Shuttling Mechanism of Peroxisome Targeting Signal Type 1 Receptor Pex5: ATP-Independent Import and ATP-Dependent Export

Abstract

Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol. To address molecular mechanisms underlying Pex5 functions, we constructed a cell-free Pex5 translocation system with a postnuclear supernatant fraction from CHO cell lines. In assays using the wild-type CHO-K1 cell fraction, 35 S-labeled Pex5 was specifically imported into and exported from peroxisomes with multiple rounds.35 S-Pex5 import was also evident using peroxisomes isolated from rat liver. ATP was not required for 35 S-Pex5 import but was indispensable for export. 35 S-Pex5 was imported neither to peroxisome remnants from RING peroxin-deficient cell mutants nor to those from pex14 cells lacking a Pex5-docking site. In contrast, 35 S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, 35 S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export. Moreover, we analyzed the 35 S-Pex5-associated complexes on peroxisomal membranes by blue-native polyacrylamide gel electrophoresis.35 S-Pex5 was in two distinct, 500-and 800-kDa complexes comprising different sets of peroxins, such as Pex14 and Pex2, implying that Pex5 transited between the subcomplexes. Together, results indicated that Pex5 most likely enters peroxisomes, changes its interacting partners, and then exits using ATP energy.

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…Little is known about the mechanistic details of translocation and cargo release, although receptor import has been shown to be an ATP-independent process Miyata and Fujiki, 2005). Evidence has suggested Pex5p may be involved in import cascades, or that a population of Pex5p may form an import pore in the membrane for a second cargoloaded species.…”

Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes

“…PK treatment was performed as described in ref. 39. Briefly, 14A2.6 cells were grown as described above.…”

Distinct Modes of Ubiquitination of Peroxisome-targeting Signal Type 1 (PTS1) Receptor Pex5p Regulate PTS1 Protein Import

“…Pex5p then releases the cargo proteins into the peroxisomal matrix, which is mediated by the association of Pex5p with a putative import machinery, including a docking complex (Pex14p and Pex13p) and a translocation complex comprising three RING peroxins, Pex2p, Pex10p, and Pex12p (6,7). Finally, Pex5p shuttles back to the cytosol in a manner dependent on ATP and AAA family peroxins, Pex1p and Pex6p (16,17).…”