Journal
TRAFFIC
Volume 12, Issue 8, Pages 1067-1083Publisher
WILEY-BLACKWELL
DOI: 10.1111/j.1600-0854.2011.01217.x
Keywords
CHO cell mutant; dominant-negative mutant; peroxin; peroxisome biogenesis; peroxisome-targeting signal; Pex5p; protein import; PTS1 receptor; ubiquitination
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Funding
- Science and Technology Agency of Japan
- Ministry of Education, Culture, Sports, Science, and Technology of Japan
- Japan Foundation for Applied Enzymology
- Takeda Science Foundation
- Grants-in-Aid for Scientific Research [19058011] Funding Source: KAKEN
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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 cysteine11 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 cysteine11 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.
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