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Vacuolar type ATPase (V-ATPase) has recently emerged as a promising novel anticancer target based on extensive in vitro and in vivo studies with archazolids, complex polyketide macrolides, which present the most potent V-ATPase inhibitors known to date. Herein, we report a biomimetic, one-step preparation of archazolid F, the most potent and least abundant archazolid, the design and synthesis of five novel, carefully selected archazolid analogues, and the biological evaluation of these antiproliferative agents, leading to the discovery of a very potent but profoundly simplified archazolid analogue. Furthermore, the first general biological profiling of the archazolids against a broad range of more than 100 therapeutically relevant targets is reported, leading to the discovery of novel and important targets. Finally, first pharmacokinetic data of these natural products are disclosed. All of these data are relevant in the further preclinical development of the archazolids as well as the evaluation of V-ATPases as a novel and powerful class of anticancer targets.

The solute carrier 45 family (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1–A4, which show only 20–30% identity of amino acid sequences among each other. All these members exhibit an identity of ∼20% to plant H+/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H+/sucrose cotransporters. In contrast with the plant proteins, SLC45 transporters recognise also the monosaccharides glucose and fructose as physiological substrates and seem to be involved in alternative sugar supply as well as in osmoregulation of several mammalian tissues. In the present study, we provide novel insights into the regulation of SLC45 transporters. By screening for interaction partners, we found a 14-3-3 protein as a promising candidate for control of transport activity. Indeed, co-expression of the gamma isoform of murine 14-3-3 protein in yeast and Xenopus oocytes led to a significant decrease in transport rates of the murine SLC45 transporters as well as of the plant H+/sucrose transporter Sut1.

In the present study, we describe the detection and analysis of a novel type of sugar transporter in mammalian spermatozoa. This transporter belongs to the SLC45 family for which two features are remarkable and distinguish it from other known families of sugar transporters. Firstly, SLC45 transporters recognise not only the monosaccharides glucose or fructose but also the disaccharide sucrose as a substrate. Secondly, the uptake of sugars is coupled to a proton gradient. Uptake experiments using radioactively labelled sucrose indicated a functional transporter of the SLC45 family in bull spermatozoa. Real-time PCR as well as Western blots demonstrated the occurrence of the SLC45 member A4 in mouse testis and sperms. Furthermore, immunocytochemical analysis of mouse tissues revealed that the signal of SLC45A4 was mainly located in the principle piece of spermatozoa. We postulate that the SLC45A4 transporter plays an important role in nutrition of spermatozoa during their maturation in the epididymis. Moreover, we suggest that knowledge about the presence of the SLC45A4 may be useful also for the methodical improvement of cryopreservation of mammalian spermatozoa.

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In the present study, we describe the detection and analysis of a novel type of sugar transporter in mammalian spermatozoa. This transporter belongs to the SLC45 family for which two features are remarkable and distinguish it from other known families of sugar transporters. Firstly, SLC45 transporters recognise not only the monosaccharides glucose or fructose but also the disaccharide sucrose as a substrate. Secondly, the uptake of sugars is coupled to a proton gradient. Uptake experiments using radioactively labelled sucrose indicated a functional transporter of the SLC45 family in bull spermatozoa. Real-time PCR as well as Western blots demonstrated the occurrence of the SLC45 member A4 in mouse testis and sperms. Furthermore, immunocytochemical analysis of mouse tissues revealed that the signal of SLC45A4 was mainly located in the principle piece of spermatozoa. We postulate that the SLC45A4 transporter plays an important role in nutrition of spermatozoa during their maturation in the epididymis. Moreover, we suggest that knowledge about the presence of the SLC45A4 may be useful also for the methodical improvement of cryopreservation of mammalian spermatozoa.

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., Graphical abstract, Highlights • Dissociation of vacuolar H+-ATPase domains deactivates its V1 motor. • V1 has one “open” catalytic site linked to the stator filament bound by subunit H. • Movement of subunit H to prevent rotary catalysis is possible. • Three stator filaments project from deactivated V1, indicating inherent stiffness. • This work gives new insight into energetic coupling and control in V-ATPases.

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.

Two structurally novel analogues of the macrolides archazolids A and B, archazolid A-15-O-β-D-glucopyranoside (archazolid E, 5) and iso-archazolid B (archazolid F, 6), were isolated from the myxobacterium Cystobacter violaceus and Archangium gephyra, respectively. Macrolactone 5 represents the first 15-O-glycoside of the archazolids. iso-Archazolid B (6) incorporates a C-3 alkene and presents the first constitutional isomer reported for this natural product class. The structures of these polyketides were determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations and by chemical degradation. iso-Archazolid B (6) demonstrated extremely high antiproliferative and V-ATPase inhibitory effects, with IC(50) values in the picomolar range, while only moderate activity was observed for glycoside 5. iso-Archazolid B presents the most potent archazolid known.

