Title: Functional Domains and Dynamic Assembly of the Peroxin Pex14p, the Entry Site of Matrix Proteins
Abstract: The 41-kDa membrane-anchored peroxin Pex14p functions as the peroxisome targeting signal (PTS) receptor-mediated, initial import site for matrix proteins. We here identify the functional domains of Pex14p involved in the assembly of import site subcomplexes. The minimal region of Pex14p required for restoring impaired protein import in pex14 Chinese hamster ovary cell mutant lies at residues 21-260 in the primary sequence. A highly conserved N-terminal region, encompassing residues 21-70, interacts with the PTS1 receptor Pex5p, Pex13p, and Pex19p that is essential for membrane biogenesis. N-terminal residues 21-140, including a hydrophobic segment at 110-138, function as a topo-genic sequence. Site-directed mutagenesis, size fractionation, and chemical cross-linking analyses demonstrate that the coiled-coil domain at residues 156-197 regulates homodimerization of Pex14p. Moreover, AXXXA and GXXXG motifs in the transmembrane segment mediate homomeric oligomerization of Pex14p, giving rise to assembly of high molecular mass complexes and thereby assuring Pex13p-dependent localization of Pex14p to peroxisomes. Pex5p, Pex13p, and Pex19p bind to Pex14p homo-oligomers with different molecular masses, whereas cargo-unloaded Pex5p apparently disassembles Pex14p homo-oligomers. Thus, Pex14p most likely forms several distinct peroxin complexes involved in peroxisomal matrix protein import. The 41-kDa membrane-anchored peroxin Pex14p functions as the peroxisome targeting signal (PTS) receptor-mediated, initial import site for matrix proteins. We here identify the functional domains of Pex14p involved in the assembly of import site subcomplexes. The minimal region of Pex14p required for restoring impaired protein import in pex14 Chinese hamster ovary cell mutant lies at residues 21-260 in the primary sequence. A highly conserved N-terminal region, encompassing residues 21-70, interacts with the PTS1 receptor Pex5p, Pex13p, and Pex19p that is essential for membrane biogenesis. N-terminal residues 21-140, including a hydrophobic segment at 110-138, function as a topo-genic sequence. Site-directed mutagenesis, size fractionation, and chemical cross-linking analyses demonstrate that the coiled-coil domain at residues 156-197 regulates homodimerization of Pex14p. Moreover, AXXXA and GXXXG motifs in the transmembrane segment mediate homomeric oligomerization of Pex14p, giving rise to assembly of high molecular mass complexes and thereby assuring Pex13p-dependent localization of Pex14p to peroxisomes. Pex5p, Pex13p, and Pex19p bind to Pex14p homo-oligomers with different molecular masses, whereas cargo-unloaded Pex5p apparently disassembles Pex14p homo-oligomers. Thus, Pex14p most likely forms several distinct peroxin complexes involved in peroxisomal matrix protein import. Peroxisome is a ubiquitous, spherical organelle present in virtually all of eukaryotes, from yeast to mammals. Peroxisome functions in a wide variety of metabolic pathways, including β-oxidation of very long chain fatty acids and biosynthesis of plasmalogens (1van den Bosch H. Schutgens R.B.H. Wanders R.J.A. Tager J.M. Annu. Rev. Biochem. 1992; 61: 157-197Crossref PubMed Scopus (733) Google Scholar). More than 30 peroxins have been identified (2Gould S.J. Valle D. Trends Genet. 2000; 16: 340-345Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 3Matsumoto N. Tamura S. Fujiki Y. Nat. Cell Biol. 2003; 5: 454-460Crossref PubMed Scopus (146) Google Scholar, 4Yan M. Rayapuram N. Subramani S. Curr. Opin. Cell Biol. 2005; 17: 376-383Crossref PubMed Scopus (100) Google Scholar). The import processes of matrix proteins include: 1) recognition of peroxisomal targeting signals 1 and 2 (PTS1 and PTS2) 2The abbreviations used are: PTS1 and PTS2, peroxisome targeting signal types 1 and 2; BS3, bis(sulfosuccinimidyl)-suberate; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; MDH, malate dehydrogenase; GST, glutathione S-transferase; PMP, peroxisomal membrane protein; PMSF, phenylmethylsulfonyl fluoride; PNS, postnuclear supernatant; TM, transmembrane; ER, endoplasmic reticulum; NTA, nitrilotriacetic acid. 2The abbreviations used are: PTS1 and PTS2, peroxisome targeting signal types 1 and 