Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes

Abstract

PEX13 and PEX14 are two core components of the so‐called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease‐protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin‐Cout topology, and that PEX13 adopts a Nout‐Cin topology, thus exposing its carboxy‐terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. Enzymes Trypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/4.html; Proteinase K, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/64.html; Tobacco etch virus protease, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/22/44.html.

The importomer is a peroxisomal membrane protein translocase

“…We observed neither species, however, when Triton X-100 was included in the reaction, nor did we see protected fragments containing the C-terminal MYC epitope under any condition tested ( Figure 1B). Our results confirm that yeast Pex14 has the expected N matrix -C cytosol topology similar to human Pex14 (Barros- Barbosa et al, 2019).…”

“…Second, it is directly targeted to peroxisomes for integration. We considered the conserved PMP Pex14 as such a model because a rigorous study established that mammalian Pex14 has a single-pass topology with an N-terminal domain (NTD) facing the matrix (Barros- Barbosa et al, 2019). Indeed, we could confirm by in vitro protease protection analysis that yeast Pex14 has the same expected topology.…”

The intrinsically disordered nature of the peroxisomal protein translocation machinery

“…Both the N-and C terminus of PEX13 have been described as being exposed into the cytosol [130,131]. However, recent data from our laboratory suggest that the C-terminal SH3 domain of mammalian PEX13 actually faces the organelle matrix [127].…”

“…[125] for a review). However, most of the available evidence suggests that PEX14 is a bitopic membrane protein with a N in -C out topology, thus possessing its NTD inside the peroxisome [122,[125][126][127].…”

Towards the molecular architecture of the peroxisomal receptor docking complex

“…Mammalian PEX14 contains only one Pex5p binding site and only a single, shortened central coiled-coil domain 25 , which has been shown to mediate dimerization, whereas the transmembrane domain is responsible for formation of higher order assemblies 26 . The topology of Pex14p at the peroxisomal membrane has been controversial, but recent results indicate that the Nterminus faces the matrix of the organelle 27 . Pex17p contains a putative transmembrane domain near the N-terminus and two short coiled-coil domains (Figure 1a).…”