Abstract
Vacuolar proton-translocating ATPases are composed of a peripheral complex, V(1), attached to an integral membrane complex, V(o). Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384-391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V(1) complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V(1) sectors in a mutant lacking V(o) sectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE small middle dotV(1) interactions are specifically disrupted in cells lacking V(1) subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE small middle dotV(1) binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly.
Highlights
Vacuolar proton-translocating ATPases (V-ATPases)1 are highly conserved proton pumps responsible for acidification of organelles such as the lysosome/vacuole, Golgi apparatus, and endosomes in all eukaryotic cells [1,2,3]
This result implicated the RAVE complex in the glucose-dependent reassembly of the V-ATPase. To probe this aspect of RAVE activity further, we examined the effects of extracellular glucose on the RAVE1⁄7V1 interaction by using a monoclonal antibody against the V1 B subunit to immunoprecipitate free V1 complexes from cytosol along with bound RAVE subunits
These immunoblots demonstrate that there are detectable levels of V1 subunits in the cytosol even in the presence of glucose (ϩ), but the level of V1 subunits in the cytosol increases markedly when the cells are deprived of glucose for 15 min (Ϫ) and falls to the original level when glucose is restored to the cells (Ϫ/ϩ). ( this immunoblot was probed only with the anti-A subunit antibody, previous results indicate that all of the V1 subunits except subunit C are present in the cytosolic V1 complexes [6, 12].) In contrast, the levels of Rav1p and Rav2p in the cytosol remain the same regardless of the level of extracellular glucose
Summary
V-ATPase, vacuolar proton-translocating ATPase; RAVE, regulator of the ATPase of vacuolar and endosomal membranes; YEPD, yeast extract-peptone-2% dextrose medium; SD, fully supplemented minimal medium; SCF, Skp1-cullin-F-box; E2, ubiquitin conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; Mes, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. The assembly pathways for V-ATPases are complicated and incompletely understood Both mammalian cells and yeast contain free V1 and Vo sectors in vivo (8 –10), and yeast mutants lacking one subunit of either sector are able to assemble the other sector [10]. The V-ATPase showed assembly defects in the rav mutants, and the V1 subunits that were assembled on the vacuolar membrane in a rav1⌬ mutant disassembled rapidly in response to glucose deprivation but reassembled much more slowly upon glucose re-addition than in wild-type cells.
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