2; BS3, bis(sulfosuccinimidyl)-suberate; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; MDH, malate dehydrogenase; GST, glutathione S-transferase; PMP, peroxisomal membrane protein; PMSF, phenylmethylsulfonyl fluoride; PNS, postnuclear supernatant; TM, transmembrane; ER, endoplasmic reticulum; NTA, nitrilotriacetic acid. of proteins by cytosolic receptors, Pex5p and Pex7p; 2) translocation and docking of the cargo protein-PTS receptor complexes to a potential import machinery on peroxisome membranes; and 3) unloading of cargoes into the matrix. In mammals, the longer isoform of Pex5p, termed Pex5pL, also functions as Pex7p-PTS2 transporter (5Otera H. Harano T. Honsho M. Ghaedi K. Mukai S. Tanaka A. Kawai A. Shimizu N. Fujiki Y. J. Biol. Chem. 2000; 275: 21703-21714Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). Potential import machinery complexes on peroxisome membranes contain several peroxins, including Pex14p, Pex13p, and RING peroxins such as Pex2p, Pex10p, and Pex12p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar, 7Agne B. Meindl N.M. Niederhoff K. Einwaechter 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 (194) Google Scholar). Pex14p plays a central role in matrix protein import (5Otera H. Harano T. Honsho M. Ghaedi K. Mukai S. Tanaka A. Kawai A. Shimizu N. Fujiki Y. J. Biol. Chem. 2000; 275: 21703-21714Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar, 8Albertini 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, 9Will 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, 10Girzalsky 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, 11Shimizu 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). Biochemical functions of other members of the import machinery, such as Pex13p and RING peroxins remain to be better defined.We used genetic phenotype complementation screening of the peroxisome-deficient Chinese hamster ovary (CHO) cell mutants, ZP110 and ZP161, to isolate mammalian PEX14 that encoded a 41-kDa integral membrane peroxin (11Shimizu 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). Pex14p comprises three distinct parts, a highly conserved N-terminal region containing a hydrophobic sequence, a typical coiled-coil domain in the middle region, and a C-terminal part. In mammals, interactions of Pex14p with Pex5p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar, 12Schliebs W. Saidowsky J. Angianian 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) and Pex13p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar, 13Fransen M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (135) Google Scholar) as well as Pex19p (14Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (237) Google Scholar, 15Fransen 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, 16Matsuzono Y. Fujiki Y. J. Biol. Chem. 2006; 281: 36-42Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) have been reported. However, it is uncertain whether these three peroxins, Pex5p, Pex13p, and Pex19p, share the same domain of the N-terminal part of Pex14p in the interaction. Moreover, Pex14p monomer interacts itself (11Shimizu 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, 17Fransen M. Brees C. Ghys K. Amery L. Mannaerts G.P. Ladant D. Van Veldhoven P.P. Mol. Cell Proteom. 2002; 1.3: 243-252Abstract Full Text Full Text PDF Scopus (63) Google Scholar), and the middle region containing the coiled-coil domain is suggested to be required for oligomerization of Pex14p (18Oliveira M.E.M. Reguenga C. Gouveia A.M.M. Guimaraes C.P. Schliebs W. Kunau W.-H. Silvaa M.T. Sa-Miranda C. Azevedo J.E. Biochim. Biophys. Acta. 2002; 1567: 13-22Crossref PubMed Scopus (40) Google Scholar).We here attempted to unravel the molecular mechanisms underlying the import of matrix proteins into peroxisomes. We searched for minimal regions of Pex14p essential for biological activities, including phenotype complementation of CHO pex14 mutants (11Shimizu 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) and interaction with other peroxins. We show that the well conserved sequence in the N-terminal portion of Pex14p is required for peroxisomal targeting and interaction with several peroxins including Pex5p and Pex13p. Moreover, Pex14p forms a homodimer via the coiled-coil domain, while the GXXXG and AXXXA motifs in the hydrophobic domain mediate formation of high