Molecular Mechanism of Microdomain Dependent Protein Trafficking

Abstract

Eukaryotic cells are organized into spatially and functionally distinct membrane-bound organelles, whose functions are defined by their composition. Accurate sorting of membrane components between these compartments is necessary for maintaining organelle identity. For most membrane proteins, the determinants of their steady-state subcellular localization remain unknown. Lateral membrane domains (lipid rafts) provide an ideal platform for membrane sorting processes. However, the structural determinants of protein association with such domains are almost entirely unknown. We developed and characterized a robust experimental system for quantitative measurements of raft affinity in intact plasma membranes and used it to explore the determinants of transmembrane protein recruitment into raft domains. We identified several structural features associated with raft affinity, and established a quantitative and functional relationship between raft association and subcellular protein localization. Specifically, we observed that raft association is fully sufficient for PM recycling of certain proteins, and that abrogation of raft partitioning for these proteins led to their degradation in the lysosomes. These findings support the conclusion that ordered membrane domains mediate recycling of specific membrane components from the endosomal compartments to the PM. We have proceeded to define the molecular machinery that mediates raft lipid and protein sorting and recycling to the PM. Using a set of orthogonal transmembrane proteins as probes of raft and non-raft domains, we developed a high throughput siRNA screen to dissect the molecular machinery and dynamics for raft-mediated sorting. We identified a number of validated hits including known players of the early endocytic traffic (Rab5 and EEA1), but also novel players that appear to define a distinct class of trafficking mediators specific for raft-associated proteins. This pathway is not dependent on the classical recycling pathways defined by Rab4 and Rab11, rather defining a novel route for PM recycling of raft-preferring cargo.