Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity

Abstract

SUMMARYV-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits. This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available. In this system, the effect of antibody on the activity of the enzyme can be analyzed. Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants. The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme. Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme. These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus. The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane. This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA. Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four. Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices.

Actin Binding Activity of Subunit B of Vacuolar H+-ATPase Is Involved in Its Targeting to Ruffled Membranes of Osteoclasts

“…(15)(16)(17) Stalk rotation is coupled to the passage of protons through the associated membrane and intraconversion of ADP and ATP. V-ATPases pump protons at the expense of ATP hydrolysis.…”

“…(1) Like the synthase, V-ATPases are rotary motors composed of mul-tiple subunits that include both integral and peripheral membrane proteins. (15)(16)(17) Stalk rotation is coupled to the passage of protons through the associated membrane and intraconversion of ADP and ATP. V-ATPases pump protons at the expense of ATP hydrolysis.…”

Mapping of C‐termini of V‐ATPase subunits by in vivo‐FRET measurements

“…VHA-G is a subunit of the peripheral stator. Binding of an antibody against VHA-G did not affect the activity of the V-ATPase [39]. VHA-G is thought to be attached to the hexamer [37], additionally interacting with VHA-E similar to subunit b in the F-ATP-synthase [25].…”

The little we know on the structure and machinery of V-ATPase

“…Moreover, sequence homology of subunit cЉ between lemon fruit and yeast, reveals high identity among α-helices 2-5 (56%) but not with helix 1, which is lacking in plants. These findings led us to suggest that in yeast, helices 2-5 of subunit cЉ are transmembrane and that the first α-helix is a cytoplasmic segment (Aviezer-Hagai et al, 2003). Taking into account the orientation of each proteolipid (Flannery et al, 2004), a series of gene fusion constructs between pairs of proteolipids was tested for assembly and activity and the results suggested a particular Grivin et al, 1998).…”