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• W2108789322 abstract "The peroxisomal protein acyl-CoA oxidase (Pox1p) ofSaccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2. Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor. The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins. The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found. By making use ofpex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p. The peroxisomal protein acyl-CoA oxidase (Pox1p) ofSaccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2. Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor. The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins. The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found. By making use ofpex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p. peroxisomal targeting signal 1 and 2, respectively peroxin tetratricopeptide repeat peroxisomal malate dehydrogenase carnitine acetyltransferase acyl-CoA oxidase glutathione S-transferase maltose-binding protein green fluorescent protein Proteins destined for import into the peroxisomal matrix are synthesized on free polyribosomes in the cytoplasm. For targeting to their proper destination, these proteins possess a peroxisomal targeting signal (PTS)1 that directs them to peroxisomes. Two different PTSs have been identified, PTS1 and PTS2. The majority of peroxisomal matrix proteins contain a PTS1 and only a few have a PTS2. The PTS1 is located at the extreme carboxyl terminus of a peroxisomal matrix protein and was first defined as three amino acids with the consensus sequence (S/C/A)(K/R/H)(L/M) (1Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar, 2Swinkels B.W. Gould S.J. Subramani S. FEBS Lett. 1992; 305: 133-136Crossref PubMed Scopus (107) Google Scholar). The PTS2 is positioned at the amino-terminal part of a protein and has the consensus sequence (R/K)(L/V/I)X 5(H/Q)(L/A) (3Swinkels B.W. Gould S.J. Bodnar A.G. Rachubinski R.A. Subramani S. EMBO J. 1991; 10: 3255-3262Crossref PubMed Scopus (515) Google Scholar, 4Glover J.R. Andrews D.W. Subramani S. Rachubinski R.A. J. Biol. Chem. 1994; 269: 7558-7563Abstract Full Text PDF PubMed Google Scholar, 5Gietl C. Faber K.N. van der Klei I.J. Veenhuis M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3151-3155Crossref PubMed Scopus (126) Google Scholar, 6Tsukamoto T. Hata S. Yokota S. Miura S. Fujiki Y. Hijikata M. Miyazawa S. Hashimoto T. Osumi T. J. Biol. Chem. 1994; 269: 6001-6010Abstract Full Text PDF PubMed Google Scholar). The PTS1 and PTS2 are recognized and bound in the cytosol by specific receptor proteins, Pex5p (peroxin-5protein) (7McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 8Van 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 (202) Google Scholar, 9Brocard C. Kragler F. Simon M.M. Schuster T. Hartig A. Biochem. Biophys. Res. Commun. 1994; 204: 1016-1022Crossref PubMed Scopus (127) Google Scholar, 10Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 11Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar, 13Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L., Li, X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 15Dodt G. Gould S.J. J. Cell Biol. 1996; 135: 1763-1774Crossref PubMed Scopus (262) Google Scholar) and Pex7p (16Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar, 17Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (121) Google Scholar, 18Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W.H. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar, 19Zhang J.W. Lazarow P.B. J. Cell Biol. 1996; 132: 325-334Crossref PubMed Scopus (80) Google Scholar, 20Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (356) Google Scholar, 21Motley A.M. Hettema E.H. Hogenhout E.M. Brites P. ten Asbroek A.L. Wijburg F.A. Baas F. Heijmans H.S. Tabak H.F. Wanders R.J. Distel B. Nat. Genet. 1997; 15: 377-380Crossref PubMed Scopus (222) Google Scholar, 22Purdue P.E. Zhang J.W. Skoneczny M. Lazarow P.B. Nat. Genet. 1997; 15: 381-384Crossref PubMed Scopus (223) Google Scholar, 23Elgersma Y. Elgersma-Hooisma M. Wenzel T. McCaffery J.M. Farquhar M.G. Subramani S. J. Cell Biol. 1998; 140: 807-820Crossref PubMed Scopus (74) Google Scholar), respectively. For Pex5p it has been shown that an array of tetratricopeptide repeats (TPR) in the carboxyl-terminal part of the protein mediates the binding of PTS1 (9Brocard C. Kragler F. Simon M.M. Schuster T. Hartig A. Biochem. Biophys. Res. Commun. 