| While several protein machineries generate membrane curvature towards the cytoplasm, there is only a single primary effector of membrane curvature away from the cytoplasm---the endosomal sorting complexes required for transport (ESCRTs). The ESCRT proteins are essential for a number of processes in the cell, including multivesicular body (MVB) formation, cytokinesis, autophagy, and viral budding. One factor essential for ESCRT protein function is, Vps4, a AAA+ ATPase. Vps4 ATP hydrolysis is directly implicated in disassembly of the ESCRT-III polymer, and may be required for the MVB sorting reaction (and viral budding, cytokinesis, etc.) to proceed to completion. Functional regulation of Vps4 occurs through interactions with substrate proteins (ESCRTs), and an additional modulating factor, Vta1. Vta1's stimulation of Vps4 activity is biologically significant, and potentiates ESCRT function in MVB sorting and viral budding. This work describes a novel element in Vta1 with Vps4 stimulatory activity (Vta1 stimulatory element; VSE). The VSE not only directly stimulates Vps4, but is also required for enhancement of Vta1-mediated stimulation of Vps4 by the ESCRT-III proteins Vps60 or Did2. Utilizing mutations in the VSE or cognate residues in Vps4 in biochemical studies of ATPase activity, we determined that Vta1-mediated stimulation of Vps4 occurs through two distinct mechanisms: 1) decreasing the concentration of Vps4 required for half-maximal activity by binding of a conserved C-terminal domain (VSL), and 2) stimulation of the Vps4 ATP hydrolysis by Vta1 VSE residues contacting the Vps4 small ATPase domain. In vivo, mutations in the VSE stabilize ESCRT-III on membranes and impact the sorting of MVB cargos, although in a manner distinct from complete loss of Vta1 protein. These results highlight the complex regulation of Vps4 function by Vta1, and provide a basis for additional structural and mechanistic investigation of Vps4 ATPase activity, and its modulation by Vta1, ESCRT-III, and other factors. |
