Fig. 5. Schematic representation of the effect of bound antibody to Vma16p-HA on V-ATPase activity. The V-ATPase is composed of two functional domains: the catalytic V₁ (A and B subunits) and the membranous V₀ (a, c, c', c'' and d subunits), which are interconnected by peripheral (C, E, G, H subunits) and central (D and F subunits) stalks, respectively. It is assumed that hydrolysis of ATP by V₁ rotates the central shaft (D and F subunits), which in turn causes the rotation of the membrane c-ring against its stator (E, G and N'-a subunits), resulting in proton translocation into the vesicle (Nelson et al., 2002; Nishi and Forgac, 2002). It is now clear that, similar to c and c' (Vma3p and Vma11p, respectively), the c'' subunit (Vma16p) contains four TMs, although in an opposite arrangement in the membrane with both N' and C' pointing to the cytoplasm as shown above. The Vma16p (red) was tagged with three HA-epitopes at its N terminus. When the resulting tagged V-ATPase was incubated with anti-HA antibody, an inhibition of activity was observed. This suggests that the binding of antibody (green) to the HA-tags interfered with the rotation of the c-ring and shaft subunits (all presumed rotating subunits are framed with a thick line). This interference is probably due to a collision with the static subunits, and primarily with subunit a, against which the rotor is turning. A similar effect was observed when antibodies were allowed to bind to the Vma7p-HA (F subunit)-containing complex. This is in contrast to the effect of antibody on the Vma10p-HA (G subunit)-containing V-ATPase. Abs, bound anti-HA antibodies.

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