Role of TLDc Proteins Oxr1 and Rtc5 in Yeast V-ATPase Reversible Disassembly

Abstract

The vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is a highly conserved, ATP hydrolysis-driven dedicated proton pump found on the membranes of intracellular organelles in virtually all eukaryotic cells and on the plasma membrane of specialized cell types. Regulation of V-ATPase activity is key to maintaining normal physiological functions, as aberrations in its activity are associated with several pathophysiological conditions. V-ATPase activity is mainly regulated by a mechanism called reversible disassembly, in which the assembly state - and hence the activity - of the enzyme is controlled by nutrient availability and extracellular cues. During the process, V-ATPase activity becomes either turned off by dissociation of the V1-ATPase from the Vo proton channel, or turned on by reassembling the two subcomplexes into an active enzyme. While the process is well-characterized at the cellular level, the molecular mechanism at the level of the enzyme remains elusive. Here, we show that two TLDc proteins, Oxr1p and Rtc5p, control the assembly state of yeast V-ATPase, with the former promoting disassembly, and the latter (re)assembly of the enzyme. Based on cryoEM analysis and in vitro and in vivo approaches, we discovered that Oxr1p is a V-ATPase disassembly factor. Oxr1p binding to V-ATPase results in autoinhibited V1 in two steps - first producing a disassembly intermediate, which, upon ATP hydrolysis, gets converted into autoinhibited V1. From in vitro experiments, we find that the second TLDc protein, Rtc5p, primes autoinhibited V1 for (re)assembly with Vo. CryoEM structures of Rtc5p bound V1 show Rtc5p's C-terminal ⍺ helix inserted into the catalytic core of the enzyme, thereby opening a second catalytic site, a conformational change that may facilitate (re)assembly of V1Vo. In vivo experiments, however, suggest that Rtc5p is not essential for V-ATPase reassembly in the cell, suggesting redundancy and/or alternative pathways. Overall, this study enhances our understanding of the molecular basis for the regulation of V-ATPase activity by reversible disassembly.