Vacuolar-type ATPases (V-ATPases) 1 are complex, heteromultimeric proteins consisting of a peripheral, catalytic V 1 complex and a membrane bound, ion translocating V o complex.

SummaryV-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.

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.

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Background:The V-ATPase has been proposed to function at the plasma membrane in tumor cell invasion. Results: Inhibition of plasma membrane V-ATPases prevented invasion of MDA-MB-231 cells. Conclusion: Activity of plasma membrane V-ATPases is critical for breast cancer cell invasion. Significance: Plasma membrane V-ATPases are a possible therapeutic target to limit metastasis.

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC 50 Vacuolar ATPases (V-ATPases)3 are heteromultimeric proteins that use the energy of ATP hydrolysis to translocate protons from the cytoplasm into intracellular compartments or across the plasma membrane of eukaryotic cells. This transport of protons is mediated by the membrane-integral V O complex, whereas the cleavage of ATP occurs at the cytoplasmatic V 1 complex (1). The V O complex is composed of single copies of subunits a, d, and e, and the ring-forming proteolipid subunits c, cЉ, and in fungi subunit cЈ also (2). Based on the crystal structure from the V O ring of K subunits, a homologue of the H ϩ -translocating subunit c in the V-type Na ϩ -ATPase from Enterococcus hirae, and a cryoelectron microscopy structure from the V-ATPase of Manduca sexta, an arrangement of 10 subunits is proposed for the V O ring (3, 4). The subunits c and cЈ are predicted to have four transmembrane helices (TM 1 to 4), whereas subunit cЉ contains an additional fifth transmembrane helix. All proteolipid subunits contain a conserved glutamate residue, subunits c and cЈ in TM4 and subunit cЉ in TM3, which are essential for proton transport across the membrane (2). This glutamate is a target for the covalent binding inhibitor N,NЈ-dicyclohexylcarbodiimide (DCCD) and its derivatives (5-8).By regulating the pH homeostasis and membrane energization of cells, V-ATPases are involved in a variety of fundamental processes like vesicular trafficking or secondary transport. In addition, plasma membrane V-ATPases are responsible for extracellular acidification, e.g. in osteoclasts or metastasing tumor cells, and therefore play an important role in severe diseases such as osteoporosis or cancer (7). For these reasons the V-ATPase is a promising therapeutic target, and inhibitors of this enzyme are the focus of biomedical research. A variety of such compounds has been discovered of which the plecomacrolide inhibitors bafilomycin and concanamycin are the best studied examples (9). With IC 50 values at low nanomolar concentrations these compounds are highly specific inhibitors of the V-ATPase (10). Throughout the past years the binding site and inhibition mechanism of the plecomacrolides has been studied in more detail. In 2002 Bowman et al. (11) identified via mutagenesis studies in Neurospora crassa amino acids in V O subunit c that contribute to the binding of bafilomycin. In the same year photoaffinity labeling studies with the radioactive concanamycin derivative 125 I-concanolid A also resulted in the *

-l-phenylalanine 2-naphthylamide; LAMP1, lysosomal-associated membrane protein 1; Leup, leupeptin; MAP1LC3, microtubule-associated protein 1 light chain 3;MTOR, mechanistic target of rapamycin; RFP, red fluorescent protein; tfLC3, tandem fluorescence-tagged LC3.Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca 2C content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. IntroductionAmong 3 types of autophagy, including microautophagy, chaperone-mediated autophagy, and macroautophagy, in mammals, macroautophagy (hereafter referred as autophagy) is the most common type. Autophagy is an evolutionarily conserved catabolic degradation cellular process in which misfolded proteins or damaged organelles are first sequestered by a doublemembrane vesicle, called an autophagosome. Autophagosomes then fuse with lysosomes to form autolysosomes, inside which the sequestered contents are digested by lysosomal enzymes and recycled to maintain cellular homeostasis. Autophagy can also be markedly induced by a wide variety of stresses, e.g., nutrient starvation, infection, and aging, for cell survival. Dysfunctional autophagy has been associated with wide ranges of human diseases, e.g., cancer and neurodegenerative diseases. [1][2][3][4][5][6] Basal autophagy activity is essential for cell homeostasis, and it is tightly controlled by a complicated interplay among several key machineries, including ULK1 or ULK2 complexes and the class III phosphatidylinositol-3 kinase complexes. The MTOR (mechanistic target of rapamycin) Ser/Thr kinase suppresses autophagy by inhibiting the ULK1/2 comple...

Background: V-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.