Turnabout is Fair Play: Mutual Regulation of Palmitoylation and Raft Partitioning

Abstract

The physical basis of protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by non‐ionic detergents. We have used Giant Plasma Membrane Vesicles (GPMVs), a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to analyze the structural determinants for raft partitioning of the transmembrane protein Linker for Activation of T‐cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines. This palmitoylation requirement for raft association was also shown for the majority of integral raft proteins. Independently, systematic mutations that disrupted raft partitioning showed a causal relationship between raft association and plasma membrane (PM) localization, with non‐raft mutants mis‐sorted to late endosomes and depalmitoylated by the lysosomal protein thioesterase PPT1. These findings implicate lipid‐driven phase separation as one mechanism for protein sorting at the late stage of the secretory pathway and suggest mutual regulation of palmitoylation and raft partitioning of LAT.