(Trifluoromethyl)diazirines are well established photoaffinity labels (PAL) used in biochemical investigations to create covalent ligand-receptor bonds. Two new diazirinylbenzoic acids 8b,c with perfluorobutyl and perfluorooctyl chains (F-PAL) were efficiently prepared from p-bromobenzaldehyde and attached to the highly potent and specific V-ATPase inhibitors 21-deoxyconcanolide A (2) and bafilomycin A 1 (5), deriving from the natural product pool from Streptomyces producer strains. The labelled derivatives 17 and 18 were efficiently purified by fluorous solid-phase extraction. Functional biochemical assays with the V-ATPase holoenzymes proofed strong inhibition activities. So far, radioactive isotopes or biotin-tags have mainly been used for tracing compounds in photoaffinity studies. The C 4 F 9 - and C 8 F 17 -fluorous tags aim to enable advantageous separation and identification of labelled peptide fragments by fluorous chromatography followed by MS analysis. Therefore, F-PAL represents an innovative new concept for binding site determination and should significantly accelerate and simplify such biochemical investigations.

Biology of bracken insects. A contribution to the problem of the biological control of bracken Pteridium aquilinum (L.) Kuhn in Central Europe. As a contribution to basic work on biological control of P. aquilinum the morphology, distribution and biology of 44 fern-insects in Central-Europe are shortly described by putting together all available information. Plantsociological, physiological and ecological aspects are discussed before this.

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.

Diazirine-tagged systems are considered reliable compounds for photoaffinity labeling (PAL) in biochemical studies as they enable investigation and understanding of biological mechanisms through covalent bonding to the target and subsequent detection. 14 C-labeled 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzoic acid (11) was prepared by a lithium-bromide exchange on the bis-silylated 4-bromophenyldiaziridine 19 with subsequent transformations with electrophiles as key steps of the synthesis. Using 14 CO2, which was generated from rather inexpensive Ba 14 CO3, the desired diaziridinylbenzoic acid 21 was obtained in 78 % yield based on the

The novel polyketide macrolide archazolid-7-O-β-D-glucopyranoside (3) has been isolated from the myxobacterium Cystobacter violaceus and the structure of this first archazolid-glycoside has been determined by spectroscopic and degradative methods. A synthesis of simplified 7-O analogues, based on regioselective derivatisation of archazolid A, was elaborated. These structurally novel archazolids of natural and synthetic origin were evaluated in detail for V-ATPase inhibition and their biological activities are discussed in terms of their solution conformations, as determined by high-field NMR studies, including J-based conformation analysis and constrained molecular dynamics simulations. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Novel analogues of the V-ATPase inhibitors archazolid A and B with modifications of the free hydroxyl groups and the side chain were designed by molecular modeling, synthesized by derivatization of the parent natural product and evaluated for V-ATPase inhibition and growth inhibition of murine connective tissue cells.

Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.

The novel macrolide cruentaren A was produced at levels up to 3.2 mg/liter by cultures of the myxobacterium Byssovorax cruenta. The new compound strongly inhibited the growth of yeasts and filamentous fungi and showed high cytotoxicity against L929 mouse fibroblast cells. A minor co-metabolite of cruentaren A, named cruentaren B, and identified as a six-membered lactone isomer of cruentaren A, showed only marginal cytotoxicity and no antifungal activity. Cruentaren A inhibited F0F1 mitochondrial ATP-hydrolysis in submitochondrial particles of yeasts and beef heart.

SUMMARYIt 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 V0complexes 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.

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-type ATPase (V-ATPase) purified from the midgut of the tobacco hornworm Manduca sexta is inhibited 50% by 10 nm of the plecomacrolide concanamycin A, the specific inhibitor of V-ATPases. To determine the binding site(s) of that antibiotic in the enzyme complex, labeling with the semisynthetic 9-O-[p-(trifluoroethyldiazirinyl)-benzoyl]-21,23-dideoxy-23-[(125)I]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15-20 microm. Upon treatment with UV light, a highly reactive carbene is generated from this concanamycin derivative, resulting in the formation of a covalent bond to the enzyme. In addition, the radioactive tracer (125)I makes the detection of the labeled subunit(s) feasible. Treatment of the V(1)/V(o) holoenzyme, the V(o) complex, and the V-ATPase containing goblet cell apical membranes with concanolide resulted in the labeling of only the proteolipid, subunit c, of the proton translocating V(o) complex. Binding of J-concanolide A to subunit c was prevented in a concentration-dependent manner by concanamycin A, indicating that labeling was specific. Binding was also prevented by the plecomacrolides bafilomycin A(1) and B(1), respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.