1994; 204: 1016-1022Crossref PubMed Scopus (127) Google Scholar, 10Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 12Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar). The details of the interaction between Pex5p and PTS1 have been resolved by an extensive mutational analysis of Pex5p (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and determination of the crystal structure of a Pex5p-PTS1 peptide complex (25Gatto G.J., Jr. Geisbrecht B.V. Gould S.J. Berg J.M. Nat. Struct. Biol. 2000; 7: 1091-1095Crossref PubMed Scopus (296) Google Scholar). Those studies revealed that the TPR domain of Pex5p forms two clusters of three TPR motifs that are close together in space and form a single binding site for the PTS1. Amino acids from both TPR clusters are interacting with the PTS1 peptide backbone and with the amino acid side chains. How binding of PTS2 by Pex7p, a WD-40 repeat protein, takes place is still unclear. The receptor-cargo complex docks on the peroxisome via the interaction with a protein complex located in the peroxisomal membrane. Although some of the details vary between different species, it has been shown that Pex13p, Pex14p, and Pex17p are part of this docking complex (26–41). Proteins implicated in the translocation over the peroxisomal membrane are Pex2p, Pex10p, and Pex12p (15Dodt G. Gould S.J. J. Cell Biol. 1996; 135: 1763-1774Crossref PubMed Scopus (262) Google Scholar, 42Chang C.C. Warren D.S. Sacksteder K.A. Gould S.J. J. Cell Biol. 1999; 147: 761-774Crossref PubMed Scopus (124) Google Scholar). However, it is still unclear how the actual translocation over the peroxisomal membrane takes place, except that protein unfolding is not a prerequisite for translocation (43Glover J.R. Andrews D.W. Rachubinski R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10541-10545Crossref PubMed Scopus (244) Google Scholar, 44McNew J.A. Goodman J.M. J. Cell Biol. 1994; 127: 1245-1257Crossref PubMed Scopus (278) Google Scholar, 45Walton P.A. Hill P.E. Subramani S. Mol. Biol. Cell. 1995; 6: 675-683Crossref PubMed Scopus (210) Google Scholar, 46Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 47Hausler T. Stierhof Y.D. Wirtz E. Clayton C. J. Cell Biol. 1996; 132: 311-324Crossref PubMed Scopus (76) Google Scholar, 48Leiper J.M. Oatey P.B. Danpure C.J. J. Cell Biol. 1996; 135: 939-951Crossref PubMed Scopus (79) Google Scholar, 49Lee M.S. Mullen R.T. Trelease R.N. Plant Cell. 1997; 9: 185-197Crossref PubMed Scopus (125) Google Scholar, 50Yang X. Purdue P.E. Lazarow P.B. Eur. J. Cell Biol. 2001; 80: 126-138Crossref PubMed Scopus (62) Google Scholar). The first PTS1 identified was that of firefly luciferase and consists of the carboxyl-terminal tripeptide SKL (1Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar,51Gould S.J. Keller G.A. Subramani S. J. Cell Biol. 1987; 105: 2923-2931Crossref PubMed Scopus (359) Google Scholar). This tripeptide proved not only essential for the import of luciferase but was also shown to be sufficient to direct other proteins to peroxisomes (1Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar, 2Swinkels B.W. Gould S.J. Subramani S. FEBS Lett. 1992; 305: 133-136Crossref PubMed Scopus (107) Google Scholar, 52Gould S.J. Keller G.A. Subramani S. J. Cell Biol. 1988; 107: 897-905Crossref PubMed Scopus (271) Google Scholar, 53Gould S.J. Keller G.A. Schneider M. Howell S.H. Garrard L.J. Goodman J.M. Distel B. Tabak H. Subramani S. EMBO J. 1990; 9: 85-90Crossref PubMed Scopus (221) Google Scholar). However, a number of observations (1Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar,54Motley A. Lumb M.J. Oatey P.B. Jennings P.R., De Zoysa P.A. Wanders R.J. Tabak H.F. Danpure C.J. J. Cell Biol. 1995; 131: 95-109Crossref PubMed Scopus (86) Google Scholar, 55Sommer J.M. Cheng Q.L. Keller G.A. Wang C.C. Mol. Biol. Cell. 1992; 3: 749-759Crossref PubMed Scopus (133) Google Scholar) suggest that the definition of a PTS1 as being both necessary and sufficient for the import of proteins into peroxisomes needs some adjustment. These studies have shown that whether or not