Academic literature on the topic 'V-ATPase proton pump'

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

Two ATP-dependent proton pumps function in plant cells. Plasma membrane H+-ATPase (PM H+-ATPase) transfers protons from the cytoplasm to the apoplast, while vacuolar H+-ATPase (V-ATPase), located in tonoplasts and other endomembranes, is responsible for proton pumping into the organelle lumen. Both enzymes belong to two different families of proteins and, therefore, differ significantly in their structure and mechanism of action. The plasma membrane H+-ATPase is a member of the P-ATPases that undergo conformational changes, associated with two distinct E1 and E2 states, and autophosphorylation during the catalytic cycle. The vacuolar H+-ATPase represents rotary enzymes functioning as a molecular motor. The plant V-ATPase consists of thirteen different subunits organized into two subcomplexes, the peripheral V1 and the membrane-embedded V0, in which the stator and rotor parts have been distinguished. In contrast, the plant plasma membrane proton pump is a functional single polypeptide chain. However, when the enzyme is active, it transforms into a large twelve-protein complex of six H+-ATPase molecules and six 14-3-3 proteins. Despite these differences, both proton pumps can be regulated by the same mechanisms (such as reversible phosphorylation) and, in some processes, such as cytosolic pH regulation, may act in a coordinated way.

Vacuolar ATPases (V-ATPases) are present in specialized proton secretory cells in which they pump protons across the membranes of various intracellular organelles and across the plasma membrane. The proton transport mechanism is electrogenic and establishes an acidic pH and a positive transmembrane potential in these intracellular and extracellular compartments. V-ATPases have been found to be practically identical in terms of the composition of their subunits in all eukaryotic cells. They have two distinct structures: a peripheral catalytic sector (V1) and a hydrophobic membrane sector (V0) responsible for driving protons. V-ATPase activity is regulated by three different mechanisms, which control pump density, association/dissociation of the V1 and V0 domains, and secretory activity. The C subunit is a 40-kDa protein located in the V1 domain of V-ATPase. The protein is encoded by the ATP6V1C gene and is located at position 22 of the long arm of chromosome 8 (8q22.3). The C subunit has very important functions in terms of controlling the regulation of the reversible dissociation of V-ATPases.

Vacuolar H+-ATPases (V-ATPases) are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (pro)renin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.

Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.

Plant cells are unique in containing large acidic vacuoles which occupy most of the cell volume. The vacuolar H+-ATPase (V-ATPase) is the enzyme responsible for acidifying the central vacuole, although it is also present on Golgi and coated vesicles. Many secondary transport processes are driven by the proton-motive force generated by the V-ATPase, including reactions required for osmoregulation, homeostasis, storage, plant defense and many other functions. However, a second proton pump, the V-PPase, serves as a potential back-up system and may, in addition, pump potassium. The plant V-ATPase is structurally similar to other eukaryotic V-ATPases and its subunits appear to be encoded by small multigene families. These multigene families may play important roles in the regulation of gene expression and in the sorting of V-ATPase isoforms to different organelles.

The vacuolar (H+)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.

The vacuolar H+-ATPase (V-ATPase) is a universal component of eukaryotic organisms. It is present in the membranes of many organelles, where its proton-pumping action creates the low intra-vacuolar pH found, for example, in lysosomes. In addition, there are a number of differentiated cell types that have V-ATPases on their surface that contribute to the physiological functions of these cells. The V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the familiar FoF1 ATP synthase (F-ATPase), having the same basic architectural construction, and many of the subunits from the two display identity with one another. All the core subunits of the V-ATPase have now been identified and much is known about the assembly, regulation and pharmacology of the enzyme. Recent genetic analysis has shown the V-ATPase to be a vital component of higher eukaryotes. At least one of the subunits, i.e. subunit c (ductin), may have multifunctional roles in membrane transport, providing a possible pathway of communication between cells. The structure of the membrane sector is known in some detail, and it is possible to begin to suggest how proton pumping is coupled to ATP hydrolysis.

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

Recent evidences highlighted that glioblastomas (GBM) secreted microvesicles (EVs), particularly exosomes (Exo) and large oncosomes (LO), play a major role in the cross-talk between tumor cell and non-neoplastic parenchyma. Recent work from our group has identified the vacuolar pump H+-ATPase (V-ATPase) as an important effector of GBM growth and glioma stem cells (GSC) maintenance. Additionally, in ExoCarta database V-ATPase subunits have been described in Exo from different cancer cell types. Taken together, these data identify V-ATPase as an important driver of gliomagenesis, and a novel, actionable therapeutic target for disease intervention. However, the role of V-ATPase in reprogramming the GBM microenvironment has not been previously investigated. The aim of this project was investigate production, biological effect and content of extracellular vesicles according to proton pump activity in glioma stem cells. Our data show that GSC are able to produce different types of EVs, which are internalized by recipient cells of different histology, such as non-neoplastic brain tumor margins, primary GBM monolayers (both differentiated and undifferentiated), and commercial glioma cultures. Exo and LO from GSC induces in recipient cells distinct effects. In particular, Exo significantly increased cell growth and cell motility, and these effects were stronger with Exo produced by NS with higher V-ATPase expression (V1G1HIGH NS). On the other hand, LO were able to strongly induce the sphere formation ability of primary GBM cultures. This effect lasted up 90 days after co-culture. In both situations, the block of V-ATPase activity by Bafilomycin A1 in NS-producing EVs completely reverted the effects. Interestingly, exosomes are able to vehiculate on their surface the V-ATPase G1 subunit, and its protein level increased in recipient cells after co-culture with EVs. At the molecular level, profiling of Exo-derived miRNAs distinguishes V1G1HIGH NS from V1G1LOW cultures. In silico analysis and annotation of miRNA target genes showed an enrichment of cancer, cell cycle and MAPK/Erk pathways. Regarding signaling pathway modulation by Exo in recipient cells, exosomes from V1G1HIGH NS activated the MAPK/Erk pathway. Altogether, these data point toward the central role of different EV types in GBM communication and suggest a role of the V-ATPase proton pump in regulating exosomes contents.

