Olga Vitavska

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

Primary proton transport by V-ATPases is regulated via the reversible dissociation of the V1V0 holoenzyme into its V1and V0 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 V1 complex detaches from the apical membrane. Binding studies, however, demonstrated that both the V1 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 V1subunits as binding partners, namely subunit B, resembling the situation in the osteoclast V-ATPase (Holliday, L. S., Lu, M., Lee, B. S., Nelson, R. D., Solivan, S., Zhang, L., and Gluck, S. L. (2000) J. Biol. Chem. 275, 32331–32337), but, in addition, subunit C, which gets released during reversible dissociation of the holoenzyme. Overlay blots and co-pelleting assays showed that the recombinant subunit C also binds to F-actin. When the V1 complex was reconstituted with recombinant subunit C, enhanced binding to F-actin was observed. Thus, subunit C may function as an anchor protein regulating the linkage between V-ATPase and the actin-based cytoskeleton.

Stimulus-induced phosphorylation of vacuolar H(+)-ATPase by protein kinase A.

Eukaryotic vacuolar-type H+-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V1V0 holoenzyme into a cytosolic V1 complex and a membrane-bound V0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V1 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 V1 complex but not as a part of the V1V0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V1 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 V1V0 holoenzyme.

Vacuolar-type proton pumps in insect epithelia

SUMMARY Active 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.

The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments

Previously, we have shown that the V-ATPase holoenzyme as well as the V1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V1 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 V1 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 ∼50 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.

Identification of an animal sucrose transporter

According 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.

The Insect Plasma Membrane H+ V-ATPase: Intra-, Inter-, and Supramolecular Aspects

The plasma membrane H+ V-ATPase from the midgut of larval Manduca sexta, commonly called the tobacco hornworm, is the sole energizer of epithelial ion transport in this tissue, being responsible for the alkalinization of the gut lumen up to a pH of more than 11 and for any active ion movement across the epithelium. This minireview deals with those topics of our recent research on this enzyme that may contribute novel aspects to the biochemistry and physiology of V-ATPases. Our research approaches include intramolecular aspects such as subunit topology and the inhibition by macrolide antibiotics, intermolecular aspects such as the hormonal regulation of V-ATPase biosynthesis and the interaction of the V-ATPase with the actin cytoskeleton, and supramolecular aspects such as the interactions of V-ATPase, K+/H+ antiporter, and ion channels, which all function as an ensemble in the transepithelial movement of potassium ions.

Vacuolar H(+)-ATPases: intra- and intermolecular interactions.

V-ATPases in eukaryotes are heteromultimeric, H(+)-transporting proteins. They are localized in a multitude of different membranes and energize many different transport processes. Unique features of V-ATPases are, on the one hand, their ability to regulate enzymatic and ion transporting activity by the reversible dissociation of the catalytic V(1) complex from the membrane bound proton translocating V(0) complex and, on the other hand, their high sensitivity to specific macrolides such as bafilomycin and concanamycin from streptomycetes or archazolid and apicularen from myxomycetes. Both features require distinct intramolecular as well as intermolecular interactions. Here we will summarize our own results together with newer developments in both of these research areas.

Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.

The SLC45 gene family of putative sugar transporters.

According to the classic point of view, transport of sugars across animal plasma membranes is performed by two families of transporters. Secondary active transport occurs via Na(+) symporters of the SLC5 gene family, while passive transport occurs via facilitative transporters of the SLC2 family. In recent years a new family appeared in the scenery which was called the SLC45 gene family of putative sugar transporters, mainly because of obvious similarities to plant sucrose transporters. The SLC45 family consists of only four members that have been denominated A1-A4. These members apparently have counterparts in all vertebrates. Moreover, their amino acid sequences reveal close homologies also to respective invertebrate proteins such as a recently detected sucrose transporter in Drosophila, and suggest a phylogenetic relationship also to corresponding proteins from plants, fungi and bacteria. This minireview describes the molecular features of its members with a focus on their possible role as sugar transporters.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.