Structural analysis of the stalk subunit Vma5p of the yeast V‐ATPase in solution

Armbrüster, Andrea; Svergun, Dmitri I.; Coskun, Ünal; Juliano, Sandra; Bailer, Susanne M.; Grüber, Gerhard

doi:10.1016/j.febslet.2004.06.029

Cited by 47 publications

(57 citation statements)

References 52 publications

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“…An intriguing result was the formation of a stable and ATPase active hybrid complex composed of Vma5p and V 1 from M. sexta, lacking endogenous subunit C. It has been demonstrated that the addition of recombinant subunit C to the V 1 (-C) complex significantly increased ATPase activity [13], supporting its functional role in regulation of ATPase activity [15,16]. Here, we report for the first time the property of nucleotide binding of subunit C, independently observed by photoaffinity labeling and fluorescence correlation spectroscopy (FCS).…”

Section: Introductionmentioning

confidence: 99%

“…Electron microscopy studies of the disassembled V 1 complex from tobacco hornworm Manduca sexta have shown that subunit C dissociates from the V 1 subcomplex [11], although it is essential for the reassembly of the functional V 1 V 0 [9,12]. Previously, the structure of the C subunit (Vma5p) from the yeast V 1 V 0 ATPase has been studied by small angle X-ray scattering, revealing that the hydrated Vma5p has an elongated bootshaped structure with a maximum size of 12.5 nm [13]. A recent 1.75 Å map from X-ray diffraction studies of Vma5p [14] confirms this feature and shows that this subunit consists of three distinct domains.…”

Section: Introductionmentioning

confidence: 99%

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Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

et al. 2005

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The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

et al. 2005

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“…A Profilin-like Sequence in the V-ATPase Subunits B and CLow resolution x-ray scattering recently revealed an elongated boot-shaped form of the yeast subunit C (34). However, up to now no high resolution structural analysis exists which is a necessary precondition for the prediction of actin binding domains.…”

Section: Fig 2 Overlay Slot-blots Reveal Binding Of Subunit C To Bomentioning

confidence: 99%

The V-ATPase Subunit C Binds to Polymeric F-actin as Well as to Monomeric G-actin and Induces Cross-linking of Actin Filaments

Vitavska

Merzendorfer

Wieczorek

2005

Journal of Biological Chemistry

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Previously, we have shown that the V-ATPase holoenzyme as well as the V 1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V 1 subunits B and C (Vitavska, O., Wieczorek, H., and Merzendorfer, H. (2003) J. Biol. Chem. 278, 18499 -18505). Since the recombinant subunit C not only enhances actin binding of the V 1 complex but also can bind separately to F-actin, we analyzed the interaction of recombinant subunit C with actin. We demonstrate that it binds not only to F-actin but also to monomeric G-actin. With dissociation constants of ϳ50 nM, the interaction exhibits a high affinity, and no difference could be observed between binding to ATP-G-actin or ADP-G-actin, respectively. Unlike other proteins such as members of the ADF/cofilin family, which also bind to G-as well as to F-actin, subunit C does not destabilize actin filaments. On the contrary, under conditions where the disassembly of F-actin into G-actin usually occurred, subunit C stabilized F-actin. In addition, it increased the initial rate of actin polymerization in a concentration-dependent manner and was shown to cross-link actin filaments to bundles of varying thickness. Apparently bundling is enabled by the existence of at least two actin-binding sites present in the N-and in the C-terminal halves of subunits C, respectively. Since subunit C has the possibility to dimerize or even to oligomerize, spacing between actin filaments could be variable in size.V-ATPases are ubiquitous and highly conserved proton pumps that acidify specific organelles such as endosomes, lysosomes, or secretory vesicles in every eukaryotic cell (1). They also are found in plasma membranes of many specialized animal cells where they either are involved in pH homeostasis or in membrane energization (2). V-ATPases consist of two complexes, a peripheral V 1 complex whose catalytic part faces the cytosol and a membrane-bound proton-conducting V 0 complex. In the midgut of the tobacco hornworm (Manduca sexta), the V 1 complex of the plasma membrane V-ATPase contains eight different subunits, A-H, whereas the V 0 complex consists of the four different subunits a and c-e (3). Under special physiological conditions V-ATPase activity is down-regulated by reversible dissociation of the V 1 complex from the membrane as was shown in the tobacco hornworm as well as in yeast (4,5).Subunit C appears to be released into the cytoplasm during this process, because the purified V 1 complex lacks most of it (3, 6).Dissociation of subunit C from the V 1 complex and its support of holoenzyme reassembly indicate that this subunit may play a crucial role in the regulation of V-ATPases. Another role, previously detected by us in the M. sexta midgut, is its ability to bind to the actin cytoskeleton (7). In feeding tobacco hornworms, actin filaments co-localize with the V-ATPase at the apical membrane of midgut goblet cells. Like in osteoclasts (8), actin binding occurs via the V 1 subunit B; however, binding to F-actin al...

