Interaction of yeast V-ATPase with TLDc protein Rtc5p

The eukaryotic vacuolar H⁺-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V₁-ATPase and V_o proton channel subcomplexes, a mode of regulation conserved from yeast to humans. While signals that govern V-ATPase assembly have been studied in the cellular context, the molecular mechanisms of the process at the level of the enzyme remain poorly understood. We recently discovered that Oxr1p, one of the two TLDc domain proteins in yeast, is essential for rapid V-ATPase disassembly in vivo. How the second TLDc protein, Rtc5p, functions in reversible disassembly, however, is less clear. Here we find that Rtc5p promotes assembly of functional holo V-ATPase from purified V₁ and V_o subcomplexes in vitro. CryoEM structures show that Rtc5p's TLDc domain binds the C-terminal domain of the V₁-B subunit, with Rtc5p's C-terminal α-helix inserting into the catalytic hexamer, thereby opening a second catalytic site distal to its binding site. Unlike Oxr1p, however, which when deleted produces a distinct phenotype, Rtc5p does not appear to be essential for glucose driven enzyme (re)assembly, hinting at the presence of multiple assembly pathways in vivo.