a carboxyl-terminal tripeptide can function as a PTS1 depends on its context. For instance, targeting of alanine:glyoxylate aminotransferase I to peroxisomes in humans depends on the carboxyl-terminal tripeptide KKL (54Motley A. Lumb M.J. Oatey P.B. Jennings P.R., De Zoysa P.A. Wanders R.J. Tabak H.F. Danpure C.J. J. Cell Biol. 1995; 131: 95-109Crossref PubMed Scopus (86) Google Scholar). However, this carboxyl-terminal KKL was not sufficient to direct the reporter protein luciferase to peroxisomes in human fibroblasts (54Motley A. Lumb M.J. Oatey P.B. Jennings P.R., De Zoysa P.A. Wanders R.J. Tabak H.F. Danpure C.J. J. Cell Biol. 1995; 131: 95-109Crossref PubMed Scopus (86) Google Scholar) and in monkey kidney CV-1 cells (1Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar) or to glycosomes in Trypanosoma brucei(55Sommer J.M. Cheng Q.L. Keller G.A. Wang C.C. Mol. Biol. Cell. 1992; 3: 749-759Crossref PubMed Scopus (133) Google Scholar). For peroxisomal malate dehydrogenase (Mdh3p), it was also shown that in the homologous context many variations that do not comply with the consensus sequence could still direct this protein to peroxisomes in Saccharomyces cerevisiae (46Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). These results can be explained by the presence of accessory sequences in a peroxisomal matrix protein that, when this protein is presented in its homologous context, contribute to the binding of the PTS1-containing protein to Pex5p. These accessory sequences can sometimes be located close to the PTS1 and can influence the binding to Pex5p in a species-dependent manner, as was shown for hexadecapeptides containing a PTS1 (56Lametschwandtner G. Brocard C. Fransen M. Van Veldhoven P. Berger J. Hartig A. J. Biol. Chem. 1998; 273: 33635-33643Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In other cases a PTS1 is not essential at all. This is most evident for carnitine acetyltransferase (Cat2p); its targeting to peroxisomes in S. cerevisiae is Pex5p-dependent, but after deletion of the PTS1 most of the carnitine acetyltransferase is still directed to peroxisomes (57Elgersma Y. van Roermund C.W. Wanders R.J. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (162) Google Scholar). Deletion of the PTS1 in Cat2p also does not affect its interaction with Pex5p in the two-hybrid system. These results suggest that in some cases accessory or alternative sequences can be used for binding to Pex5p and that these can function as a targeting signal. Import of proteins in a PTS1- or PTS2-independent way can be explained in various ways. In genetically constructed S. cerevisiae strains import into peroxisomes can take place by formation of homo-oligomers between subunits without a PTS and subunits with a PTS (43Glover J.R. Andrews D.W. Rachubinski R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10541-10545Crossref PubMed Scopus (244) Google Scholar, 44McNew J.A. Goodman J.M. J. Cell Biol. 1994; 127: 1245-1257Crossref PubMed Scopus (278) Google Scholar, 46Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 49Lee M.S. Mullen R.T. Trelease R.N. Plant Cell. 1997; 9: 185-197Crossref PubMed Scopus (125) Google Scholar). Similarly, it has been shown that S. cerevisiae Δ3,Δ2-enoyl-CoA isomerase (Eci1p) can hetero-oligomerize with Δ3,5-Δ2,4-dienoyl-CoA isomerase (Dci1p) resulting in the import of Eci1p from which the PTS1 had been deleted (50Yang X. Purdue P.E. Lazarow P.B. Eur. J. Cell Biol. 2001; 80: 126-138Crossref PubMed Scopus (62) Google Scholar). In a natural context, there are several peroxisomal matrix proteins that are not equipped with a recognizable PTS1 or PTS2. Examples of such proteins are Hansenula polymorpha malate synthase (58Bruinenberg P.G. Blaauw M. Kazemier B. Ab G. Yeast. 1990; 6: 245-254Crossref PubMed Scopus (17) Google Scholar) and acyl-CoA oxidases of the yeasts Candida tropicalis (59Small G.M. Szabo L.J. Lazarow P.B. EMBO J. 1988; 7: 1167-1173Crossref PubMed Scopus (139) Google Scholar), Candida maltosa (60Hill D.E. Boulay R. Rogers D. Nucleic Acids Res. 1988; 16: 365-366Crossref PubMed Scopus (29) Google Scholar), S. cerevisiae (61Dmochowska A. Dignard D. Maleszka R. Thomas D.Y. Gene (Amst.). 