Abstract:
The evolutionarily conserved vacuolar protein sorting (VPS) pathway prescribes a mechanism by which newly synthesized proteins are delivered from the Golgi apparatus to the lysosomes/vacuole and back via endosomes. Originally designed based on monitoring cargo trafficking to the yeast vacuole, the VPS screen discovered the Vacuolar Protein Sorting 1 (Vps1) protein, which is a member of the large GTPase dynamin superfamily. Loss of Vps1 elicits phenotypes that range from defects in cargo trafficking to the vacuole to growth defects at high temperature. Despite these insights, the intrinsic functions of Vps1 remain poorly understood. Here, we employ a suite of in vitro assays to probe for protein function in membrane binding and remodelling. We find Vps1 to bind the endosomal PI(3)P lipid, which in turn stimulates its GTPase activity. Furthermore, reconstitution of Vps1 functions on tubular membranes with GTP reveal that PI(3)P binding causes Vps1 to self-assemble into scaffolds that cause membrane fission. Self-assembly and GTPase-defective Vps1 mutants are unable to cause fission and display defects in cargo trafficking to the yeast vacuole. Unlike classical dynamins, Vps1 lacks a folded lipid-binding domain and instead features an unstructured InsertB region. The precise function of this domain remains unclear. We find that deletion of InsertB impairs Vps1 defective in membrane binding and, consequently, membrane fission. Together, these results signify the first report of characterizing the intrinsic functions of Vps1. Expanding on these insights, we analyzed the global impact of loss of Vps1 functions on cargo trafficking pathways from a proteomic analysis of isolated vacuoles. Results from such experiments identify distinct sets of cargo that are enriched or depleted in the vacuole in the absence of Vps1 thereby expanding the repertoire of proteins that require Vps1 for their trafficking to the vacuole. Enrichment at the vacuole can be explained by defects in fission and vesicle release out of the vacuole, while depletion could be caused by defects in trafficking to the vacuole. Thus, these results inform of Vps1-independent fission mechanisms involved in VPS. To address this, we screened yeast lysates for membrane fission using tubular membranes as a template. Preliminary results indicate the existence of proteins that manage membrane fission in a phosphoinositide-specific manner. Together, these findings not only advance our understanding of Vps1 functions in cargo trafficking to and from the vacuolar but also open up avenues for identifying novel Vps1-independent vesicular transport pathways.
