Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol

Abstract

<p class="first" id="d8935555e350">PEX1 and PEX6 are two members of the <span style="text-decoration: underline">A</span>TPases <span style="text-decoration: underline">a</span>ssociated with diverse cellular <span style="text-decoration: underline">a</span>ctivities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1–PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5–interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free <i>in vitro</i> system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a <i>bona fide</i> substrate of the PEX1–PEX6 complex. </p>