1990; 88: 247-252Crossref PubMed Scopus (77) Google Scholar), and Yarrowia lipolytica (62Wang H., Le Clainche A., Le Dall M.T. Wache Y. Pagot Y. Belin J.M. Gaillardin C. Nicaud J.M. Yeast. 1998; 14: 1373-1386Crossref PubMed Scopus (38) Google Scholar). How targeting of these proteins to peroxisomes takes place, via piggy-backing or via alternative targeting sequences in these proteins, is not known (59Small G.M. Szabo L.J. Lazarow P.B. EMBO J. 1988; 7: 1167-1173Crossref PubMed Scopus (139) Google Scholar). Remarkably, in human (63Fournier B. Saudubray J.M. Benichou B. Lyonnet S. Munnich A. Clevers H. Poll-The B.T. J. Clin. Invest. 1994; 94: 526-531Crossref PubMed Scopus (65) Google Scholar), rat (64Miyazawa S. Osumi T. Hashimoto T. Ohno K. Miura S. Fujiki Y. Mol. Cell. Biol. 1989; 9: 83-91Crossref PubMed Scopus (186) Google Scholar), mouse (65Nohammer C., El- Shabrawi Y. Schauer S. Hiden M. Berger J. Forss-Petter S. Winter E. Eferl R. Zechner R. Hoefler G. Eur. J. Biochem. 2000; 267: 1254-1260Crossref PubMed Scopus (29) Google Scholar), and in the yeast Pichia pastoris (66Koller A. Spong A.P. Luers G.H. Subramani S. Yeast. 1999; 15: 1035-1044Crossref PubMed Scopus (13) Google Scholar) acyl-CoA oxidase is imported via its PTS1. Here we show that S. cerevisiae acyl-CoA oxidase (Pox1p) binds directly to Pex5p and that binding is not dependent on the carboxyl-terminal 17 amino acids of Pox1p. By using a pex5mutant that is specifically disturbed in the interaction with and the import of PTS1 proteins, we show that S. cerevisiae Pox1p is imported into peroxisomes in a PTS1-independent manner. The site of Pox1p interaction on Pex5p was identified and shown to be located in a region outside of the TPR domain. A pex5 mutant containing a Y253N substitution within the Pox1p-binding region is specifically disturbed in the interaction with and the import of Pox1p. These results demonstrate a novel, non-PTS1-mediated import route for Pox1p that is dependent on Pex5p. The yeast strains used in this study are as follows: S. cerevisiaeBJ1991 (MATα, leu2, trp1, ura3-251, prb1-1122, pep4-3, gal2); BJ1991pex5Δ (MATα, pex5::LEU2, leu2, trp1, ura3-251, prb1-1122, pep4-3, gal2); BJ1991pex3Δ and BJ1991pex7Δ were described previously (67Hettema E.H. Girzalsky W. van Den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (223) Google Scholar); HF7c (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, gal4-542, gal80-538, LYS2::GAL1UAS-GAl1TATA-HIS3, URA3::GAL4 17-mer(3×) - CyC1TATA-lacZ); and PCY2 (MATα, Δgal4, Δgal80, URA3::GAL1-lacZ, lys2-801, his3-Δ200, trp1-Δ63, leu2, ade2-101). The Escherichia coli strain DH5α (recA, hsdR, supE, endA, gyrA96, thi-1, relA1, lacZ) was used for all transformations and plasmid isolations. Yeast transformations were carried out as described (68Gietz D., St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2883) Google Scholar). Transformants were selected and grown on minimal medium containing 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose, and amino acids as needed. Cell culture conditions are as follows: cells were pre-grown overnight on minimal 0.3% glucose medium (0.3% glucose, 0.67% yeast nitrogen base (YNB; Difco) and amino acids (20–30 μg/ml) as required). These cultures were inoculated in fresh 0.3% glucose medium and further grown to log phase. For induction on oleate these cultures were inoculated 1:10 in fresh oleate medium (0.5% potassium phosphate buffer, pH 6.0, 0.5% peptone and 0.3% yeast extract, 0.1% oleate, 2% Tween 40) and grown overnight at 28 °C. Standard techniques for DNA manipulations were used (69Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The following plasmids have been described previously: pGST-Pex5p, encoding a fusion of glutathioneS-transferase (GST) with Pex5p (39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar); pAN4, encoding a fusion of the Gal4 trans-activating domain (Gal4AD) with Pex5p (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); pDBMDH3, encoding a fusion of the Gal4 DNA-binding domain (Gal4BD) with Mdh3p (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); pEL128, encoding a fusion of Gal4BD with ΔN-Cat2-ΔC (57Elgersma Y. van Roermund C.W. Wanders R.J. