Some reassembly required: Requirements for RAVE‐mediated reassembly of the yeast V‐ATPase
Authors: Jaskolka Michael C , Kane Patricia 
Published: June 2021

Lysosomes and vacuoles are the most acidic organelles in eukaryotic cells and play a central role in cellular metabolism – serving as a location that coordinates anabolic and catabolic processes. This coordination requires the activity of a highly conserved proton pump, the Vacuolar H+‐ATPase (V‐ATPase). Interestingly, both V‐ATPase activity and a cell's metabolic state are dependent on glucose levels. Glucose deprivation induces the V‐ATPase to disassemble into 3 distinct subcomplexes: V1, V1C, and Vo. V1 and V1C are released from Vo at the vacuolar membrane, and this disassembly silences both ATP hydrolysis in V1 and proton transport through Vo. Reassembly occurs rapidly but requires both glucose readdition and a conserved V‐ATPase‐specific assembly factor known as the RAVE (Regulator of the H+‐ATPase of Vacuolar and Endosomes) complex in yeast. The RAVE complex, composed of Rav1, Rav2, and Skp1, interacts with each V‐ATPase subcomplex and, upon glucose readdition, recruits cytosolic V1 and V1C to Vo at the vacuolar membrane to allow V‐ATPase reassembly (Smardon et al., (2015) J. Biol. Chem 290:27511). Although RAVE is essential for efficient V‐ATPase reassembly, how RAVE targets the vacuolar membrane in a glucose‐dependent manner and promotes V‐ATPase reassembly is not understood.Like the V1 subcomplex and V1C subunit, the RAVE complex is reversibly recruited to the vacuolar membrane in response to glucose. Interestingly, we found that RAVE requires the presence of Vo, but neither V1 nor V1C, for its glucose‐dependent vacuolar localization. We identified a 6‐amino acid, conserved motif within Rav1 that is essential for RAVE's vacuolar localization in vivo. In vitro, deletion of this motif diminishes binding between Rav1 and the cytosolic N‐terminal domain of Vo subunit Vph1. These data suggest that this motif is essential for RAVE to identify Vo subunit Vph1 at the vacuolar membrane, but they do not explain the release of RAVE from the membrane upon glucose deprivation. We seek to determine the signaling mechanism and structural changes involved in RAVE's glucose‐dependent activities. V‐ATPase activity, glycolytic enzymes, the Ras/cAMP pathway, PI(3,5)P2 levels, and cytoskeletal elements have all been implicated in V‐ATPase assembly. We are genetically or chemically silencing the activity of each of these factors and assessing the glucose‐dependent localization of GFP‐tagged RAVE subunits. Initial results indicate that RAVE cycles on and off the vacuolar membrane even in the presence of an assembled but inactive V‐ATPase mutant that is incapable of disassembly. Similar experiments will determine the effects of other factors. Detailed biochemical characterization of the RAVE complex has been thwarted by low expression levels of RAVE subunits. However, we can now express and purify milligram quantities of the RAVE complex alone or bound to V1. This will allow us to identify glucose‐sensitive interactions with RAVE in vitro and to test the hypothesis that RAVE undergoes a glucose‐sensitive conformation change that reversibly exposes the Rav1‐Vph1 binding site. This work addresses the molecular mechanisms governing RAVE‐mediated V‐ATPase reassembly and is essential to understanding the central role of the V‐ATPase in cellular metabolism.Support or Funding InformationNIH GM127364This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.