Structural and Functional Studies of Peroxisome Matrix Protein Import

Abstract

Compartmentalization by intracellular membrane systems is a hallmark of eukaryotic cells. While membranes form barriers to many molecules, transport systems facilitate the exchange of material between compartments (organelles) and thereby establish and maintain their unique compositions. Many organelle-specific transport systems translocate proteins across membranes, such as the Sec61 translocon at the endoplasmic reticulum and the protein import system of the mitochondria. The machinery of peroxisome matrix protein import is unique, but mechanistically poorly understood. Its importance is illustrated by devastating genetic diseases caused by defects in peroxisome biogenesis. Here we report on two studies with a goal of understanding peroxisome protein import. We targeted two systems to define their functions in peroxisome biogenesis. The first centers on Pex1 and Pex6, members of the conserved AAA ATPase family. To understand how these proteins might function, we obtained structures of the Pex1/Pex6 ATPase complex by cryo-electron microscopy. Using new computational modeling methods, we were able to place the domains of these ATPases into subnanometer resolution density maps. We showed that the Pex1/Pex6 complex is arranged in a unique, triangular, double-ring structure in which the two proteins alternate around the hexameric ring. Our results illuminate the mechanism and function of this ATPase. The second study remedies the lack of a robust and reproducible assay of peroxisome protein import, which has been a major hindrance to mechanistic studies in the field. We developed a powerful new in vitro assay. In this assay, we added fluorescent proteins with peroxisomal import signals to Xenopus oocyte extracts and followed import by fluorescence microscopy. Our data show that these proteins are specifically imported into the peroxisome. We provide evidence that the “folded protein hypothesis” is valid, showing that folded proteins can be imported into the peroxisome without first being unfolded. Our work also raises questions about the prevailing hypotheses for the driving force in peroxisome protein import. This assay provides the basis for future mechanistic dissection of peroxisome protein import.