molecular mass complexes of Pex14p, thereby ensuring peroxisomal localization of Pex14p. We also discuss dynamic assembly of Pex14p in peroxisome biogenesis.EXPERIMENTAL PROCEDURESBiochemicals—Restriction enzymes and DNA-modifying enzymes were purchased from Nippon Gene (Tokyo, Japan) and TaKaRa (Kyoto, Japan). We used rabbit antibodies to human catalase (3Matsumoto N. Tamura S. Fujiki Y. Nat. Cell Biol. 2003; 5: 454-460Crossref PubMed Scopus (146) Google Scholar), PTS1 peptide (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar), 3-ketoacyl-CoA thiolase (19Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), Pex5p (5Otera H. Harano T. Honsho M. Ghaedi K. Mukai S. Tanaka A. Kawai A. Shimizu N. Fujiki Y. J. Biol. Chem. 2000; 275: 21703-21714Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), Pex13p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar), Pex14p (11Shimizu 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), Pex19p (20Matsuzono Y. Kinoshita N. Tamura S. Shimozawa N. Hamasaki M. Ghaedi K. Wanders R.J.A. Suzuki Y. Kondo K. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2116-2121Crossref PubMed Scopus (187) Google Scholar), 70-kDa peroxisomal membrane protein (PMP70) (19Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar), and malate dehydrogenase (MDH) (21Otera H. Nishimura M. Setoguchi K. Mori T. Fujiki Y. J. Biol. Chem. 2001; 276: 2858-2864Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Rabbit antibodies to green fluorescent protein (Molecular Probes) and calreticulin (Santa Cruz Biotechnology), mouse monoclonal antibody to His6 tag (Qiagen), and guinea pig antibody to Pex14p (22Mukai S. Ghaedi K. Fujiki Y. J. Biol. Chem. 2002; 277: 9548-9561Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) were also used. MitoTracker was from Molecular Probes. Escherichia coli strain XL-1 Blue was used for amplification of plasmids and expression of recombinant proteins.Cell Culture, DNA Transfection, and Morphological Analysis—Wild-type and pex mutant CHO cells and human fibroblasts were cultured as described (19Tsukamoto T. Yokota S. Fujiki Y. J. Cell Biol. 1990; 110: 651-660Crossref PubMed Scopus (186) Google Scholar, 20Matsuzono Y. Kinoshita N. Tamura S. Shimozawa N. Hamasaki M. Ghaedi K. Wanders R.J.A. Suzuki Y. Kondo K. Fujiki Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2116-2121Crossref PubMed Scopus (187) Google Scholar). DNA transfection to CHO cells was done by lipofection (11Shimizu 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). Peroxisomes in cells were visualized by indirect immunofluorescence light microscopy with monospecific rabbit antibodies and fluorescein isothiocyanate- or Texas Red-labeled goat anti-rabbit immunoglobulin G (IgG) antibody (Cappel) (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). As second antibodies, fluorescein isothiocyanate-labeled sheep anti-mouse IgG (Amersham Biosciences) and goat anti-guinea pig IgG (Molecular Probes) antibodies were also used. Enhanced green fluorescent protein (EGFP)-fused proteins were monitored by EGFP fluorescence.Construction of Pex14p Fusion Proteins and Their Variants—Construction of expression plasmids for glutathione S-transferase (GST)-fused Pex14p mutants and His6-tagged PEX14, and introduction of various amino acid substitutions in Pex14p were performed by conventional recombinant DNA methods and described under supplementary information.Purification of Recombinant Proteins and in Vitro Binding Assay— E. coli XL-1 Blue cells were transformed with PEX14 cDNAs in pGEX6P-2 coding for GST and GST fusion proteins of wild-type and mutant GST-Pex14p in pGEX6P-2 and conventionally cultured. GST-Pex14p variants were purified as described (11Shimizu 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). Recombinant Pex5pL, Pex13p, and Pex19p were likewise isolated from respective GST fusion proteins (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). His6-tagged proteins expressed in E. coli were purified as done for GST fusion proteins, except for suspending buffer: 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride (PMSF). Binding of GST-Pex14p variants to Pex5pL, Pex13p, and Pex19p was assayed as described (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). Binding assays using His6-Pex14p and its mutants were similarly done, except that the binding buffer 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 10 mm imidazole, 1 mm PMSF was used.Sedimentation Analysis of Recombinant and Endogenous Pex14p—5 μg each of His6-Pex14p or its mutants were incubated with or without other peroxins and EGFP-PTS1 for 2 h at 4°C in the in vitro binding buffer. The reaction mixture was applied onto the top of continuous 1-30% glycerol gradient in the same buffer and was centrifuged at 30,000 rpm for 18 h at 4 °C in a Beckman SW41Ti rotor. Organelle fraction from CHO-K1 cells were lysed with Triton X-100 in the in vitro binding buffer and analyzed. 