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Peroxisomal Monoubiquitinated PEX5 Interacts with the AAA Atpases PEX1 and PEX6 and Is Unfolded During Its Dislocation Into the Cytosol Ana G

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Peroxisomal Monoubiquitinated PEX5 Interacts with the AAA Atpases PEX1 and PEX6 and Is Unfolded During Its Dislocation Into the Cytosol Ana G JBC Papers in Press. Published on June 8, 2018 as Manuscript RA118.003669 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.003669 Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol Ana G. Pedrosa1,2,3,#, Tânia Francisco1,2,#, Diana Bicho1,2, Ana F. Dias1,2,3, Aurora Barros- Barbosa1,2, Vera Hagmann4, Gabriele Dodt4, Tony A. Rodrigues1,2,3, Jorge E. Azevedo1,2,3,* From the 1Instituto de Investigação e Inovação em Saúde (i3S), 2Instituto de Biologia Molecular e Celular (IBMC) and 3Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; 4Interfakultäres Institut für Biochemie, Universität Tübingen, , Hoppe Seyler Str.4, 72076 Tübingen, Germany Running title: Ub-PEX5 is a substrate of the PEX1.PEX6 complex # These authors contributed equally to this work. * Corresponding author: Jorge E. Azevedo, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; tel: +351220408800; Downloaded from Email: [email protected] Keywords: ATPases associated with diverse cellular activities (AAA); Peroxisome; PEX5; PEX1; PEX6; ubiquitin; protein sorting; protein translocation http://www.jbc.org/ ABSTRACT INTRODUCTION PEX1 and PEX6 are two members of the Peroxisomes are cytoplasmic organelles ATPases Associated with diverse cellular delimited by a single membrane found in almost at UNIVERSIDADE DO PORTO on November 2, 2018 Activities (AAA) family and the core all eukaryotes (1). In mammals, they harbor a set components of the receptor export module of approximately 100 different proteins and are (REM) of the peroxisomal matrix protein import involved in several metabolic pathways such as machinery. Their role is to extract β-oxidation of fatty acids, synthesis of monoubiquitinated PEX5, the peroxisomal plasmalogens and bile acids, and detoxification protein shuttling receptor, from the peroxisomal of glyoxylate (2–6). Despite their simplicity, membrane docking/translocation module peroxisomes play important roles in human (DTM), so that a new cycle of protein health and development, as demonstrated by a transportation can start. Recent data have shown group of inherited metabolic disorders in which that PEX1 and PEX6 form a heterohexameric peroxisomes are partially or even completely complex which unfolds substrates by processive defective, the peroxisomal biogenesis disorders threading. However, whether the natural (7). These diseases are caused by mutations in substrate of the PEX1.PEX6 complex is genes encoding proteins mechanistically monoubiquitinated PEX5 (Ub-PEX5) itself or involved in several aspects of peroxisome some Ub-PEX5-interacting component(s) of the biogenesis – the so-called peroxins or PEX DTM remains unknown. In this work, we used proteins (6–8). In mammals 16 peroxins are an established cell-free in vitro system coupled presently known, 10 of which are components of with photoaffinity crosslinking and protein the machinery that sorts newly synthesized PEGylation assays to address this problem. We proteins to the organelle matrix (reviewed in provide evidence suggesting that DTM- (9)). embedded Ub-PEX5 interacts directly with both Proteins destined to the peroxisomal PEX1 and PEX6 through its ubiquitin moiety matrix are synthesized in the cytosol and and that the PEX5 polypeptide chain is globally transported to the organelle membrane by PEX5, unfolded during the ATP-dependent extraction the peroxisomal matrix protein shuttling receptor event. These findings strongly suggest that (10–20). There, cargo-loaded PEX5 interacts DTM-embedded Ub-PEX5 is a bona fide with the docking/translocation module (DTM), a substrate of the PEX1.PEX6 complex. multisubunit transmembrane complex comprising the core components PEX2, PEX10, PEX12, PEX13 and PEX14 (21, 22). This 1 Ub-PEX5 is a substrate of the PEX1.PEX6 complex interaction culminates with the insertion of substrates in a pore loop-dependent manner PEX5 into the DTM with the concomitant using a processive threading mechanism were translocation and release of the cargo protein reported very recently (42). Surprisingly, the into the organelle matrix (23–26). No ATP unfolding/threading activity of the PEX1.PEX6 hydrolysis or a membrane potential is needed for complex was detected using a soluble fragment these steps; the driving force for the complete of PEX15 as substrate, but whether or not cargo transport process resides in the strong PEX15 is a natural substrate for these ATPases protein-protein interactions that are established remains unknown, as thoroughly discussed by between PEX5 and DTM components (23, 25, the authors of that work ((42); see also 27). Discussion). After releasing its cargo, PEX5 has to be Here, we asked whether or not DTM- extracted from the DTM so that a new protein embedded Ub-PEX5 displays properties of a transport cycle can be