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (162) Google Scholar); pGB17, encoding a fusion of Gal4BD with the Pex13pSH3 domain (39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar); pGB47, encoding a fusion of Gal4BD with Pex14p (39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar). The plasmid for expression of Pex5p in yeast (pTI98) was created by subcloning thePEX5 insert of pAN1 (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) behind the PEX5 promoter in pEL91 (39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar) using BamHI and PstI.pex5 mutants were subcloned in pEL91 in the same way. pGB37, encoding NH-tagged Mdh3p was generated by subcloning theSacI-HindIII fragment of pEL143 (46Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) behind theCTA1 promoter in pEW111 (70Hettema E.H. Ruigrok C.C.M. Koerkamp M.G. van den Berg M. Tabak H.F. Distel B. Braakman I. J. Cell Biol. 1998; 142: 421-434Crossref PubMed Scopus (68) Google Scholar). pAN81, encoding a fusion of Gal4BD with Pox1p, was constructed by a PCR on genomic DNA of S. cerevisiae with primers pr34 and pr35. The PCR product was cloned in pGEM-T (Promega) without A-tailing, generating pAN74, which was used as template in a second PCR with primers pr34 and pr52. This PCR product was cloned SalI-SpeI in pPC97 (71Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (479) Google Scholar). pAN82, encoding a fusion of Gal4BD with Pox1p from which the last 3 amino acids had been deleted, was made by a PCR on pAN74 with primers pr34 and pr53. The PCR product was clonedSalI-SpeI in pPC97. pAN83, encoding a fusion of Gal4BD with Pox1p from which the last 17 amino acids had been deleted, was made by a PCR on pAN74 with primers pr34 and pr54. The PCR product was cloned SalI-SpeI in pPC97. pAN88, encoding a fusion of maltose-binding protein (MBP) with Pox1p, was generated by subcloning the XbaI-SpeI insert of pAN81 in theXbaI site of pMAL-c2 (New England Biolabs Inc.). For the construction of pAN87, encoding a MBP fusion with ΔN-Cat2-ΔC, theSacI-HindIII fragment of pEL99 (57Elgersma Y. van Roermund C.W. Wanders R.J. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (162) Google Scholar) was subcloned in pUC19 (New England Biolabs Inc.) generating pAN85. TheEcoRI-HindIII insert of pAN85 was subsequently subcloned in pMAL-c2. pMAL-c2 was used for expression of MBP. pAN37, encoding a fusion of Gal4AD with amino acids 252–612 of Pex5p, was made by PCR on pTI98 with primers p184 and p403. The PCR product was cloned EcoRI-SpeI in pPC86 (71Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (479) Google Scholar). pAN39, encoding a fusion of Gal4AD with amino acids 307–612 of Pex5p, was made by PCR on pTI98 with primers p184 and p405. The PCR product was clonedEcoRI-SpeI in pPC86. pHZ3, encoding a fusion of Gal4AD with amino acids 307–612 of Pex5p, was made by PCR on pTI98 with primers pex5-1 and pex5-427. The PCR product was clonedSalI-SpeI in pPC86. pAN92, encoding a Gal4AD fusion with amino acids 239–300 of Pex5p was generated by PCR on pAN4 with primers pr66 and pr68. The PCR product was clonedEcoRI-SpeI in pPC86. pAN94, encoding a GST fusion with amino acids 239–300 of Pex5p, was generated by PCR on pAN4 with primers pr66 and pr68. The PCR product was clonedEcoRI-SpeI in pRP265nb (38Barnett P. Bottger G. Klein A.T. Tabak H.F. Distel B. EMBO J. 2000; 19: 6382-6391Crossref PubMed Scopus (80) Google Scholar). pRP265nb was used for expression of GST. For introducing single amino acid substitutions, the QuickChange site-directed mutagenesis kit (Stratagene) was used. The oligonucleotides pr64 and pr65 were used for introducing the D262G substitution, and pr62 and pr63 were used for introducing the I264T substitution (see Table I).Table IPrimer compositions Open table in a new tab Subcellular fractionation experiments were performed as described previously (39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar). Protease protection was performed on oleate-grown cells (200 OD units) that were spheroplasted and lysed in hypotonic buffer similar as described for the preparation of homogenates for subcellular fractionation. 