16 or 17 aliquots of 600 μl each were collected from the bottom of the tube. Typically 5 μl each from each fraction was analyzed by SDS-PAGE and immunoblot.Cross-linking Experiment—Protein cross-linking was performed using an amino group-reacting reagent, bis(sulfosuccinimidyl)-suberate (BS3, Pierce) (23Partis M.D. Griffiths D.G. Roberts G.C. Beechey R.B. J. Prot. Chem. 1983; 2: 263-277Crossref Scopus (146) Google Scholar). Purified recombinant proteins in PBS, 1% Triton X-100, 1 mm PMSF were incubated at various concentrations of BS3 for 2 h at 4 °C with gentle mixing. Excess BS3 was quenched with 250 mm of Tris-HCl, pH 6.8.Other Methods—Immunoblot analysis was performed as described (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar), using enhanced chemiluminescence Western blotting detection reagent ECL (Amersham Biosciences).RESULTSFunctional Domain Mapping—To define functional domains of Pex14p, we constructed various deletion mutants of hexaHis (His6)-tagged Pex14p (Fig. 1A, upper left), expressed them in CHO pex14 ZP161 cells (Fig. 1A, panel b), and verified for complementation of the impaired matrix protein import by immunofluorescence microscopy. His6-Pex14p expression clearly restored PTS1 import in ZP161 cells, where numerous Pex14p-positive particles were superimposable with PTS1 particles, indicative of peroxisomes (Fig. 1A, panels a-d). An N-terminal deletion mutant His6-Pex14p21-376 showed the PTS1 import-restoring activity as the wild type, whereas further truncated mutants such as His6-Pex14p41-376 showed cytosolic PTS1 proteins (Fig. 1A, panels e-h and Table 1), suggesting that the N-terminal residues at 21-40 is required for the activity. Expression of mutants, His6-Pex14p1-260 and His6-Pex14p1-200 complemented the PTS1 import defect in ZP161, but less efficiently as compared with the wild type (Fig. 1A, panels i and j and Table 1), whereas longer deletions such as His6-Pex14p1-140 and His6-Pex14p1-106 eliminated the activity (Fig. 1A, panels l and n and Table 1). His6-Pex14p21-200, was as potent as His6-Pex14p1-200 (Fig. 1A, panel p), whereas His6-Pex14p121-260 apparently formed inactive aggregates (Table 1). It is noteworthy that catalase remained in the cytosol in ZP161 cells expressing His6-Pex14p1-200 or His6-Pex14p21-200, where PTS1 and PTS2 import was rescued (Table 1). All of the Pex14p constructs competent in restoring the impaired matrix protein import were detected in PTS1-positive, re-established peroxisomes, indicating peroxisomal localization (Fig. 1A and Table 1). Collectively, the minimal sequence of Pex14p competent in complementing the impaired protein import in pex14 cells and peroxisomal membrane targeting comprised the residues at 21-260, including the transmembrane (TM) and coiled-coil domains.TABLE 1Summary of functional domain mapping of Pex14p Peroxisome-restoring activity was verified with CHO pex14 mutant ZP161. Peroxisome targeting was assessed in CHO-K1 cells. Open table in a new tab To assess this issue in regard to the TM and coiled-coil regions, we expressed in ZP161 cells four constructs: His6-Pex14pΔ141-240, deleted in the coiled-coil region; His6-Pex14pΔ110-138, lacking the sequence containing TM segment; His6-Pex14pΔ110-123, deleted in the N-terminal part of TM; and His6-Pex14pΔ131-138. Only His6-Pex14pΔ131-138 complemented the mutant phenotype of ZP161, whereas the other three showed no restoring activity (Fig. 1A, panels q-v and Table 1). Thus, it is apparent that a hydrophobic segment and its downstream coiled-coil domain are essential for the biological activity of Pex14p.Pex14p Requires the N-terminal Portion for Peroxisomal Localization—To search for the peroxisomal targeting sequence