Functional domains and dynamic assembly of the peroxin Pex14p, the entry site of matrix proteins.
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 topogenic 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.
Itoh
Ryota
R
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Fujiki
Yukio
Y
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002374:Catalase / Q000378:metabolism; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; C101884:PEX13 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; C499193:Pex14 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; C087343:PEX19 protein, human; D002374:Catalase