of Pex5p to the N and C termini of Pex14p as well as an interaction of Pex7p with the C terminus of Pex14p are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The C Terminus of Pex14p Also Interacts with Pex7p—To determine the binding region of Pex7p, we assayed the same truncated versions of ScPex14p as described above using again the two-hybrid system. Fig. 1B shows that the C-terminal fragment Pex14-(235-341)-p (which contains a binding site of the PTS1 receptor) also interacts with the PTS2 receptor. All other ScPex14p fragments did not bind to Pex7p (Fig. 1B; data not shown). An analogous pull-down experiment as described above for the binding of Pex5p (Fig. 2, A and B) was carried out with Myc-Pex7p and GST-Pex14-(235-341)-p. As Myc-Pex7p could not be stably overexpressed in E. coli, a yeast lysate containing Myc-Pex7p but no wild-type Pex14p was used. The results shown in Fig. 2C demonstrate the specific interaction of the PTS2 receptor with the C terminus of Pex14p. These data suggest that, although Pex5p directly interacts with ScPex14p through two distinct regions, Pex7p possesses only one binding site in the C terminus of Pex14p.To determine whether Pex5p and Pex7p bind to the same amino acid sequence within the C terminus of ScPex14p, we shortened the Pex14-(235-341)-p fragment further on both ends and tested these constructs in the yeast two-hybrid assay. The results (Fig. 1C) can be summarized in two points. First, the smallest fragment that interacted with Pex7p was Pex14-(235-325)-p. Second, Pex5p binds to the even smaller regions Pex14-(235-308)-p and Pex14-(250-341)-p. In conclusion, the amino acid residues in the C terminus of ScPex14p that interact with the two PTS receptors overlap, but they are not identical.Deletion of the N-terminal 58 Amino Acid Residues of ScPex14p Leads to a Partial Import Defect—The overall import activity of ScPex14p can be assayed by the ability of pex14Δ cells expressing wild-type Pex14p to grow on oleate as the sole energy and carbon source or by determining the presence and/or properties of peroxisomes using electron microscopy, immunofluorescence, and cell fractionation. All of these techniques were used to assess the functional significance of the identified PTS receptor binding sites within ScPex14p.We first tested pex14Δ cells expressing Pex14p without the N-terminal 58 amino acid residues under the endogenous promoter of Pex14p. The steady state concentration of this fragment in these cells was similar to that of Pex14p in wild-type cells (Fig. 3A). We observed that pex14Δ cells expressing the N-terminal deletion mutant Pex14-(59-341)-p grew significantly less efficiently on oleate than wild-type cells (Fig. 3C) but that they do contain peroxisomes, which according to their morphology and size, are indistinguishable from those of cells expressing wild-type Pex14p (Fig. 4). As the reduced ability to grow on oleate suggested a partial impairment of the protein import, we explored this possibility in more detail. Immunofluorescence microscopy and cell fractionation studies clearly confirmed this assumption for both the PTS1 and surprisingly the PTS2 import pathway. Antibodies against the PTS1 protein Pcs60p and the PTS2 protein thiolase revealed, besides a punctate immunofluorescence pattern, also a distribution of both proteins over the entire range of cells (Fig. 5). Moreover, differential centrifugation experiments showed that catalase and thiolase could only partially be pelleted with the peroxisomes at 25,000 × g, and significant portions were found in the supernatant fractions (Fig. 6). Interestingly, the same result was obtained with Fox1p (Acyl-CoA oxidase). This protein possesses neither a PTS1 nor a PTS2, but as recently shown, its import is nevertheless dependent on Pex5p (20Schäfer A. Kerssen D. Veenhuis M. Kunau W.H. Schliebs W. Mol. Cell. Biol. 2004; 24: 8895-8906Crossref PubMed Scopus (86) Google Scholar, 33Klein A.T. van Den Berg M. Bottger G. Tabak H.F. Distel B. J. Biol. Chem. 2002; 277: 25011-25019Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).FIGURE 3Expression of Pex14p fragments and phenotypical analysis of cells expressing Pex14-(59-341)-p or Pex14-(1-234)-p by oleate growth test. A and B, equal fractions of yeast wild-type cells, pex14Δ cells, as well as mutant cells expressing Pex14p full-length and (A) Pex14-(59-341)-p (A) or Pex14-(1-234)-p (B) were analyzed by SDS-PAGE and Western blotting. For immunodecoration, anti-Pex14p antibodies or anti-Pex14-(1-58)-p-His6 antibodies were used, respectively. C and D, cells of indicated yeast strains were grown overnight on glucose minimal media. Subsequently, d" @default.
• W2085967013 created "2016-06-24" @default.
• W2085967013 creator A5001675658 @default.
• W2085967013 creator A5008819336 @default.
• W2085967013 creator A5012995576 @default.
• W2085967013 creator A5018758279 @default.
• W2085967013 creator A5037962658 @default.
• W2085967013 creator A5038194850 @default.
