Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex. In this work, a cross-linking mass-spectrometry map of protein-protein interactions of yeast vacuoles identifies Rtc5 as a novel interactor of the V-ATPase, a complex required for vacuole acidification. Rtc5 and its paralog Oxr1 regulate in vivo assembly of the V-ATPase complex and subcellular localization of the Golgi-specific V-ATPase isoform.A cross-linking mass-spectrometry-based interactome of yeast vacuoles reproduces known interactions with high fidelity and identifies novel interactions. The TLDc-domain-containing protein of unknown function, Rtc5, is a novel interactor of the vacuolar V-ATPase. The yeast TLDc-domain proteins Oxr1 and Rtc5 promote disassembly of the V-ATPase complex in vivo and counteract the function of the RAVE complex. Oxr1 is required for the retention of the Golgi-specific isoform of the V-ATPase subunit a (Stv1) in pre-vacuolar compartments. Cross-linking mass spectrometry of yeast vacuoles identifies many novel protein-protein interactions, among them a novel regulator of the V-ATPase complex.