Studies on the Role of Subunit d for V‐ATPase Reversible Disassembly

Abstract

V1V0‐ATPase proton pumps are multisubunit proteins consisting of integral and peripheral subunits regulated by reversible disassembly in response to the extracellular concentration of glucose; it is hypothesized that disassembly is an energy conserving strategy to preserve ATP due to limited glucose. In the absence of glucose, the peropheral sub‐complex (V1) is released into the cytoplasm and the membrane sub‐complex (Vo) remains integral. The disassembled enzyme is catalytically inactive but addition of glucose to glucose‐deprived cells yields reassembly of a functional holoenzyme. This project studies subunit d, a key subunit coupling ATP hydrolysis to proton transport. Subunit d remains bound to the membrane upon disassembly and interacts with different V1 and Vo subunits depending on the V‐ATPase's state of assembly. We used computational models of subunit d to introduce cysteine residues that will be photo‐chemically cross‐linked to neighboring V‐ATPase subunits using the sulfhydryl reagent MBP. This study will map subunit interactions involving subunit d in the assembled and disassembled states. It will generate new information regarding the roles of subunit d for V‐ATPase function and the mechanism of glucose‐dependent dissociation.Supported by NSF