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

Abstract

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...

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

“…3F-H). Consistent with earlier observations linking embryo-wide asymmetry with subcellular organization (Bunney et al, 2003), and recent work showing that H + -V-ATPase subunits interact directly with the actin cytoskeleton (Chen et al, 2004;Lee et al, 1999;Vitavska et al, 2003), we found that the asymmetric targeting of the H + -V-ATPase is dependent on actin, but not microtubule, organization. We propose that some as yet uncharacterized aspect of subcellular targeting (AlAwqati, 1996; Brown, et al, 1992) [perhaps oriented by a nucleation center which fits the role of Wolpert and Brown's 'F-molecule' (Brown and Wolpert, 1990)] is upstream of the H + -V-ATPase in the LR pathway.…”

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

“…Strikingly, in AE2-depleted cells, Golgi stacks display numerous fenestrations and swollen cisternae [Holappa et al, 2004]. On the other hand, it has also been reported that actin-binding activity is a requirement for transport of (V)H þ -ATPase to and from the plasma membrane [Vitavska et al, 2003;Chen et al, 2004b] and therefore, it is reasonable to assume a role for microfilaments and actin dynamics in the delivery and/or activation of (V)H þ -ATPase in endomembrane systems, including the Golgi complex. To this respect, Baf and ConcA treatments induced very similar cisternae ultrastructural alterations (Fig.…”

“…The former are electrochemically neutral because exchanges cations (NHEs) or anions (AEs), whilst the latter actively pumps H þ resulting in lumen or extracellular acidification. Ion exchangers and (V)H þ -ATPases can be both Golgi-resident and en route to the plasma membrane [Kellokumpu et al, 1988;Kopito, 1990;Moriyama and Nelson, 1998;Nishi and Forgac, 2002;Vitavska et al, 2003;Chen et al, 2004b]. By analogy to NHEs isoforms localized to the plasma membrane (NHE1) and endocytic compartments (NHE3 and NHE5) [Szaszi et al, 2000;Denker and Barber, 2002], Golgi-associated NHEs isoforms (NHE7 and NHE8) [Nakamura et al, 2005] might be regulated by the state of F-actin in the Golgi complex.…”

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

“…In addition, V-ATPases have been shown to bind to actin filaments and to be transported to the polarized ruffled border membrane by actin-myosin II contraction (41). More recently, V 1 subunits B and C have also been shown to directly associate with filamentous actin in osteoclasts (44,45). Whether Ac45 targeting is also actin-dependent will be the focus of future investigations.…”