Cysteine-directed Cross-linking to Subunit B Suggests That Subunit E Forms Part of the Peripheral Stalk of the Vacuolar H+-ATPase

Arata, Yoichiro; Baleja, James D.; Forgac, Michael

doi:10.1074/jbc.m109967200

Cited by 85 publications

(107 citation statements)

References 74 publications

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“…However, recent cross-linking experiments have suggested that the Esubunit occupies a peripheral position (58). Such an exposed position agrees with several regulatory protein-protein interactions described for the E-subunit (59,60).…”

Section: Discussionsupporting

confidence: 60%

“…Therefore, we reasoned that the faint stalk in V-ATPases was probably partly formed by one or two (63) copies of the G-subunit and the E-subunit. The E-subunit was shown to be associated with the G-subunit (62)(63)(64) and is located at the periphery of V 1 (58).…”

Section: Discussionmentioning

confidence: 99%

“…The Model-In combining our three-dimensional maps with previous studies (5,16,49,50,58,(62)(63)(64)(65)68), we propose the following tentative model of the V-ATPase, as depicted in Fig. 7.…”

Section: Discussionmentioning

confidence: 99%

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

Domgall

Venzke

Lüttge

et al. 2002

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

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

Domgall

Venzke

Lüttge

et al. 2002

Journal of Biological Chemistry

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“…Using yeast two-hybrid assay and co-immunoprecipitation, Landolt-Marticorena et al (10) showed that the a subunit of V-ATPase interacts with the H and A subunits. Arata et al (11) showed that the B and E subunits of V-ATPase are in close proximity to each other by introducing unique cysteine residues into a cysteine-less form of the B subunit of V-ATPase and cross-linking purified vacuolar membranes. Their findings suggest that the E subunit forms part of the peripheral stalk of V-ATPase.…”

Section: Vacuolar Hmentioning

confidence: 99%

The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

Lu¹,

Vergara²,

Zhang³

et al. 2002

Journal of Biological Chemistry

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Vacuolar H؉ -ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F 1 F 0 -ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominantnegative mutants resulted in dramatic decreases in bafilomycin A 1 -sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase. Vacuolar Hϩ -ATPases (V-ATPases) 1 energize and acidify intracellular compartments of the vacuolar system of eukaryotic cells. They are essential for the normal function of secretory vesicles, the trans-Golgi network, endosomes, lysosomes, the yeast vacuole, and other intracellular membrane compartments (1, 2). In some specialized cells such as the intercalated cells of the kidney and the osteoclasts, V-ATPases reside at high levels on the plasma membrane, where they are responsible for transepithelial or cellular proton transport required for normal acid-base homeostasis and bone remodeling (2). Despite their wide range of physiological functions, V-ATPases share a highly conserved structure and common enzymatic properties that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol (3). They contain two macrodomains or sectors: V 1 , a catalytic domain composed of peripheral membrane proteins, and V 0 , a transmembrane domain composed of intrinsic membrane proteins that transmits protons through the lipid bilayer (4). The V 1 domain attaches to the V 0 domain at the cytoplasmic face of the membrane. In Saccharomyces cerevisiae, the V 1 domain is composed of eight distinct polypeptide chains, and the V 0 domain contains five (4).V-ATPases are evolutionarily related and structurally similar to F 1 F 0 -ATPases (F-ATPases) of bacteria, chloroplasts, and mitochondria (3). F-ATPases also have two sectors: F 1 , a peripherally attached complex composed of a catalytic head and a stalk, and F 0 , composed of intrinsic membrane subunits and a stator arm. F-ATPases have a rotary catalytic mechanism (5-7). The proton electrochemical gradient across the membrane drives translocation of protons through a pathway composed of the a and c subunits in the F 0 sector,...

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“…Subunit E forms an elongated structure sitting in the cavity of the V 1 headpiece and subunit C a spherical structure that provides the connection with the rotor in V 0 (Chaban et al 2002). The assignment of subunit E to the central stalk is a matter of debate (e.g., Yokohama et al 2003;Chaban et al 2004;Arata et al 2002). Building subcomplexes from isolated subunits is likely to be the best way to resolve this issue.…”

Section: Introductionmentioning

confidence: 99%

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

et al. 2006

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The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na + pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and F, G) that have very similar mobilities on SDS-PAGE of the puriWed complex (24.3 and 22.7 kDa, and 12.3 and 11.6 kDa) was established by tryptic digestion of the protein bands followed by mass spectrometric analysis of the peptides.

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Cysteine-directed Cross-linking to Subunit B Suggests That Subunit E Forms Part of the Peripheral Stalk of the Vacuolar H+-ATPase

Cited by 85 publications

References 74 publications

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

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

The Amino-terminal Domain of the E Subunit of Vacuolar H+-ATPase (V-ATPase) Interacts with the H Subunit and Is Required for V-ATPase Function

The ntp operon encoding the Na+ V-ATPase of the thermophile Caloramator fervidus

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