Abstract
Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.
Highlights
Protein ubiquitination at cysteine residues is a puzzling phenomenon for two reasons
Data on the half-lives of ubiquitin-protein thiol ester conjugates under physiologically relevant conditions are scarce, it is known that ubiquitin thiol esters are disrupted by nucleophiles such as GSH [13], raising the possibility that, to some degree, proteins subjected to this kind of conjugation may undergo futile ubiquitination/deubiquitination cycles
Substitution of Cys11 of Pex5p by a Lysine Results in a Peroxisomal Import/Export-competent Protein—To characterize the properties of the Ub-Pex5p thiol ester conjugate, we started our work by generating a mutant Pex5p protein, Pex5(C11K)p, in which the conserved cysteine residue at position 11 was replaced by a lysine
Summary
Protein ubiquitination at cysteine residues is a puzzling phenomenon for two reasons. Monoubiquitin-Pex5p Thiol Ester Properties results in the insertion of Pex5p into this machinery (stage 2) with the concomitant translocation of the cargo protein across the peroxisomal membrane. Pex5p is monoubiquitinated at a conserved cysteine residue present near the N terminus of the peroxin [7, 10] This monoubiquitinated Pex5p (Ub-Pex5p; stage 3) is dislocated in an ATP-dependent process by Pex1p and Pex6p, two members of the AAA (ATPases associated with various cellular activities) family, yielding a soluble Ub-Pex5p species (stage 4) [7, 22, 23]. The soluble Ub-Pex5p thiol ester conjugate (stage 4) is still a substrate for the DTM, suggesting that ubiquitination of Pex5p does not change its cargo protein binding properties. Our data suggest that treatment of cell cultures with MG132, a proteasome inhibitor [30], leads to a block of the peroxisomal import machinery
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
