V-ATPases are highly conserved proton pumps responsible for organelle acidification in all eukaryotic cells and for proton transport across plasma membranes in some settings. V-ATPase activity is associated with multiple disease states including osteoporosis, cancer, and neurodegeneration, making regulation of V-ATPase activity an attractive therapeutic target. However, the therapeutic promise of V-ATPase regulation has not been realized, in part because of the difficulty of targeting specific V-ATPase subpopulations. Reversible disassembly of the V1 and Vo sectors is a major mechanism of V-ATPase regulation. Disassembly inactivates the ATPase activity of V1 and closes the proton pore in Vo, effectively inhibiting ATP-driven proton transport, but is rapidly reversible in vivo. The molecular mechanisms governing this process are not well understood. The yeast RAVE (Regulator of the ATPase of Vacuoles and Endosomes) complex is required for efficient reassembly of V-ATPases. Rabconnectin complexes in higher eukaryotes are functional and structural homologues of yeast RAVE that are involved in assembly and activation of certain V-ATPase subpopulations. We propose to elucidate structural and mechanistic features of the yeast RAVE complex in order to better understand the mechanism of reversible disassembly and the involvement of RAVE/rabconnectins in V- ATPase regulation.
Aim 1 builds on recent advances that allow purification of milligram quantities of yeast RAVE and RAVE-V1 complexes for the first time. We will address the molecular architecture of cytosolic RAVE and RAVE-V1 complexes through crosslinking-mass spectrometry, single particle cryo-EM, and structural analysis of core sub-complexes. These data will be integrated with preliminary data mapping regions of interaction within the RAVE complex and between RAVE and V1 subunits.
Aim 2 focuses on the isoform specificity and glucose sensitivity of interactions between the RAVE complex and the Vo membrane domain. The yeast RAVE complex distinguishes between the two Vo a-subunit isoforms (Vph1 and Stv1) in yeast, and only V-ATPases containing the Vph1 isoform require RAVE for assembly and function. We will dissect how RAVE distinguishes Vph1 from Stv1, in order to better predict which mammalian V-ATPases might depend on rabconnectins for assembly. We will also use chemically induced heterodimerization to assess whether RAVE actively catalyzes V-ATPase reassembly or simply brings V1 and Vo subcomplexes into proximity in a glucose- dependent manner. Finally, we will test the mechanism through which glucose dictates the timing of V-ATPase reassembly and controls the RAVE-Vph1 interaction, and further dissect the kinetics and order of assembly events in vitro. Little is known about the structural underpinnings or mechanism of RAVE/rabconnectin- induced V-ATPase assembly. These experiments address these gaps and lay the groundwork for control of activity in defined V-ATPase subsets through manipulation of rabconnectin activity.
V-ATPases present in all cells help control pH, degrade defective proteins, and respond to different types of environmental stress. Their dysfunction is associated with neurodegenerative diseases, cancer, and osteoporosis, but it has been difficult to specifically target subpopulations of V-ATPases therapeutically. We are characterizing a conserved assembly complex that regulates specific V-ATPase subpopulations and has also been implicated in cancer and neurological disease; targeting this complex could allow specific regulation of V-ATPase populations involved in disease.