Structure of the Vacuolar ATPase by Electron Microscopy

Abstract

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergentsolubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V 1 and V 0 , with overall dimensions of 28 ؋ 14 ؋ 14 nm. The V 1 is a more or less spherical molecule with a central cavity. The V 0 has the shape of a flattened sphere or doughnut with a radius of about 100 Å. The V 1 and V 0 are joined by a 60-Å long and 40-Å wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V 1 , and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30 -50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V 1 -V 0 interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F 1 F 0 -ATP synthase, confirming their common origin.A vacuolar ATPase (or V-ATPase) 1 is found in the membrane of subcellular compartments of eucaryotic cells, where it functions to acidify the interior and at the same time energize the membranes of organelles such as clathrin-coated vesicles, endosomes, lysosomes, chromaffin granules, and Golgi-derived vesicles (1, 2). ATP hydrolysis-driven acidification of these organelles plays an important role in processes like receptormediated endocytosis, neurotransmitter release, protein trafficking, pH maintenance, and storage of metabolites. Early electron microscopic images (3-5) show the V-ATPase complex organized in two parts, a membrane extrinsic V 1 and a membrane-embedded V 0 , named after the related F 1 F 0 -type ATP synthase. As in F 1 F 0 , ATP hydrolysis on the membrane extrinsic domain is coupled to proton translocation across the membrane bilayer, but unlike F 1 F 0 , the vacuolar enzyme cannot utilize the potential energy of a proton gradient to synthesize ATP. The V 1 V 0 -ATPase is composed of at least 12 different subunits. The V 1 contains subunits A-H with molecular weights of 73,000, 58,000, 40,000, 34,000, 33,000, 14,000, 10,000, and 50,000 -57,000, and the V 0 contains subunits a, cЈ, cЉ, and d having molecular weights of 100,000, 17,000, 19,000 and 38,000, respectively. The subunit stoichiometry of the complex is A 3 B 3 CDEF x G y H z ac (1) 6 cЉd, giving a calculated molecular weight of approximately 840,000 (assuming one copy of subunits F, G, and H each), in good agreement...

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

“…This feature is not observable in the final averaged images. Similar fine structure between central stalk and the peripheral stalk was also observed for the V 1 V o -ATP synthase [42,43].…”

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

“…Ultrastructural analysis of the V-ATPase complex has previously suggested that Ac45 may represent a globular density orientated toward the luminal side V 0 domain subcomplex (51). This proposed structure was found to be associated and centered in the asymmetric ring composed of six c subunits and one cЉ subunit, with the a subunit seen as peripheral density next to ring of c/cЉ subunits (51). In this study, our immunoprecipitation data showed that both Ac45 and Ac45⌬C co-precipitated with these V 0 subunits in vitro.…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…Similarly to the more familiar and related F-ATP synthase (FATPase) there are 3 catalytic sites, here for ATP hydrolysis, in the water soluble V 1 domain (F 1 domain in F-ATPase), and trans-membrane proton transport takes places in hydrophilic channels (or sacks) in the interface between the "c ring" and subunit a of the membrane bound V o (F o ) domain (Wilkens et al 1999;Grabe et al 2000;KawasakiNishi et al 2001;Wang et al 2004;Beyenbach and Wieczorek 2006) (Fig. 1).…”