Abstract
The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.
Highlights
Of certain specialized cells where they pump protons from the cytosol out of the cell [1, 5]
We found that Rav1p interacts with Rav2p and Skp1p of the RAVE complex and Vma4p, Vma10p, and Vma5p of the V1 complex
The Vma2p-GFP data described above indicate that V1/V0 is able to assemble in rav1⌬ and vma5⌬ mutants in vivo, these complexes are highly unstable in vitro because very small percentages of V1 subunits are found in isolated vacuolar vesicles from these strains, and V-ATPase activity is extremely low in isolated vesicles from these mutants. rav1⌬ V-〈TPase activity is 5.3%, and vma5⌬ is 2.0% of wild type activity
Summary
7.5 and 60 mM CaCl2 at elevated temperatures (33–37 °C) [18]. Consistent with this phenotype, rav1⌬ mutants have a loss of quinacrine staining of the vacuole at elevated temperatures (37 °C), indicative of a loss of vacuolar acidification. Even at the permissive temperature of 30 °C, the rav1⌬ mutant shows a kinetic delay in reassembly of V-ATPase complexes after the readdition of glucose to glucose-deprived cells [18]. Vacuoles isolated from cells lacking RAVE subunits have very low levels of V1 subunits and V-ATPase activity even when cells are grown in glucose at the permissive temperature (30 °C) [17]. The interaction between RAVE and V1 is lost in mutants lacking two V1 subunits, E and G [17] These two subunits, present in at least two copies/V1 complex, are believed to form one or more peripheral stalks connecting the catalytic head group and the V0 sector. V-ATPase peripheral stalk subunit interactions via a chaperone type assembly function
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