Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain

Mohammad H Fakieh,Joanne M Munck,Ewald H Hettema,Joanne Lacey,Peter J M Drake,Alison M Motley

doi:10.1242/bio.20134788

Abstract

SummaryPex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway.

Highlights

Membrane proteins of the endomembrane system are first inserted into the ER before they are sorted to their cellular destinations
Summary Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation
It is inserted into the ER membrane and sorted via an ER subdomain to peroxisomes

Summary

Introduction

Membrane proteins of the endomembrane system are first inserted into the ER before they are sorted to their cellular destinations. After entry into the ER, most of these proteins follow the secretory pathway (Fig. 1A), i.e. are incorporated into COPII transport vesicles and are transported to the Golgi. Many of these proteins have specific signals that bind either directly or via a cargo receptor to COPII components. Some proteins are sorted to subdomains of the ER or to compartments that originate from this organelle, for example, lipid bodies and peroxisomes (Lynes and Simmen, 2011). Intra-ER sorting is a poorly understood process but signals and protein elements that mediate these sorting processes are being identified (Castillon et al, 2009; Ingelmo-Torres et al, 2009; Lynes and Simmen, 2011; Ronchi et al, 2008; Watanabe and Riezman, 2004)

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