In lepidopteran larvae, three transport mechanisms are involved in the active and electrogenic K(+) secretion that occurs in the epithelial goblet cells of the midgut. These consist of (i) basolateral K(+) channels, allowing K(+) entry from the haemolymph into the cytosol, (ii) apical electrogenic K(+)/2H(+) antiporters, which are responsible for secondary active extrusion of K(+) from the cell into the gut lumen via the goblet cavity and (iii) apical V-ATPase-type proton pumps. The latter energize apical K(+) exit by building up a large, cavity-positive electrical potential that drives the antiporters. Net K(+) secretion (I(K)) can be measured as short-circuit current (I(sc)) across the in vitro midgut mounted in an Ussing chamber. We investigated the influence of protons on the transepithelial I(K) and the partial reactions of the basolateral K(+) permeability (P(K)) and the apical, lumped 'K(+) pump' current (I(P)) at various extra- and intracellular pH values. In particular, we wanted to know whether increased cellular acidity could counteract the reversible dissociation of the V-ATPase into its V(1) and V(o) parts, as occurs in yeast after glucose deprivation and in the midgut of Manduca sexta during starvation or moulting, thus possibly enhancing K(+) transport. When intact epithelia were perfused with high-[K(+)] (32 mmol l(-1)) salines with different pH values, I(K) was reversibly reduced when pH values fell below 6 on either side of the epithelium. Attempts to modify the intracellular pH by pulsing with NH(4)(+) or propionate showed that intracellular acidification caused a reduction in I(K) similar to that obtained in response to application of external protons. Treatment with azide, a well-known inhibitor of the mitochondrial ATP synthase, had the same effect as pulsing with ammonium or propionate with, however, much faster kinetics and higher reversibility. Breakdown of the basolateral or apical barrier using the antibiotic nystatin allowed the intracellular pH to be clamped to that of the saline facing the nystatin-treated epithelial border. Cell acidification achieved by this manipulation led to a reduction in both apical I(P) and basolateral P(K). The transepithelial I(K) showed an approximately half-maximal reduction at external pH values close to 5 in intact tissues, and a similar reduction in I(P) and P(K) values was seen at an intracellular pH of 5 in nystatin-permeabilised epithelia. Thus, the hypothesized V(1)V(o) stabilization by cell acidity is not reflected in the pH-sensitivity of I(P). Moreover, all components that transport K(+) are synchronously inhibited below pH 6. The significance of our findings for the midgut in vivo is discussed.

Background Granulomatous reactions to poly- l -lactic acid (PLLA)-based filler have been described previously. Neither the biological background of these partly late-onset reactions or the desired augmenting effect of PLLA has been studied to date. Histological studies have revealed foreign body reactions and foreign body giant cell formation. Objective The aim of this study was to increase our knowledge about the biological mechanisms behind the augmenting effect of PLLA-based filler. Methods We characterised the cell infiltrate and collagen type of PLLA-treated tissue by immunofluorescence staining. The expression of genes related to collagen metabolism was determined. Results CD68+ macrophages were found next to PLLA. CD90+ fibroblasts were found alongside. αSMA-positive structures indicated myofibroblasts and neovascularisation. Substantial collagen type III deposition was detected next to PLLA particles and collagen type I was found at the periphery of PLLA encapsulations. mRNA expression for collagen type I and III transcripts, as well as for TGFβ1 and TIMP1, was upregulated significantly. Conclusion PLLA-induced augmentation is most likely based on capsule formation orchestrating macrophages, (myo-)fibroblasts, and collagen type I and III fibres. We observed considerably slower degradation of PLLA particles than described previously. Thus PLLA particles were still retrievable 28 months after subcutaneous application.

Summary Vacuolar H+-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases., Graphical Abstract, Highlights • Subnanometer V-ATPase EM structure gives new insights into mechanism • Comparison of two distinct catalytic states in a complete rotary ATPase • Describes a conserved electrostatic bearing that supports high motor efficiency • Proposes how different c ring stoichiometries are accommodated, Rawson et al. solve a high-resolution structure of vacuolar ATPase. The complex rests in a catalytic state different from those previously reported. The work gives new insights into the organization, mechanism, and the basis for functional properties such as high thermodynamic efficiency.

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. [14C]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.

Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V1 subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V1 and Vo even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.

