Abstract
Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.
Highlights
Peroxisomes are ubiquitous eukaryotic organelles bounded by a single membrane
Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import
We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway
Summary
Pex4p1⁄7Pex22p complexes [29, 30]. for Pex5p, it was proposed that ubiquitination results in proteasomal degradation. Ubiquitination of proteins requires the sequential action of at least three types of enzymes; they are a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (for review, see Ref. 31). Polyubiquitinated proteins (i.e. with a chain of at least four ubiquitin molecules) are usually recognized and degraded by the proteasome [32]. We show for the first time that Pex5p is a monoubiquitinated and stable protein in wild type cells. Pex5p monoubiquitination takes place at the peroxisome and is blocked in cells lacking functional docking or RING finger complexes, suggesting that Pex5p monoubiquitination is a late event in peroxisomal matrix protein import. Monoubiquitination of Pex5p in wild type cells is not dependent on Ubc4p, and peroxisome biogenesis is not affected in cells lacking Ubc4p. On the basis of these findings we propose distinct functions for Pex5p monoubiquitination and polyubiquitination
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