initiated. Recycling of bona fide REM substrate. Our data shows that PEX5 involves three steps. First, DTM- DTM-embedded Ub-PEX5 interacts directly embedded PEX5 is monoubiquitinated at a with PEX1.PEX6 and is globally unfolded conserved cysteine residue (Cys11 in the human during the ATP-dependent extraction step. This Downloaded from protein) (28, 29). Then, monoubiquitinated suggests that Ub-PEX5 is indeed a natural PEX5 (Ub-PEX5) is extracted into the cytosol in substrate of the REM. an ATP hydrolysis-dependent manner by the so- called receptor export module (REM) (27, 30, RESULTS 31). This is a protein complex comprising PEX1 http://www.jbc.org/ and PEX6, two members of the AAA ATPases The results reported below were family, plus a poorly conserved peroxisomal obtained with a cell-free in vitro system that membrane protein (PEX26 in mammals, recapitulates all the steps of the PEX5-mediated APEM9 in plants, or PEX15 in yeasts/fungi) protein import pathway. This experimental which anchors the ATPases to the organelle system was thoroughly described recently (43) at UNIVERSIDADE DO PORTO on November 2, 2018 membrane (32–34). Finally, Ub-PEX5 is but a brief explanation is provided here for deubiquitinated in the cytosol probably by a clarity. The core of the system is a rat liver post- combination of enzymatic and non-enzymatic nuclear supernatant (PNS; the source of events (35–37). peroxisomes and cytosolic proteins), which is Although the general properties of the incubated with an in vitro synthesized PEX5-mediated protein import pathway are radiolabeled PEX5 protein under appropriate known, the mechanistic details of each of its conditions. During this incubation, a fraction of steps remain mostly unclear. In this work, we focused on the mechanism used by the REM to the radiolabeled PEX5 protein becomes inserted extract Ub-PEX5 from the DTM. into the peroxisomal DTM, where it is An abundance of structural/functional subsequently monoubiquitinated at cysteine 11, data was recently reported for the yeast REM. and rapidly exported into the cytosol by the These include electron microscopy structures of REM in an ATP-dependent manner. A number the PEX1.PEX6 complex, an X-ray structure of of strategies can be used to block this pathway the cytosolic domain of PEX15, and the (43). For instance, AMP-PNP and ATPγS, two structure of a trimeric complex containing these ATP analogs used in this work, are excellent three proteins (reviewed in (38); (39–42)). These substrates for the ubiquitin-conjugating cascade studies revealed that PEX1 and PEX6 assemble (36, 44) but potent inhibitors of the REM (36). into a ring-shaped heterohexameric complex, Thus, in vitro assays containing one of these best described as a trimer of PEX1.PEX6 nucleotides will reveal an accumulation of Ub- heterodimers, displaying a relatively large pore PEX5 at the peroxisomal DTM, particularly so if at its center. These data, together with mutational analyses of PEX1 and PEX6 showing the PNS was previously “primed”, i.e., pre- that the conserved hydrophobic residues of the incubated with a small amount of ATP to release so-called pore loops are important for endogenous PEX5 from the DTMs (25). Two PEX1.PEX6 function, might suggest that the types of in vitro assays are used here: the so- PEX1.PEX6 complex uses its pore to handle called single-step and the two-step import/export substrates (39, 40, 42). Robust data showing that assays. The first involves a single incubation of the PEX1.PEX6 complex indeed unfolds the reaction mixture. If such an assay is 2 Ub-PEX5 is a substrate of the PEX1.PEX6 complex performed in the presence of ATP then all the material represents non-ubiquitinated PEX5 steps of the pathway will occur, i.e., specifically interacting with the DTM, the radiolabeled PEX5 will continuously interact majority may represent PEX5 protein non- with the DTM, where it is monoubiquitinated specifically adsorbed to the organelles, some of and subsequently exported into the soluble phase which may even appear in the supernatant of the assay. Thus, if at the end of the incubation fraction of a two-step assay. Thus, no period the PNS is centrifuged to separate conclusions are drawn from the behavior of organelles from soluble proteins, most these non-ubiquitinated PEX5 species. monoubiquitinated PEX5 will be detected in the soluble fraction. In the two-step import/export Neither the N- nor the C-terminus of DTM- embedded Ub-PEX5 is important for the assay, a radiolabeled PEX5 protein is first export step. accumulated at the DTM in the presence of AMP-PNP or ATPγS (the first step of the assay); Many members of the AAA family of the organelles are then isolated by mechanoenzymes explore the presence of centrifugation, ressuspended and incubated in extended N- or C-termini in their substrates to Downloaded from buffer containing ATP (the second step), to unfold or pull them out of protein complexes allow export of the Ub-PEX5 protein into the (46, 47). For instance, katanin, a member of the AAA microtubule-severing family, extracts soluble phase of the assay (36).
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