of Pex14p, we expressed various deletion mutants of His6-Pex14p (Table 1) in wild-type CHO-K1 cells and verified for their subcellular localization, as in Fig. 1A. His6-Pex14p1-140 was localized to numerous particles, which were coincided with PMP70-stained peroxisomes (Fig. 1B, panels a and e), thereby indicating localization to peroxisomes, despite being incompetent in complementing pex14 ZP161 cells (Fig. 1A, panels k and l). Moreover, His6-Pex14p1-140 was integrated into membranes as His6-Pex14p (supplementary data, Fig. S1). In contrast, His6-Pex14p1-106 showed a diffused staining pattern (Fig. 1A, panel m). Taken together, the N-terminal region at 1-140 was apparently sufficient for targeting to peroxisomes. Distinct from His6-Pex14p21-376, His6-Pex14p41-376 and His6-Pex14p65-376 were observed in larger structures (Fig. 1B, panel d and Table 1), which were coincided with those with MDH, a mitochondrial matrix protein (Fig. 1B, panel h), suggesting that both mutants were mislocalized to mitochondria. Thus, the N-terminal part encompassing residues 21-140 of Pex14p was essential for peroxisomal localization. A middle region mutant His6-Pex14p121-260 apparently aggregated, and His6-Pex14p241-376 showed cytosolic staining (Table 1).His6-Pex14pΔ110-123 and His6-Pex14pΔ110-138 were not colocalized with PMP70, indicating a defect of peroxisomal targeting, whereas His6-Pex14pΔ131-138 maintained the targeting activity (Fig. 1B, panels b and f and Table 1). Pex14pΔ141-240 was detected as numerous small punctate-stained particles, not colocalized with PMP70 (Fig. 1B, panels c and g). These results strongly suggested that both the cluster of hydrophobic residues at 110-123 and the coiled-coil region are essential for Pex14p anchoring to peroxisomal membranes.Next, we generated several EGFP-fused N-terminal parts of Pex14p and expressed in CHO-K1 cells. EGFP-Pex14p1-140 was clearly localized to peroxisomes, as assessed by colocalization with PTS1 proteins (Fig. 1C, panels b and f), whereas EGFP was located in nucleus and cytoplasm (Fig. 1C, panel a). EGFP-Pex14p21-140 was mostly targeted to peroxisomes, but EGFP-Pex14p41-140 was detected in the cytosol and perinuclear region (Fig. 1C, panels c, d, g, and h). These results indicated that the N-terminal residues 21-140 of Pex14p play an indispensable role in translocation to peroxisomes.Pex5p, Pex13p, and Pex19p Bind to the N-terminal Region of Pex14p—To search for Pex14p-interacting partners, we performed a pull-down assay using His6-Pex14p and pex14 ZP161 cell lysates. Pex5p, Pex13p, and Pex19p were specifically detected, where mitochondrial MDH was not bound (Fig. 2A). Other peroxins such as Pex10p, Pex12p, Pex1p, and PMP70 were under the detectable level (not shown). The findings were consistent with other reports describing that Pex14p interacts with three peroxins, Pex5p, Pex13p, and Pex19p (5Otera H. Harano T. Honsho M. Ghaedi K. Mukai S. Tanaka A. Kawai A. Shimizu N. Fujiki Y. J. Biol. Chem. 2000; 275: 21703-21714Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 13Fransen M. Terlecky S.R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8087-8092Crossref PubMed Scopus (135) Google Scholar, 14Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (237) Google Scholar, 24Brocard C. Lametschwandtner G. Koudelka R. Hartig A. EMBO J. 1997; 16: 5491-5500Crossref PubMed Scopus (107) Google Scholar). We therefore determined the precise regions involved in such interaction with three peroxins, Pex5pL, Pex13p, and Pex19p, using various truncated recombinant Pex14p variants fused to GST (Fig. 2B, left panel). Upon incubation, Pex5pL was specifically bound to the full-length and various truncated mutants of GST-Pex14p such as GST-Pex14p31-70 (Fig. 2B, center panel). Similarly, Pex13p was bound to GST-Pex14p and several Pex14p mutants including Pex14p21-70. Together, the sequences at 31-70 and 21-70 in the N-terminal region of Pex14p are required for binding to Pex5p and Pex13p, respectively. Moreover, normal and mutant His6-Pex14p constructs containing the N-terminal residues at 21-70 specifically bound to Pex19p (Fig. 2B, center).FIGURE 2Pex14p interacts with Pex5p, Pex13p, and Pex19p. A, cell lysates of CHO pex14 ZP161 (107 cells) were incubated with mock- and His6-Pex14p-bound Ni-NTA beads (lanes 2 and 3). Bound proteins