Three-dimensional Map of a Plant V-ATPase Based on Electron Microscopy

Abstract

V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V 0 ), and a soluble catalytic (V 1 ) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V 0 and V 1 were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V 0 changed with respect to V 1 , and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly. V-ATPases1 are found in all eukaryotic cells. They hydrolyze ATP to pump protons into various intracellular compartments (1). In plant tonoplasts the proton motive force generated by the V-ATPase is used for secondary transport processes contributing to osmoregulation, ion and pH homeostasis, nutrient and remnant storage, and plant defense (2).V-ATPases are highly conserved among species, and their gross architecture is similar to that of the well characterized F-ATPases. In the V-ATPases, the soluble V 1 subcomplex is known to carry the catalytic nucleotide-binding sites and to be connected via a thinner stalk region to the membrane-integrated V 0 subcomplex, which contains the proton-translocating machinery. The exact subunit composition and stoichiometry of V-ATPases, however, is still controversial. In yeast, the V 1 subcomplex is probably formed by the subunits (AB) 3 , C-H, and the V 0 subcomplex by the subunits c, cЈ, cЉ, a, and d (for review see Ref.3). Homologues of the cЈ-and cЉ-subunits have not been identified in plants as of yet (4).Among the subunits known to comprise V-ATPases, several share significant sequence homology to subunits of F-ATPases.The catalytic A-subunits of V-ATPase are homologous to the catalytic ␤-subunits in F-ATPase (5) and the B-subunits of V-ATPase to the non-catalytic ␣-subunits in F-ATPase (6). The membrane-integrated V-ATPase c-subunit has probably emerged by gene duplication (7) from a common ancestor of Fand V-ATPases. The G-subunit of V-ATPases has sequence similarity to the hydrophilic part of the membrane-anchored F-ATPase b-subunit (8). Other components of the F-ATPase machinery do not have any homologues in V-ATPase. Furthermore, V-ATPases contain various subunits (C, F, H, a, and d) whose functions and relationships to F-ATPases still need to be elucidated. This divergence might reflect an adaptation to the different physiological requirements of F-and V-ATPases. Unlike F-ATPases, V-AT...

Differential Expression of Vacuolar H+-ATPase Subunit c Genes in Tissues Active in Membrane Trafficking and Their Roles in Plant Growth as Revealed by RNAi

“…Yet the specific subcellular functions of V-ATPases in distinct cell types and their consequences on a developing multicellular plant are largely unknown. Thus, from the perspectives of molecular, cell, and developmental biologists, many questions remain.Based on extensive biochemical and structural studies from animal, yeast and plant V-ATPases, and the complete set of V-ATPase genes in Arabidopsis (Sze et al, 2002), a eukaryote V-ATPase consists of at least 12 distinct subunits organized in two large subcomplexes: the cytosolic V 1 and membrane V o (Arata et al, 2002b;Domgall et al, 2002). The large cytosolic V 1 complex of subunits A through H catalyzes the hydrolysis of ATP that is coupled to the pumping of protons into a compartment via the membrane-bound V o complex.…”

Building the Stator of the Yeast Vacuolar-ATPase

“…In particular, electron microscopy has revealed significant protrusions in the connecting elements linking V 0 to V 1 (14,15), indicating a more complex structure for this region. In addition, extensive experimental characterization of these connecting elements using different techniques such as chemical cross-linking and low resolution imaging has led to the proposal of several models for their topological arrangement (16 -20) confirming the higher complexity of these elements in V-ATPases compared with FATPases.…”

“…Recently we have proposed a topology for the assembly of a plant V-ATPase based on a three-dimensional electronic microscopy reconstruction model (15) and published cross-linking data. According to this model, subunits E and G would interact to form a peripheral stalk in analogy to subunits I and II of the chloroplast ATP-synthase (29), subunit b of E. coli F-ATPase (22,30), and the subunit b of mitochondrial ATPase.…”

Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase

“…ATP hydrolysis is catalyzed in V 1 , which is peripherally bound to the cytosolic side of the membrane and consists of subunits A-H. Integral to the membrane is V o , which forms the proton transporting domain and consists of subunits a, c, cЈ, cЉ, d, and e (2,3,12). Connecting V 1 to V o are one central stalk made of subunits D and F and one to three peripheral stalks consisting of subunits C, E, G, H, and the amino terminus domain of the V o subunit a (13)(14)(15)(16)(17).…”