The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms1The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL data bank with accession numbers AJ249388, AJ249389, AJ249390 and AJ251992.1

Merzendorfer, Hans; Reineke, Stephan; Zhao, Xiao‐Fan; Jacobmeier, Birgit; Harvey, William R.; Wieczorek, Helmut

doi:10.1016/s0005-2736(00)00233-9

Cited by 45 publications

(17 citation statements)

References 47 publications

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“…For labeling of the V 1 complex, the cryosections were treated with a 1:10 dilution of monoclonal antibody 221-9 to subunit A of the Manduca V-ATPase (22). Soluble subunit C was labeled with a 1:2000 dilution of monospecific, polyclonal antibodies to the recombinant protein (6). Visualization of the primary antibodies was performed with a 1:1000 dilution of Cy3-or TRITC-conjugated anti-mouse and anti-guinea pig F(abЈ) 2 -fragments (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant subunit C was expressed in Escherichia coli BL21 cells and purified by nickel-nitriloacetic acid (Ni-NTA) affinity chromatography as described previously (6). Protein concentrations were determined by the Amido Black method (26).…”

Section: Methodsmentioning

confidence: 99%

“…3C). Based on the observed molecular mass, we assumed that the labeled polypeptide was the V 1 subunit C, which we had identified previously to be part of the Manduca V-ATPase (6). Because this subunit is present in preparations of the Manduca V 1 complex in substoichiometric amounts at the most (7), we were not astonished to observe labeling only in the V 1 V 0 holoenzyme.…”

Section: V-atpase and F-actin Co-localize At The Apical Membranes Of mentioning

confidence: 97%

“…They comprise at least 12 subunits that are part of two different subcomplexes, i.e. a peripheral, catalytic V 1 complex with subunits A 3 B 3 CDE X FG Y H and a membrane-bound, proton-conducting V 0 complex with subunits ac 6 de. In the midgut of the tobacco hornworm (Manduca sexta) they are localized in the apical membrane of goblet cells where they exclusively energize all secondary active transport processes across the epithelium (3).…”

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A Novel Role for Subunit C in Mediating Binding of the H+-V-ATPase to the Actin Cytoskeleton

Vitavska

Wieczorek

Merzendorfer

2003

Journal of Biological Chemistry

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126

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Primary proton transport by V-ATPases is regulated via the reversible dissociation of the V 1 V 0 holoenzyme into its V 1 and V 0 subcomplexes. Laser scanning microscopy of different tissues from the tobacco hornworm revealed co-localization of the holoenzyme and F-actin close to the apical membranes of the epithelial cells. In midgut goblet cells, no co-localization was observed under conditions where the V 1 complex detaches from the apical membrane. Binding studies, however, demonstrated that both the V 1 complex and the holoenzyme interact with F-actin, the latter with an apparently higher affinity. To identify F-actin binding subunits, we performed overlay blots that revealed two V V-ATPases are ubiquitous proton pumps that are found in the endomembranes of all and the plasma membranes of many specialized eucaryotic cells (1, 2). They comprise at least 12 subunits that are part of two different subcomplexes, i.e. a peripheral, catalytic V 1 complex with subunits A 3 B 3 CDE X FG Y H and a membrane-bound, proton-conducting V 0 complex with subunits ac 6 de. In the midgut of the tobacco hornworm (Manduca sexta) they are localized in the apical membrane of goblet cells where they exclusively energize all secondary active transport processes across the epithelium (3). During starvation or molt, pump activity abolishes due to the dissociation of the V 1 complex from the membrane (4). In the tobacco hornworm as well as in yeast, where reversible dissociation occurs upon the withdrawal of glucose, subunit C seems to be released into the cytoplasm because the purified V 1 complex contains not more than substoichiometric amounts of this polypeptide (5-7). The regulatory mechanisms responsible for reversible V-ATPase dissociation are not understood, although it seems likely that interactions of V-ATPase subunits with other cellular proteins control this process. In fact, several proteins have already been identified that bind to subunits of the V-ATPase and thus may link this enzyme to a comprehensive cellular network. Among recently discovered candidates is the yeast RAVE complex (8), which associates with the cytosolic V 1 complex via its rav1p component and is apparently essential for stable assembly of the V-ATPase holoenzyme (9). Another example is the plasma membrane V-ATPase in osteoclasts, which may be linked to glycolysis via the interaction of its subunit E with aldolase, an enzyme of the glycolytic pathway (10).The involvement of proteins interacting with V-ATPases may also be relevant to sorting processes within the cell. In clathrin-coated vesicles, V-ATPase seems to be associated with the 50-kDa polypeptide of the AP-2 complex (11), which is involved in the internalization of proteins from the plasma membrane (12). Even more, this interaction may be necessary for activity and in vitro reassembly of the V-ATPase (13). Binding of V-ATPase to AP-2, however, may also be linked to the mechanisms of pathogenicity in human immunodeficiency virus (HIV) 1 infection. Direct interaction between the HIV accessory p...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: V-atpase and F-actin Co-localize At The Apical Membranes Of mentioning

confidence: 97%

mentioning

confidence: 99%

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A Novel Role for Subunit C in Mediating Binding of the H+-V-ATPase to the Actin Cytoskeleton

Vitavska

Wieczorek

Merzendorfer

2003

Journal of Biological Chemistry

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140

126

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“…Subunit C appears to be released into the cytoplasm during this process, because the purified V 1 complex lacks most of it (3,6).…”

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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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Validation of RNA interference in western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) adults

Rangasamy

Siegfried

2011

Pest Management Science

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The multigene family of the tobacco hornworm V-ATPase: novel subunits a, C, D, H, and putative isoforms1The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL data bank with accession numbers AJ249388, AJ249389, AJ249390 and AJ251992.1

Cited by 45 publications

References 47 publications

A Novel Role for Subunit C in Mediating Binding of the H+-V-ATPase to the Actin Cytoskeleton

A Novel Role for Subunit C in Mediating Binding of the H+-V-ATPase to the Actin Cytoskeleton

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

Validation of RNA interference in western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) adults

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