Pathways and Molecular Mechanisms of Microdomain-Dependent Membrane Trafficking

Abstract

The plasma membrane is the physical barrier and communication interface between the cell and its environment. The lipid and protein composition of this membrane is tightly controlled by endocytic recycling; however, the sorting and trafficking mechanisms mediating efficient recycling are not known. One hypothesis implicates membrane microdomains known as lipid rafts. Such domains are inherently selective for certain proteins and serve to organize and concentrate many distinct cargoes, including lipids. We have developed and characterized a robust experimental system for direct, quantitative measurements of raft affinity in intact plasma membranes and used it to explore the determinants of protein recruitment into raft domains and its consequences on subcellular traffic. We identified several structural features associated with raft affinity, and established that raft association was fully sufficient for PM recycling of certain proteins. Abrogation of raft partitioning for these proteins led to their degradation in lysosomes. These findings strongly support a model wherein ordered membrane domains mediate PM recycling in the endosomal system. Using a set of proteis probes for raft and non-raft domains, we developed a high throughput screen to dissect the molecular machinery and pathways of raft-mediated sorting in endocytic and biosynthetic pathways. We identified several known players of the early endocytic traffic, but also novel players that define a distinct class of trafficking mediators specific for raft-associated proteins. We implicate Rab3 as a central regulator of this pathway, and show that it is essential for PM homeostasis, as abrogation of Rab3 or other pathway effectors dramatically disrupts PM lipidomes and proteomes. Thus, our findings reveal a fundamental role for raft microdomains in endocytic sorting and recycling and support a novel role for Rab3 as a central regulator of this previously unrecognized mechanism for PM and endosome homeostasis.