Pex6 and ubiquitination regulate topological remodeling of the peroxisomal membrane protein Pex14.

Pex14 is a membrane peroxin that plays a central role in matrix protein import by mediating the docking of the cytosolic receptor Pex5, which delivers cargo harboring a peroxisome targeting signal 1. We previously reported the crystal structure of the conserved N-terminal domain of Pex14, which harbors the primary binding site for Pex5. However, the mechanistic contribution of this domain to the import process, particularly regarding its membrane orientation, remains unclear. In this study, we investigated the role of the AAA+ ATPase peroxin Pex6 in regulating the membrane topology of the Pex14 N-terminal domain. By a combination of immunofluorescence microscopy and protease protection assays, we show that the orientation of the Pex14 N terminus is dynamically modulated in an ATP- and ubiquitination-dependent topological remodeling. Under normal culture conditions, the N-terminal domain of Pex14 is oriented toward the peroxisomal lumen. Deficiency of Pex6 or its membrane-recruiting partner Pex26, as well as pharmacological inhibition of AAA+ ATPases, resulted in a marked topological remodeling, exposing the Pex14 N terminus to the cytoplasm. Conversely, inhibition of ubiquitin activation using MLN-7243 prevented this reorientation, likely by blocking Pex5 ubiquitination and its subsequent extraction from the membrane. These findings support a model in which Pex14 undergoes reversible, ATP-dependent topological remodeling during Pex5 recycling, functioning as a molecular reset mechanism for the docking-translocation complex. Our study reveals an additional mechanism of regulation in peroxisomal protein import and highlights the coordinated roles of Pex6 and Pex5 ubiquitination in maintaining the structural organization of the translocation machinery.