RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase

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.Vacuolar proton-translocating ATPases (V-ATPases) 2 are conserved in all eukaryotic cells where they function to acidify internal organelles such as the lysosome/vacuole, Golgi apparatus, secretory vesicles, and endosomes. V-ATPases couple hydrolysis of cytoplasmic ATP to transport of protons from the cytosol into intracellular compartments. Organelle acidification is essential for a wide range of cellular processes including protein sorting in the biosynthetic and endocytic pathways; protein processing, activation, and degradation; cellular ion homeostasis; and coupled transport of small molecules (1-4). V-ATPases also have been identified in the plasma membrane of certain specialized cells where they pump protons from the cytosol out of the cell (1, 5). The structure and subunit composition of V-ATPases is very similar in all organisms from yeast to humans. They are multisubunit complexes composed of two domains. The V 1 domain is a peripheral cytoplasmic complex composed of eight different subunits (subunits A-H), and it contains the sites of ATP hydrolysis. The V 0 domain is an integral membrane complex that is composed of six different subunits (subunits a, d, e, c, cЈ, and cЉ). It comprises the proton pore. The V 1 and V 0 domains must be structurally and functionally coupled for ATP-driven proton translocation to occur. V 1 complexes that are not attached to V 0 at the membrane cannot hydrolyze MgATP, and V 0 complexes in the membrane that are not attached to V 1 are not able to transport protons (6, 7).The biosynthetic assembly pathway for V-ATPases is not completely understood. Free V 1 and V 0 complexes exist in vivo in both yeast and mammalian cells (8 -11). Independent ...

An update in the structure, function, and regulation of V-ATPases: the role of the C subunit

“…Similarly, in the absence of RAVE, the C subunit has been found to be incapable of assembling into V-ATPase, resulting in the loss of enzyme activity (Smardon and Kane, 2007). These data are supported by findings by Keenan et al (2002), which showed a reduction of over 48% in catalytic activity that did not affect enzyme assembly in experimental models with different mutations in the C subunit.…”

“…In an in vitro study, Smardon and Kane (2007), found that while V1V0 assembly took place in the absence of the C subunit, the resulting complex was highly unstable and that V-ATPase activity was extremely low; this would suggest that the C subunit is exclusively responsible for regulating the assembly of the V1V0 complex. Similarly, in the absence of RAVE, the C subunit has been found to be incapable of assembling into V-ATPase, resulting in the loss of enzyme activity (Smardon and Kane, 2007).…”

“…The RAVE complex (a regulator of V-ATPases and endosomal membranes) needs to be present for optimal C subunit and V-ATPase assembly to occur (Forgac, 2007;Smardon and Kane, 2007).…”

Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase

“…Further complicating the understanding of this mechanism are the number of cellular binding partners for subunits likely to be involved in enzyme regulation. Subunit C, for example, has been shown to interact with kinases (47,50), actin (51), ATP (52), and the Regulator of the H ϩ -ATPase of vacuoles and endosomal membranes complex (53). Interestingly, the C subunit x-ray crystal structure was solved in two different conformations, but it remains unclear whether these changes may be linked to release of subunit C from the enzyme during regulated disassembly (18).…”

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

“…Moreover, the dynamic interaction observed with the c subunit and trafficking to the plasma membrane suggests a role for HRG-1 in assembly or targeting of the V 0 domain to specific membranes. Regulation of V-ATPase assembly is not completely understood but has previously been shown to involve several different proteins, including the protein complex RAVE that regulates assembly of the V 1 domain in yeast (13), and chaperones for V 0 domain assembly (9).…”