Masashi Toei

The vacuolar (H(+))-ATPases are ATP-dependent proton pumps that acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in normal physiological processes such as receptor-mediated endocytosis, intracellular membrane trafficking, pro-hormone processing, protein degradation, and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, and various disease processes, including renal tubular acidosis, osteopetrosis, and tumor metastasis. V-ATPases are composed of a peripheral V(1) domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V(0) domain containing six different subunits that translocates protons. In mammalian cells, most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V(0) subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V(1) and V(0) domains, control of the tightness of coupling of proton transport and ATP hydrolysis, and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both via the control of coupling and via selective targeting. This review will begin with a brief introduction to the function, structure, and mechanism of the V-ATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity.

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (ΔpH) and the electrical potential differences (ΔΨ) across membranes. The optimum rate of synthesis reached 85 s −1 , and the H + /ATP ratio of 4.0 ± 0.1 was obtained. ATP was synthesized at a considerable rate solely by ΔpH, indicating ΔΨ was not absolutely required for synthesis. Consistent with the H + /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 V o -c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases.

Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.