A mechanism for retromer endosomal coat complex assembly with cargo

Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot–Marie–Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.Sorting of cargo within the endosome determines whether it will be retained and ultimately degraded via lysosome-mediated turnover, or exported via a plasma membrane recycling or retrograde pathway that directs cargo to the TGN or recycling endosome. Genetic dissection of endosomal retrograde pathways in budding yeast (Saccharomyces cerevisiae) led to the identification of an endosome-associated protein complex termed retromer, composed of a Vps5–Vps17 heterodimer and a trimeric complex of the Vps26, Vps29, and Vps35 proteins (1). The retromer trimer, also called the “cargo recognition complex,” is the core functional unit of retromer, serving as a platform for recruiting many other factors to the endosome (2), and we shall refer herein to the trimer as retromer. It is now appreciated that retromer constitutes an ancient, evolutionarily conserved protein sorting complex that operates in multiple endosomal cargo export pathways (2, 3). Hence, elucidating the molecular mechanisms that underlie retromer function is key for understanding the endosomal system.The formation of a vesicular transport carrier is typically initiated by a GTPase module that elicits recruitment of a coat protein from the cytosol to a particular site on the membrane. Retromer is an effector of RAB7A henceforth referred to as RAB7 (human) or Ypt7 (yeast)], a GTPase regulator of endosome dynamics and depletion of RAB7-GTP in cells results in a substantial loss of endosome-associated retromer (4–9). In addition to GTPase signaling modules, interactions of coat proteins with membrane lipids, such as phosphoinositides, contribute to coat assembly by increasing the avidity of membrane binding. There is no evidence that retromer directly recognizes membrane lipids (10, 11). Instead, retromer membrane recruitment is attributed to its association with any of several different sorting nexins (3), which are peripheral membrane proteins defined by the presence of a Phox homology (PX) domain that recognizes phosphatidylinositol 3-phosphate (PtdIns3P), a signature component of endosomal membranes. However, a formal test of this hypothesis is lacking. Retromer binds sorting nexin 3 henceforth referred to as SNX3 (human) or Snx3 (yeast)] (12–15), and in SNX3 knockdown cells, less retromer is associated with endosomes (14). In this study, we show that SNX3 and RAB7 are coordinately recognized by retromer and that these interactions are sufficient to recruit retromer to a membrane where they poise retromer to capture integral membrane retrograde cargo.