We determined the structure of the V(1)-ATPase from Manduca sexta to a resolution of 1.8 nm, which for the first time reveals internal features of the enzyme. The V(1)-ATPase consists of a headpiece of 13.5 nm in diameter, with six elongated subunits, A(3) and B(3), of approximately equal size, and a stalk of 6 nm in length that connects V(1) with the membrane-bound domain, V(O). At the center of the molecule is a cavity that extends throughout the length of the A(3)B(3) hexamer. Inside the cavity the central stalk can be seen connected to only two of the catalytic A subunits. The structure was obtained by a combination of the Random Conical Reconstruction Technique and angular refinements. Additional recently developed techniques that were used include methods for simultaneous translational rotational alignment of the 0 degrees images, contrast transfer function correction for tilt images, and the Two-Step Radon Inversion Algorithm.

The shape and overall dimensions of the oxidized and reduced form of the V1 ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6.20 ± 0.06 and 5.84 ± 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 ± 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V1 molecule provide the first strong evidence for major structural changes in the V1 ATPase because of redox modulation.

The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.

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. © 1999 John Wiley & Sons, Inc.

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3–4 nm in diameter.

Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel Vo subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta.

The midgut plasma membrane V-ATPase from larval Manduca sexta,the tobacco hornworm, is the sole energizer of any epithelial ion transportin this tissue and is responsible for the alkalinization of the gut lumen upto a pH of more than 11. This mini-review deals with those topics of researchon this enzyme which may have contributed or are expected to contribute noveland general aspects to the field of V-ATPases. Topics dealt with includenovel subunits or the quaternary structure of the V1 complex, aswell as the regulation of the enzyme's function by reversible dissociation ofthe V1 from the V0 complexes and by genetic control onthe transcriptional and posttranscriptional level.

The V1 ATPase from the tobacco hornworm Manduca sexta and the Escherichia coli F1 ATPase were characterized by small-angle X-ray scattering (SAXS). The radii of gyration (Rg) of the complexes were 6.2 +/- 0.1 and 4.7 +/- 0.02 nm, respectively. The shape of the M. sexta V1 ATPase was determined ab initio from the scattering data showing six masses, presumed to be the A and B subunits, arranged in an alternating manner about a 3-fold axis. A seventh mass with a length of about 11.0 nm extends perpendicularly to the center of the hexameric unit. This central mass is presumed to be the stalk that connects V1 with the membrane domain (V(O)) in the intact V1V(O)-ATPase. In comparison, the shape of the F1 ATPase from E. coli possesses a quasi-3-fold symmetry over the major part of the enzyme. The overall asymmetry of the structure is given by a stem, assumed to include the central stalk subunits. The features of the V1 and F1 ATPase reveal structural homologies and diversities of the key components of the complexes.

SYNOPSIS. H+ V-ATPases are well known energizers of endomembranes; thus they play a key role in the acidification of vacuoles and vesicles. More recently it has become clear that they energize many plasma membranes as well. In epithelial cells H+ V-ATPases usually energize apical plasma membranes in the same sense that Na+/K+ P-ATPases usually energize basolateral plasma membranes. Examples of four fundamental processes so energized will be reviewed—Na+ and Cl− absorption by the frog skin, K+ secretion by the caterpillar midgut, fluid secretion by insect Malpighian tubules, and fluid absorption by insect ovarian follicle cells. It is likely that apical membranes of fresh water fish and other animals that live in media in which the concentration of Na+ is low, are also energized by H+ V− ATPases.

The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ∼30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies. © 2013 Song et al.

The spatial and temporal distribution of the plasma membrane V-ATPase and its encoding mRNA in the midgut of Manduca sexta were investigated during the moult from the fourth to the fifth larval instar. Digoxigenin-labelled RNA probes were used for in situ hybridization of V-ATPase mRNA of both peripheral and integrated subunits; monoclonal antibodies to subunits of the peripheral sector of the purified plasma membrane V-ATPase were used for immunocytochemistry. Extensive mRNA labelling was found in both mature columnar and goblet cells of intermoult and moulting larvae. Hybridization screening in several tissues suggested that only cells with increased V-ATPase biosynthesis were labelled by our hybridization method. Mature goblet cells contain a large amount of V-ATPase in the apical plasma membrane and were therefore expected to contain V-ATPase mRNA. The intense mRNA signal found in mature columnar cells was unexpected. However, after refining the techniques of tissue preparation, immunolabelling in apical blebs of columnar cells was demonstrated. Since this immunoreactivity did not appear to be membrane-associated, it suggested a cytosolic localization of peripheral V1 subunits. The mRNA encoding subunit A of the peripheral V1 sector was distributed unevenly in columnar cells with a strong apical preference, whereas the mRNA for the proteolipid of the integral V0 sector was evenly distributed in the cytosol. This spatial pattern reflected the distribution of free ribosomes and rough endoplasmic reticulum in the cell, supporting the view that V1 subunits are synthesized at free ribosomes, whereas the V0 subunits are synthesized at the rough endoplasmic reticulum. All undifferentiated cells exhibited intense mRNA signals for V-ATPase subunits of both holoenzyme sectors from the start of proliferation and thus precursors of columnar and goblet cells could not be distinguished.

