title: Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling.
abstract: Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Kanji
authors.initials: K
authors.lastname: Okumoto
authors.forename: Sachi
authors.initials: S
authors.lastname: Misono
authors.forename: Non
authors.initials: N
authors.lastname: Miyata
authors.forename: Yui
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Satoru
authors.initials: S
authors.lastname: Mukai
authors.forename: Yukio
authors.initials: Y
authors.lastname: Fujiki
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D008565:Membrane Proteins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; C499194:Pex14 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D003545:Cysteine; D004229:Dithiothreitol