were analyzed by SDS-PAGE and immunoblotting using specific antibodies. One-tenth of input was loaded in lane 1. B, left, schematic representation of GST-Pex14p variants as in Fig. 1A. Center, interaction of GST- or His6-Pex14p variants with the longer isoform of Pex5p (termed Pex5pL), Pex13p, and Pex19p. GST-Pex14p variants (1 μg each) bound to glutathione-Sepharose beads were incubated with His6-tagged Pex5pL (0.2 μg) or Pex13p (0.2 μg). His6-Pex14p mutants linked to Ni-NTA agarose were also incubated with Pex19p. Bound proteins were analyzed as in A. Lane 1, inputs. Right, Coomassie Blue staining of purified GST-Pex14p mutants (1 μg each); molecular size markers are on the top. C, Pex19p does not affect the import complexes. Left, in vitro binding assays were performed as in B, using His6-Pex14p (10 ng), Pex5pL (0.1μg), Pex13p (0.1μg), and Pex19p (0.1 μg) as indicated at the top. Bound peroxins were detected by immunoblotting. Lanes 1-3, Ni-NTA beads only. Right, binding assays using GST-Pex14p (20 ng) and each set of Pex13p (20 ng) and Pex19p (20 ng and 2 μg), as indicated. D, formation of hetero-oligomer complexes comprising Pex14p, Pex13p, cargo-loaded Pex5p, and Pex19p. Binding assays were performed using GST-Pex14p (10 ng), Pex5p (0.1 μg), Pex13p (0.1 μg), EGFP-His6-SKL (0.1 μg), and increasing amounts of Pex19p. EGFP-His6-SKL was detected with anti-GFP antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pex19p in the Pex14p Matrix Import Subcomplexes—Pex14p forms subcomplexes with cargo-loaded Pex5p and Pex13p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). Pex19p also interacts with Pex14p (Fig. 2, A and B) (14Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (237) Google Scholar, 15Fransen 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, 17Fransen M. Brees C. Ghys K. Amery L. Mannaerts G.P. Ladant D. Van Veldhoven P.P. Mol. Cell Proteom. 2002; 1.3: 243-252Abstract Full Text Full Text PDF Scopus (63) Google Scholar). However, whether Pex19p plays a role in peroxisomal matrix protein import remains elusive. We first examined by in vitro binding assays whether Pex19p alters Pex14p binding to Pex5p and Pex13p. His6-Pex14p bound to Pex19p (Fig. 2C, left panel, lane 5). His6-Pex14p equally bound to Pex5pL in the presence or absence of Pex19p (lanes 6 and 7), where Pex19p binding to His6-Pex14p was not detectable. Pex14p-Pex5pL interaction likely occurred with an affinity much higher than that between Pex14p and Pex19p, as noted between Pex13p and Pex14p (6Otera H. Setoguchi K. Hamasaki M. Kumashiro T. Shimizu N. Fujiki Y. Mol. Cell. Biol. 2002; 22: 1639-1655Crossref PubMed Scopus (171) Google Scholar). In contrast, a slightly reduced level of Pex13p bound to His6-Pex14p was detected in the presence of Pex19p (Fig. 2C, left, lanes 8 and 9) and apparently remained at the same level even with 100-fold excess of Pex19p to Pex13p (Fig. 2C, right). We next verified whether Pex19p binding to Pex14p modulates the complexes comprising Pex14p, Pex13p, and cargo-loaded Pex5p. GST-Pex14p was incubated with a fixed amount of Pex5p, Pex13p, PTS1-cargo EGFP-His6-SKL, and increasing amounts of Pex19p (Fig. 2D). Pex5p was moderately elevated with increasing amounts of Pex19p, whereas EGFP-His6-SKL and Pex13p were slightly decreased. Thus, it is plausible that Pex19p may regulate the assembly of import subcomplexes comprising at least Pex14p, Pex13p, and PTS1-Pex5p.Highly Conserved N-terminal Region of Pex14p—We found that the N-terminal region is indispensable for peroxisomal localization of Pex14p (Fig. 1) and that its highly conserved domain comprising residues at 21-70 is involved in interaction with Pex5p, Pex13p, and Pex19p (Fig. 2B). Sequence comparison of Pex14p from various species (11Shimizu 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, 12Schliebs W. Saidowsky J. Angianian 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, 25Nito K. Hayashi M. Nishimura M. Plant Cell Physiol. 2002; 43: 355-366Crossref PubMed Scopus (83) Google Scholar) showed that the N-terminal region was highly conserved (Fig. 3A). We constructed two His6-Pex14p mutants with Ala substitution of the most conserved residues, Phe35-Leu36 and Phe52-Leu53, termed FL35/36AA and FL52/53AA, respectively (Fig. 3B). Both were completely eliminated in binding to Pex13p, but