VARP binds SNX27 to promote endosomal supercomplex formation on membranes.

Multiple essential membrane trafficking pathways converge at endosomes to maintain cellular homeostasis by sorting critical transmembrane cargo proteins to the plasma membrane or the trans-Golgi network (TGN). The Retromer heterotrimer (VPS26/VPS35/VPS29 subunits) binds multiple sorting nexin (SNX) proteins on endosomal membranes, but molecular mechanisms regarding formation and regulation of metazoan SNX/Retromer complexes have been elusive. Here, we combine biochemical and biophysical approaches with AlphaFold2 Multimer modeling to identify a direct interaction between the VARP N-terminus and SNX27 PDZ domain. VARP and SNX27 interact with high nanomolar affinity using the binding pocket established for PDZ binding motif (PDZbm) cargo. Specific point mutations in VARP abrogate the interaction in vitro. We further establish a full biochemical reconstitution system using purified mammalian proteins to directly and systematically test whether multiple endosomal coat complexes are recruited to membranes to generate tubules. We successfully use purified coat components to demonstrate which combinations of Retromer with SNX27, ESCPE-1 (SNX2/SNX6), or both complexes can remodel membranes containing physiological cargo motifs and phospholipid composition. SNX27, alone and with Retromer, induces tubule formation in the presence of PI(3)P and PDZ cargo motifs. ESCPE-1 deforms membranes enriched with Folch I and CI-MPR cargo motifs, but surprisingly does not recruit Retromer. Finally, we find VARP is required to reconstitute a proposed endosomal supercomplex containing SNX27, ESCPE-1, and Retromer on PI(3)P-enriched membranes. These data suggest VARP functions as a key regulator in metazoans to promote cargo sorting out of endosomes.