Helmut Wieczorek

The V-type H+ ATPase: molecular structure and function,physiological roles and regulation

SUMMARY It was nearly 30 years before the V-type H+ ATPase was admitted to the small circle of bona fide transport ATPases alongside F-type and P-type ATPases. The V-type H+ ATPase is an ATP-driven enzyme that transforms the energy of ATP hydrolysis to electrochemical potential differences of protons across diverse biological membranes via the primary active transport of H+. In turn, the transmembrane electrochemical potential of H+ is used to drive a variety of (i) secondary active transport systems via H+-dependent symporters and antiporters and (ii) channel-mediated transport systems. For example, expression of Cl- channels or transporters next to the V-type H+ ATPase in vacuoles of plants and fungi and in lysosomes of animals brings about the acidification of the endosomal compartment, and the expression of the H+/neurotransmitter antiporter next to the V-type H+ ATPase concentrates neurotransmitters in synaptic vesicles. First found in association with endosomal membranes, the V-type H+ ATPase is now also found in increasing examples of plasma membranes where the proton pump energizes transport across cell membranes and entire epithelia. The molecular details reveal up to 14 protein subunits arranged in (i) a cytoplasmic V1 complex, which mediates the hydrolysis of ATP, and (ii) a membrane-embedded V0 complex, which translocates H+ across the membrane. Clever experiments have revealed the V-type H+ ATPase as a molecular motor akin to F-type ATPases. The hydrolysis of ATP turns a rotor consisting largely of one copy of subunits D and F of the V1 complex and a ring of six or more copies of subunit c of the V0 complex. The rotation of the ring is thought to deliver H+ from the cytoplasmic to the endosomal or extracellular side of the membrane, probably via channels formed by subunit a. The reversible dissociation of V1 and V0 complexes is one mechanism of physiological regulation that appears to be widely conserved from yeast to animal cells. Other mechanisms, such as subunit-subunit interactions or interactions of the V-type H+ ATPase with other proteins that serve physiological regulation, remain to be explored. Some diseases can now be attributed to genetic alterations of specific subunits of the V-type H+ ATPase.

Animal plasma membrane energization by proton-motive V-ATPases.

Proton‐translocating, vacuolar‐type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na+ gradients, imposed by Na+/K+ ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H+ V‐ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V‐ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V‐ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na+ uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V‐ATPases. Awareness of plasma membrane energization by V‐ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture. BioEssays 21:637–648, 1999.

Inhibitors of V-ATPases: old and new players

SUMMARY V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic endomembranes and plasma membranes, they energize many different transport processes. Currently, a handful of specific inhibitors of the V-ATPase are known, which represent valuable tools for the characterization of transport processes on the level of tissues, single cells or even purified proteins. The understanding of how these inhibitors function may provide a basis to develop new drugs for the benefit of patients suffering from diseases such as osteoporosis or cancer. For this purpose, it appears absolutely essential to determine the exact inhibitor binding site in a target protein on the one side and to uncover the crucial structural elements of an inhibitor on the other side. However, even for some of the most popular and long known V-ATPase inhibitors, such as bafilomycin or concanamycin, the authentic structures of their binding sites are elusive. The aim of this review is to summarize the recent advances for the old players in the inhibition game, the plecomacrolides bafilomycin and concanamycin, and to introduce some of the new players, the macrolacton archazolid, the benzolactone enamides salicylihalamide, lobatamide, apicularen, oximidine and cruentaren, and the indolyls.

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.

Archazolid and apicularen: Novel specific V-ATPase inhibitors

BackgroundV-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.ResultsArchazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative.ConclusionThe myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

Stoichiometry of K^+/H^+ antiport helps to explain extracellular pH 11 in a model epithelium

The stoichiometry of K+/H+ antiport was measured fluorometrically by the static head method in highly purified vesicles from goblet cell apical membranes of larval lepidopteran midgut. The measured stoichiometry of 1 K+/2 H+ explains how the antiport results in electrophoretic exchange of extracellular H+ for intracellular K+, driven by the voltage component of the proton‐motive force of an H+ translocating V‐ATPase that is located in the same membrane. In turn, the exchange of K+ for H+ helps to explain how the midgut contents are alkalinized to a pH of 11.

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.

ISOLATION OF GOBLET CELL APICAL MEMBRANE FROM TOBACCO HORNWORM MIDGUT AND PURIFICATION OF ITS VACUOLAR-TYPE ATPASE

Publisher Summary This chapter focuses on the isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase. The only known method for isolation of the goblet cell apical membrane from larval lepidopteran midgut is described in this chapter. This chapter also described is the purification of the ATPase associated with this membrane as well as procedures for measuring both membrane-bound and purified solubilized ATPase activity. The chapter also discusses a detailed description of reliable and sensitive colorimetric methods for measurement of inorganic phosphate and protein is included. This chapter presents a discussion on the isolation of the goblet cell apical membrane. The isolated posterior midgut segment is placed lumen side down and the Malpighian tubules are removed.this chapter concludes with the discussion on Purification of the goblet cell apical membrane ATPase.