• W2085967013 creator A5070980886 @default.
• W2085967013 date "2005-10-01" @default.
• W2085967013 modified "2023-10-16" @default.
• W2085967013 title "Yeast Pex14p Possesses Two Functionally Distinct Pex5p and One Pex7p Binding Sites" @default.
• W2085967013 cites W1492622409 @default.
• W2085967013 cites W1967390330 @default.
• W2085967013 cites W1967503648 @default.
• W2085967013 cites W1977186059 @default.
• W2085967013 cites W1983711543 @default.
• W2085967013 cites W1993691427 @default.
• W2085967013 cites W2006861434 @default.
• W2085967013 cites W2007739884 @default.
• W2085967013 cites W2008708118 @default.
• W2085967013 cites W2019672179 @default.
• W2085967013 cites W2065699737 @default.
• W2085967013 cites W2068367980 @default.
• W2085967013 cites W2081712820 @default.
• W2085967013 cites W2084938165 @default.
• W2085967013 cites W2090286778 @default.
• W2085967013 cites W2100057937 @default.
• W2085967013 cites W2106462570 @default.
• W2085967013 cites W2108789322 @default.
• W2085967013 cites W2111792196 @default.
• W2085967013 cites W2116488364 @default.
• W2085967013 cites W2117935792 @default.
• W2085967013 cites W2118176667 @default.
• W2085967013 cites W2130350654 @default.
• W2085967013 cites W2137528144 @default.
• W2085967013 cites W2137938732 @default.
• W2085967013 cites W2146795350 @default.
• W2085967013 cites W2152015543 @default.
• W2085967013 cites W2157841878 @default.
• W2085967013 cites W2161223616 @default.
• W2085967013 cites W2212472563 @default.
• W2085967013 cites W4211100528 @default.
• W2085967013 doi "https://doi.org/10.1074/jbc.m502460200" @default.
• W2085967013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16107331" @default.
• W2085967013 hasPublicationYear "2005" @default.
• W2085967013 type Work @default.
• W2085967013 sameAs 2085967013 @default.
• W2085967013 citedByCount "48" @default.
• W2085967013 countsByYear W20859670132012 @default.
• W2085967013 countsByYear W20859670132013 @default.
• W2085967013 countsByYear W20859670132014 @default.
• W2085967013 countsByYear W20859670132016 @default.
• W2085967013 countsByYear W20859670132017 @default.
• W2085967013 countsByYear W20859670132018 @default.
• W2085967013 countsByYear W20859670132019 @default.
• W2085967013 countsByYear W20859670132020 @default.
• W2085967013 countsByYear W20859670132021 @default.
• W2085967013 countsByYear W20859670132022 @default.
• W2085967013 countsByYear W20859670132023 @default.
• W2085967013 crossrefType "journal-article" @default.
• W2085967013 hasAuthorship W2085967013A5001675658 @default.
• W2085967013 hasAuthorship W2085967013A5008819336 @default.
• W2085967013 hasAuthorship W2085967013A5012995576 @default.
• W2085967013 hasAuthorship W2085967013A5018758279 @default.
• W2085967013 hasAuthorship W2085967013A5037962658 @default.
• W2085967013 hasAuthorship W2085967013A5038194850 @default.
• W2085967013 hasAuthorship W2085967013A5070980886 @default.
• W2085967013 hasBestOaLocation W20859670131 @default.
• W2085967013 hasConcept C185592680 @default.
• W2085967013 hasConcept C2779222958 @default.
• W2085967013 hasConcept C55493867 @default.
• W2085967013 hasConcept C86803240 @default.
• W2085967013 hasConceptScore W2085967013C185592680 @default.
• W2085967013 hasConceptScore W2085967013C2779222958 @default.
• W2085967013 hasConceptScore W2085967013C55493867 @default.
• W2085967013 hasConceptScore W2085967013C86803240 @default.
• W2085967013 hasIssue "42" @default.
• W2085967013 hasLocation W20859670131 @default.
• W2085967013 hasOpenAccess W2085967013 @default.
• W2085967013 hasPrimaryLocation W20859670131 @default.
• W2085967013 hasRelatedWork W1531601525 @default.
• W2085967013 hasRelatedWork W2319480705 @default.
• W2085967013 hasRelatedWork W2384464875 @default.
• W2085967013 hasRelatedWork W2606230654 @default.
• W2085967013 hasRelatedWork W2607424097 @default.
• W2085967013 hasRelatedWork W2748952813 @default.
• W2085967013 hasRelatedWork W2899084033 @default.
• W2085967013 hasRelatedWork W2917539084 @default.
• W2085967013 hasRelatedWork W2948807893 @default.
• W2085967013 hasRelatedWork W2778153218 @default.
• W2085967013 hasVolume "280" @default.
• W2085967013 isParatext "false" @default.
• W2085967013 isRetracted "false" @default.
• W2085967013 magId "2085967013" @default.
• W2085967013 workType "article" @default.