class="head no_bottom_margin" id="sec1title">IntroductionThe rotary ATPase family includes the F1Fo-ATPase (ATP synthase) of mitochondria, chloroplasts and eubacteria, the vacuolar ATPase (V-ATPase), and the A-ATPases present in archaea and some bacteria (). A common feature of this family is an ATP hydrolyzing/synthesizing motor asymmetrically coupled to a membrane-bound ion pump. In V-ATPases, ATP hydrolysis in the cytoplasmic V1 domain drives rotation of a central rotor axle, which transmits torque to the proton pump in the membrane domain (Vo). Conversely, in F- and A-ATPases operating in synthase mode, torque generated by ion flow through the membrane domain is transmitted to the soluble domain, driving ATP synthesis. Proton translocation is proposed to occur at the interface between rotating and static components in Vo/Fo/Ao, the static part (the stator) forming a continuous structure with the ATP hydrolyzing apparatus. Comparisons of ATP free energy with electrochemical membrane potential or directly measured work output indicate thermodynamic efficiency close to 100% ().V-ATPases are found in all eukaryotic cells () driving acidification essential to the function of endosomes, lysosomes, and the Golgi apparatus (). Inhibition blocks endosomal transit and arrests recycling of receptor-ligand complexes. Disease mutations cause dysfunctional glycosylation and aberrant protein sorting by the Golgi (). The V-ATPase also energizes secondary active transport processes such as neurotransmitter uptake into secretory vesicles (). V-ATPases are active at the plasma membrane of some cells, for example, in tumor cells () and in osteoclasts where acid extrusion is essential for resorption of mineralized bone ().In the V-ATPase V1 domain, alternating A and B subunits form a pseudohexameric arrangement, with three catalytic sites located at the B-A interfaces (). ATP hydrolysis moves a helix-loop-helix “lever arm” in subunit A with sequential hydrolysis imposing torque on the central axle comprising the helical coiled-coil D subunit linked at its base to subunit F. The end of the axle is connected to a ring of c subunits in the membrane via subunit d, allowing concerted c ring rotation. The rotor carries ∼35 pN nm of torque (), which is not rigid and can flex along its length (). Proton translocation is thought to occur at the interface between the rotating c ring and the static large integral membrane a subunit. Models propose that the proton boards the c ring via a half channel in a, with subsequent stepping of the rotor, allowing proton exit on the opposite side of the membrane via a second channel (). Detailed structural information for the membrane domain of subunit a is lacking, although the soluble domain of a bacterial subunit a homolog has been solved (). Biochemical studies indicate an N-terminal soluble cytoplasmic domain linked to eight transmembrane helices, with residues in helices 7 and 8 involved in proton movement (). Another small integral membrane subunit, e, is poorly characterized, but is reported to be heavily glycosylated and associated with subunit a ().Futile rotation of a with the c ring is prevented by the stator attached to the (AB)3 motor. In V-ATPase, the stator has three subunit E/G filaments attached to the top of the B subunits and converging with a horizontal collar structure that surrounds most of the midsection of the rotor axle (reviewed in href="#bib37" rid="bib37" class=" bibr popnode">Muench et al., 2011). This collar comprises the a subunit soluble domain and subunits C and H. In contrast, the A-ATPase (which lacks C and H) has only two EG filaments (href="#bib27" rid="bib27" class=" bibr popnode">Lau and Rubinstein, 2010), and in F-ATPase the stator is a single filament (href="#bib48" rid="bib48" class=" bibr popnode">Rubinstein et al., 2003). The additional complexity in V-ATPase appears to be an adaptation for more sophisticated control. Low-energy status, such as occurs during larval molt in Manduca sexta or as a result of glucose depletion in Saccharomyces cerevisiae, causes dissociation of V1 from Vo in vitro (href="#bib56" rid="bib56 bib23" class=" bibr popnode">Sumner et al., 1995; Kane, 1995). Although in vivo experiments suggest a more subtle rearrangement rather than complete separation (href="#bib57" rid="bib57" class=" bibr popnode">Tabke et al., 2014), the functional effects are well defined: catalytic silencing in V1 (href="#bib44" rid="bib44" class=" bibr popnode">Parra et al., 2000) and proton impermeability in Vo (href="#bib4" rid="bib4" class=" bibr popnode">Beltran and Nelson, 1992). Subunit C may be the receptor for the dissociation signal, and subunit H has been proposed to then prevent ATP cycling by fixing the rotor axle to the stator (href="#bib44" rid="bib44 bib38" class=" bibr popnode">Parra et al., 2000; Muench et al., 2013).Recent technical advances, including more stable microscopes, high-sensitivity direct electron detectors, and new image processing algorithms, have substantially improved the resolution attainable by electron cryomicroscopy (cryo-EM) (href="#bib52" rid="bib52" class=" bibr popnode">Smith and Rubinstein, 2014). Now, cryo-EM of even relatively small and nonsymmetrical membrane protein complexes can resolve structures to below 5 Å (href="#bib29" rid="bib29" class=" bibr popnode">Lu et al., 2014) and conformational changes linked to mechanisms (href="#bib9" rid="bib9" class=" bibr popnode">Cao et al., 2013). In this study, we report the complete structure of a eukaryotic V-ATPase at nanometer resolution, using cryo-EM. Our model provides insights into the organization of its proton-translocating apparatus and the basis for the extraordinarily high efficiency of rotary ATPases. The complex rests in a different catalytic state from previously reported yeast structures, providing new insights into the rotary mechanism.
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