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“…SAXS, for example, has been used to describe the structure and morphology of the C subunit and its role in V-ATPase regulation (Svergun et al, 1998;Armbruster et al, 2004) (Figure 3).…”

Section: The C Subunitmentioning

confidence: 99%

An update in the structure, function, and regulation of V-ATPases: the role of the C subunit

et al. 2012

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Vacuolar ATPases (V-ATPases) are present in specialized proton secretory cells in which they pump protons across the membranes of various intracellular organelles and across the plasma membrane. The proton transport mechanism is electrogenic and establishes an acidic pH and a positive transmembrane potential in these intracellular and extracellular compartments. V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic sector (V1) and a hydrophobic membrane sector (V0) responsible for driving protons. V-ATPase activity is regulated by three different mechanisms, which control pump density, association/dissociation of the V1 and V0 domains, and secretory activity. The C subunit is a 40-kDa protein located in the V1 domain of V-ATPase. The protein is encoded by the ATP6V1C gene and is located at position 22 of the long arm of chromosome 8 (8q22.3). The C subunit has very important functions in terms of controlling the regulation of the reversible dissociation of V-ATPases.Keywords:V-ATPases, proton transport, ATP6V1C1,V1 domain, V0 domain, C subunit. Uma atualização na estrutura, função e regulação das V-ATPases:o papel das subunidades C ResumoAs Vacuolar ATPases (V-ATPases) estão presentes nas células especializadas em secreção de protões, nas quais eles são bombeados através das membranas de vários organelos intracelulares e da membrana plasmática. O mecanismo de transporte de protões é eletrogênico e estabelece um pH ácido e um potencial transmembrana positivo nestes compartimentos intracelulares e extracelulares. As V-ATPases foram encontradas em todas as células eucarióticas, praticamente idênticas em termos de composição das suas subunidades. Elas têm duas estruturas distintas: um setor periférico catalítico (V1) e uma membrana hidrofóbica (V0), responsável pela condução de protões. A atividade das V-ATPases é regulada por três mecanismos diferentes, os quais controlam a densidade de bomba, associação/dissociação de domínios V1 e V0, e a atividade secretora. A subunidade C é uma proteína de 40-kDa, localizada no domínio V1 da V-ATPase. Essa proteína é codificada pelo gene ATP6V1C e está localizada na posição 22 do braço longo do cromossomo 8 (8q22.3). A subunidade C tem funções muito importantes em termos de controle do regulamento da dissociação reversível da V-ATPase.Palavras-chave: V-ATPases, transporte de protões, ATP6V1C1, domínio V1, domínio V0, subunidade C.

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Structural analysis of the stalk subunit Vma5p of the yeast V‐ATPase in solution

Cited by 47 publications

References 52 publications

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The V-ATPase Subunit C Binds to Polymeric F-actin as Well as to Monomeric G-actin and Induces Cross-linking of Actin Filaments

An update in the structure, function, and regulation of V-ATPases: the role of the C subunit

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