SummaryActive transepithelial cation transport in insects was initially discovered in Malpighian tubules, and was subsequently also found in other epithelia such as salivary glands, labial glands, midgut and sensory sensilla. Today it appears to be established that the cation pump is a two-component system of a H + -transporting V-ATPase and a cation/nH + antiporter. After tracing the discovery of the V-ATPase as the energizer of K + /nH + antiport in the larval midgut of the tobacco hornworm Manduca sexta we show that research on the tobacco hornworm V-ATPase delivered important findings that emerged to be of general significance for our knowledge of V-ATPases, which are ubiquitous and highly conserved proton pumps. We then discuss the V-ATPase in Malpighian tubules of the fruitfly Drosophila melanogaster where the potential of post-genomic biology has been impressively illustrated. Finally we review an integrated physiological approach in Malpighian tubules of the yellow fever mosquito Aedes aegypti which shows that the V-ATPase delivers the energy for both transcellular and paracellular ion transport.

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...

Eukaryotic vacuolar-type H؉ -ATPases (V-ATPases) are regulated by the reversible disassembly of the active V 1 V 0 holoenzyme into a cytosolic V 1 complex and a membrane-bound V 0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V 1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V 1 complex but not as a part of the V 1 V 0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V 1 complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3,5-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V 1 V 0 holoenzyme. Vacuolar type Hϩ -ATPases (V-ATPases) 3 are the most versatile proton pumps, being common to all eukaryotic organisms, and are found in endomembrane systems and in the plasma membrane (1-3). V-ATPases are multi-subunit transporters composed of a catalytic ATP-hydrolyzing V 1 complex (Ϸ550 kDa), which resides on the cytoplasmic side of the membrane, and a membrane-bound proton-translocating V 0 complex (Ϸ250 kDa). V-ATPase-dependent proton pumping is essential for cellular pH homeostasis and creates an electrochemical proton gradient that energizes secondary transport mechanisms in a wide variety of organelles and membrane systems. Acidification of organelles by V-ATPase activity is crucial to various cellular processes such as neurotransmitter uptake into synaptic vesicles, intracellular protein trafficking, and the secretion and activation of lysosomal enzymes for protein processing and degradation (4 -7). Located in the plasma membrane of specialized cells, V-ATPases are involved in processes such as cation secretion, bone resorption, renal acidification, and osmoregulation (8 -16). With respect to this diversity of function, mutations in genes encoding V-ATPase subunits obviously lead to several diseases, e.g. osteopetrosis (17) or renal tubular acidosis (18).Several mechanisms have been proposed for the regulation of V-ATPase activity (3). The most prominent and physiologically relevant mechanism is the reversible disassembly of the V-ATPase holoenzyme i...

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...

SummaryAccording to a classic tenet, sugar transport across animal membranes is restricted to monosaccharides. Here, we present the first report of an animal sucrose transporter, SCRT, which we detected in Drosophila melanogaster at each developmental stage. We localized the protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells. The fact that knockdown of SCRT expression results in significantly increased lethality demonstrates an essential function for the protein.Experiments with Saccharomyces cerevisiae as a heterologous expression system revealed that sucrose is a transported substrate. Because the knockout of SLC45A2, a highly similar protein belonging to the mammalian solute carrier family 45 (SLC45) causes oculocutaneous albinism and because the vesicular structures in which SCRT is located appear to contain melanin, we propose that these organelles are melanosome-like structures and that the transporter is necessary for balancing the osmotic equilibrium during the polymerization process of melanin by the import of a compatible osmolyte. In the hindgut epithelial cells, sucrose might also serve as a compatible osmolyte, but we cannot exclude the possibility that transport of this disaccharide also serves nutritional adequacy.