Abstract
Accurate localization of membrane proteins is essential for proper cellular functioning and the integrity of cellular membranes. Post-translational targeting of peroxisomal membrane proteins (PMPs) is mediated by the cytosolic chaperone PEX19 and its membrane receptor PEX3. However, the molecular mechanisms underlying PMP targeting are poorly understood. Here, using biochemical and mass spectrometry analysis, we find that a conserved PEX19 helix, αd, is critical to prevent improper exposure of the PEX26 transmembrane domain (TMD) to cytosolic chaperones. Furthermore, the αd helix of PEX19 interacts with the cytosolic domain of the PEX3 receptor, thereby triggering PEX26 release at the correct destination membrane. The peroxisome-deficient PEX3-G138E mutant completely abolishes this secondary interaction, leading to lack of PEX3-induced PEX26 release from PEX19. These findings elucidate a dual molecular mechanism that is essential to membrane protein protection and destination-specific release by a molecular chaperone.