Defined Sites of Interaction between Subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the Stator Structure of the Vacuolar H⁺-ATPase

Abstract

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

“…The latter contains the C, E, G, and H subunits and the N terminus of the a subunit (1-4). Interactions within the V 1 domain between subunits E, G, and C have previously been demonstrated by cross-linking and in vitro pulldown techniques in yeast (17) and cow (18). To date, the only previous reports of interactions that connect the V 0 and V 1 domains within the peripheral stalk describe interactions in yeast between H and the N-terminal part of the a subunit, shown by two hybrid binding assays (19) and the ability of the a, G, and E subunits in T. thermophilus to form a stable complex (20).…”

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

“…The V 1 -V o interface is comprised of a rotor connection, formed by V 1 subunits DF binding to the V o subunit d, and a stator connection, composed of the single copy C subunit, the N-terminal domains of the three subunit EG heterodimers (peripheral stators EG1-3), the soluble cytoplasmic domain of V o subunit a (a NT ), and subunit H (see Fig. 1A) (14,35,36,40,41). A critical role in the reversible disassembly process is played by subunit C, the only subunit that does not bind to either V 1 or V o after enzyme dissociation, and subunit a, whose soluble cytoplasmic domain provides an anchor to the membrane for the other stator subunits in the assembled complex.…”

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

“…Jones et al (2005), reported that the C and G subunits (Vma5p and Vma10p, respectively) are capable of strongly interacting with the E subunit (Vma4p). The N-terminus of the E subunit appears to be key not only in the regulation of the rotary molecular structure of V-ATPase but also in …”

“…Table 3. Structure, function and regulation of human V-ATPase (Torigoe et al, 2002 interactions with possible regulatory subunits (Jones et al, 2005). According to Jefferies and Forgac, 2008, interaction between the H subunit of the free V1 domain and the F subunit (subunit of rotary part), inhibits the ATP catalytic ability of V-ATPase.…”