Vps1 drives membrane constriction and fission necessary for endosomal protein sorting.

The trafficking of cargo between endosomes and the Golgi apparatus uses both retromer-dependent and retromer-independent routes. Disruptions to these routes lead to the mis-sorting of lysosomal cargo, and associated metabolic and neurological disorders. The yeast dynamin Vps1 is essential for these trafficking pathways; however, it is not clear whether it directly causes membrane fission. Using cell-free reconstitution and live-cell assays, here we demonstrate that Vps1 assembles into scaffolds on membrane tubules, and uses GTP hydrolysis to force tubule constriction and fission. Vps1 mutants that are unable to assemble or to hydrolyze GTP fail to achieve fission in vitro and cause cargo mis-sorting in vivo. Furthermore, we identify two essential motifs, a lysine-rich phosphoinositide-binding motif and a phenylalanine-rich self-assembly motif, which, when mutated, render Vps1 dysfunctional. Finally, quantitative proteomics revealed a broad range of Golgi and plasma membrane proteins that mis-sort to the vacuole without Vps1. These findings define the Vps1-dependent retrograde pathway's cargo repertoire and confirm Vps1's mechanochemical role in membrane fission.