Abstract
Endosomes are vital cellular hubs for sorting protein cargoes. Retromer (VPS26/VPS35/VPS29) binds multiple sorting nexin (SNX) proteins on endosomal membranes, but assembly mechanisms of metazoan SNX/Retromer complexes remain elusive. We combine biochemical and biophysical approaches with AlphaFold modeling to identify a previously unidentified direct interaction between SNX27 and VARP. A full biochemical reconstitution system using purified proteins systematically tests how and when coats are recruited to membranes to generate tubules. We demonstrate and measure how specific combinations of Retromer with SNX27, ESCPE-1 (SNX2/SNX6), or both complexes, remodel membranes containing physiological cargo and phospholipids. SNX27, alone and with Retromer, remodels membranes with PI(3)P and PDZbm cargo. ESCPE-1 deforms membranes with bis-phosphoinositides and CI-MPR cargo but surprisingly does not recruit Retromer. VARP co-immunoprecipitates all coat components in cells and is required to reconstitute a proposed endosomal "supercomplex" (SNX27, ESCPE-1, and Retromer) in vitro. These data suggest VARP regulates metazoan endosomal coat assembly to promote cargo sorting out of endosomes.