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.

The plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K+/nH+ antiport and thus K+ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K+ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V1 sector, whereas the membrane integral V0 subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V1 domain.

SummaryTransepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes. In parallel, membraneassociation of the V1 subunits C and D increases, consistent with the assembly of the holoenzyme. The protein kinase A inhibitor H-89 abolishes all cAMP-induced effects, consistent with PKA being involved in V-ATPase activation. Metabolic inhibition induced by KCN, azide and 2,4-dinitrophenol, respectively, also induces assembly of functional V-ATPases at the membrane without protein kinase A involvement, indicating a phosphorylation independent activation mechanism.

The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex. By photoaffinity labeling using derivatives of apicularen and of the plecomacrolides bafilomycin and concanamycin, each coupled to (14)C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid, we verified that apicularen binds at the interface of the V(O) subunits a and c. The binding site is in the vicinity to those of the plecomacrolides and of the archazolids, a third family of V-ATPase inhibitors. Expression of subunit c homologues from Homo sapiens and Manduca sexta, both species sensitive to benzolactone enamides, in a Saccharomyces cerevisiae strain lacking the corresponding intrinsic gene did not transfer this sensitivity to yeast. Therefore, the binding site of benzolactone enamides cannot be formed exclusively by subunit c. Apparently, subunit a substantially contributes to the binding of the benzolactone enamides.

The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.

Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the sub- nanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular dock- ing, molecular dynamics simulations, and biochemical experi- ments, resulting in a sound model for the inhibitory mech- anism of this class of putative anticancer agents. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded VO-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.

Both organic and inorganic solutes can be transported across biological membranes against their electrochemical gradient, by coupling to the downhill movement of another species. In animal cells, the underlying mechanism most often involves symport (cotransport) or antiport (countertransport) with either sodium or hydrogen ions. The energy invested in the uphill translocation of solutes is ultimately provided by hydrolysis of ATP by Na+or H+-transporting ATPases and is transiently stored as potential energy in the form of an electrochemical gradient of these ions. This introductory review describes the properties of systems that catalyze both Na+or H+-driven countertransport of inorganic cations, present in vertebrates and invertebrates. Although great strides have been made towards the biochemical identification and characterization of vertebrate transporters, the molecular analysis of the invertebrate systems is still in its infancy. Only a brief comparative overview is provided for general reference in this introductory section. More specific descriptions of the individual systems and recent developments are described more fully in the following chapters. To shorten the reference list and to avoid redundancy with the following sections, comprehensive reviews were cited wherever possible and references to original work were kept to a minimum.

A cDNA clone encoding a hydrophilic protein with a calculated molecular mass of 13,839 Da was isolated by shotgun screening with an anti-V-ATPase holoenzyme serum. The deduced amino acid sequence showed no significant homology to any other known protein. Southern blots revealed the existence of only one gene encoding the 14-kDa protein. Monospecific antibodies purified by affinity to the recombinant protein demonstrated the presence of a 14-kDa protein in the highly purified goblet cell apical membrane and inhibited ATP-dependent proton transport as well as V-ATPase activity to the same extent. Thus, the 14-kDa protein was shown to be a part of the V-ATPase holoenzyme. Binding of the monospecific antibodies to the ATPase seemed to require an ATP-dependent conformational change of the enzyme, since inhibition only occurred when ATP was present during the antibody binding step. The 14-kDa subunit could be stripped from the membrane by treatment with the chaotropic agent KI, confirming it to be part of the soluble complex of the V-ATPase. In immunoblots, the 14-kDa-specific antibodies showed no cross-reaction with several xenic V-ATPases.

This work presents the first invertebrate cDNA sequence encoding subunit E of a V-ATPase. It was cloned by immuno-shot-gun screening of a Manduca sexta (Insecta, Lepidoptera, Sphingidae) posterior larval midgut cDNA library. The amino acid sequence was 64% identical to that of the mammalian E-subunit and 34% to that of yeast. Southern and Northern blots suggested the existence of only one gene encoding the insect subunit E.

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.

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.