20 μg of proteinase K (Roche Molecular Biochemicals) was added to 50 μg of protein sample and incubated with or without Triton X-100 (final concentration 0.15%) at room temperature for 5, 10, 15, and 30 min. Protease activity was stopped by addition of an equal volume of 20% trichloroacetic acid, and proteins were precipitated on ice for a minimum of 1 h. Samples were centrifuged for 30 min at 20,000 × g, and pellets were washed with acetone and resuspended in Laemmli sample buffer (69Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The GST and MBP fusion proteins were expressed and isolated as described previously (38Barnett P. Bottger G. Klein A.T. Tabak H.F. Distel B. EMBO J. 2000; 19: 6382-6391Crossref PubMed Scopus (80) Google Scholar, 39Bottger G. Barnett P. Klein A.T. Kragt A. Tabak H.F. Distel B. Mol. Biol. Cell. 2000; 11: 3963-3976Crossref PubMed Scopus (89) Google Scholar). The in vitro binding assay has also been described before (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Catalase A enzyme activity was measured as described by Lucke (72Lucke H. Bergmeyer H.U. Methods of Enzymatic Analysis. Academic Press, New York1963: 885-894Google Scholar), and β-galactosidase enzyme activity was determined as described before (56Lametschwandtner G. Brocard C. Fransen M. Van Veldhoven P. Berger J. Hartig A. J. Biol. Chem. 1998; 273: 33635-33643Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 73Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). Western blots were incubated with rabbit polyclonal antibodies raised against catalase A, 3-ketoacyl-CoA thiolase, Pex5p (all raised in our own laboratory), Pox1p (a kind gift from Dr. J.M. Goodman, Dallas), NH (a kind gift from Dr. P. van der Sluijs, Utrecht, The Netherlands), GST (Sigma), and mouse monoclonal antibodies against MBP (Sigma). Secondary antibodies used were goat anti-rabbit Ig-conjugated alkaline phosphatase or goat anti-mouse Ig-conjugated alkaline phosphatase. Thepex5 mutant library and the screening procedure forpex5 mutants have been described before (24Klein A.T. Barnett P. Bottger G. Konings D. Tabak H.F. Distel B. J. Biol. Chem. 2001; 276: 15034-15041Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Candida albicans sequences homologous to S. cerevisiae Pex5p and Pox1p were retrieved from the Stanford Genome Technology Center by performing a blast search with these proteins at sequence-www.stanford.edu/group/candida. Contig6–2210 and contig6–2346 contain the C. albicans sequences homologous to S. cerevisiae Pox1p and Pex5p, respectively. S. cerevisiae Pox1p does not contain any recognizable peroxisomal targeting sequence. It is therefore unclear how this protein is imported into the peroxisomal matrix and whether it uses one of the known import receptors, Pex5p or Pex7p. To investigate this we examined the targeting of Pox1p to peroxisomes in wild type, pex5Δ, and pex7Δ cells. Cells were homogenized, and a post-nuclear supernatant was centrifuged at 17,500 ×g. Equivalent volumes of the organellar pellet and the supernatant fractions were analyzed by Western blotting with antibodies specific for Pox1p, the NH tag to detect NH-Mdh3p (a PTS1 protein expressed from a co-transformed plasmid) and 3-ketoacyl-CoA thiolase (a PTS2 protein) (Fig. 1 A). The distribution of catalase A (a PTS1 protein) was determined by measuring the enzyme activity (Fig. 1 B). In wild type cells Pox1p, catalase A, NH-Mdh3p, and thiolase were located in the pellet fraction, indicating that each of these proteins was targeted to peroxisomes. Inpex5Δ cells Pox1p, catalase A and NH-Mdh3p were mislocalized to the supernatant fraction indicating that peroxisomal targeting of Pox1p, like the PTS1 proteins catalase A and NH-Mdh3p, is dependent on Pex5p. Although a significant fraction of NH-Mdh3p was recovered in the organellar pellet, this does not represent peroxisomal import (see below). The localization of the PTS2 protein thiolase was not affected in pex" @default.
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• W2108789322 date "2002-07-01" @default.
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• W2108789322 title "Saccharomyces cerevisiae Acyl-CoA Oxidase Follows a Novel, Non-PTS1, Import Pathway into Peroxisomes That Is Dependent on Pex5p" @default.
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• W2108789322 doi "https://doi.org/10.1074/jbc.m203254200" @default.
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