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O acÃmulo de Na+ no vacÃolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulaÃÃo dos volumes e conteÃdos dos vacÃolos de cÃlulas vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prÃtons (V-ATPase e V-PPase), aquoporinas e vÃrios sistemas de transportes ativos e/ou secundÃrios, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prÃtons transmembranares funcionam como sistemas de transporte primÃrio nas cÃlulas vegetais e ambas as enzimas geram uma diferenÃa de potencial eletroquÃmico de prÃtons atravÃs da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquÃmico de prÃtons gerado pelos transportadores primÃrios para transportar Na+ para dentro do vacÃolo. No presente trabalho inicialmente foram determinados os conteÃdos de Ãons Na+ e K+ em raÃzes, hipocÃtilos e folhas e em seguida a anÃlise da expressÃo dos genes das bombas de prÃtons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plÃntulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmÃtico. As plÃntulas foram crescidas em meio nutritivo na ausÃncia de NaCl e PEG (controle), na presenÃa de 100 mM de NaCl (estresse salino) ou na presenÃa de 200,67 g/L de PEG (estresse osmÃtico). O conteÃdo de Ãons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteÃdo de Ãons K+ diminuiu na mesma condiÃÃo. A expressÃo dos genes das bombas de prÃtons e dos contra-transportadores vacuolares de folhas e de raÃzes no estresse salino aumentou em todas as condiÃÃes estudadas, porÃm o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulaÃÃo paralela entre esses genes. JÃ no estresse osmÃtico, os resultados para as folhas mostraram que a expressÃo dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes nÃo sofreram mudanÃas significativas. Nossos resultados sugerem que o estresse salino e o estresse osmÃtico induziram uma regulaÃÃo diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmÃtico.
The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress

SOBREIRA, Alana Cecília de Menezes. Estudo da expressão dos genes das bombas de prótons (V-ATPase e V-PPase) e dos contra-transportadores vacuolares (NHX) de Vigna unguiculata (L.) Walp submetidos a estresses abióticos.2009 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, 2009.
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The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress.
O acúmulo de Na+ no vacúolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulação dos volumes e conteúdos dos vacúolos de células vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prótons (V-ATPase e V-PPase), aquoporinas e vários sistemas de transportes ativos e/ou secundários, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prótons transmembranares funcionam como sistemas de transporte primário nas células vegetais e ambas as enzimas geram uma diferença de potencial eletroquímico de prótons através da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquímico de prótons gerado pelos transportadores primários para transportar Na+ para dentro do vacúolo. No presente trabalho inicialmente foram determinados os conteúdos de íons Na+ e K+ em raízes, hipocótilos e folhas e em seguida a análise da expressão dos genes das bombas de prótons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plântulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmótico. As plântulas foram crescidas em meio nutritivo na ausência de NaCl e PEG (controle), na presença de 100 mM de NaCl (estresse salino) ou na presença de 200,67 g/L de PEG (estresse osmótico). O conteúdo de íons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteúdo de íons K+ diminuiu na mesma condição. A expressão dos genes das bombas de prótons e dos contra-transportadores vacuolares de folhas e de raízes no estresse salino aumentou em todas as condições estudadas, porém o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulação paralela entre esses genes. Já no estresse osmótico, os resultados para as folhas mostraram que a expressão dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes não sofreram mudanças significativas. Nossos resultados sugerem que o estresse salino e o estresse osmótico induziram uma regulação diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmótico.

Vacuolar H + -ATPases (V-ATPase), is an ATP-dependent proton transporter that transports protons across intracellular and cellular plasma membranes. V-ATPase is a multi-protein complex, which functions as an ATP-driven proton pump and is involved in maintaining pH homeostasis. The V-ATPase is a housekeeping proton pump and is highly conserved during evolution. The proton-pumping activity of V-ATPases allows acidification of intracellular compartments and influences a diverse range of cellular and biological processes. Thus, V-ATPase aberrant overexpression, mis-localization, and mutations in the genes for subunits are associated with several human diseases. This chapter focuses on a detailed view of V-type ATPase, and how V-ATPase contributes to human health and disease.

Exposure of the yeast Saccharomyces cerevisiae to environmental stress can influence cell growth, physiology and differentiation, and thus result in a cell’s adaptive response. During the course of an adaptive response, the yeast vacuoles play an important role in protecting cells from stress. Vacuoles are dynamic organelles that are similar to lysosomes in mammalian cells. The defect of a lysosome’s function may cause various genetic and neurodegenerative diseases. The multi-subunit V-ATPase is the main regulator for vacuolar function and its activity plays a significant role in maintaining pH homeostasis. The V-ATPase is an ATP-driven proton pump which is required for vacuolar acidification. It has also been demonstrated that phospholipid biosynthetic genes might influence vacuolar morphology and function. However, the mechanistic link between phospholipid biosynthetic genes and vacuolar function has not been established. Recent studies have demonstrated that there is a regulatory role of Pah1p, a phospholipid biosynthetic gene, in V-ATPase disassembly and activity. Therefore, in this chapter we will use Saccharomyces cerevisiae as a model to discuss how Pah1p affects V-ATPase disassembly and activity and how Pah1p negatively affect vacuolar function. Furthermore, we propose a hypothesis to describe how Pah1p influences vacuolar function and programmed cell death through the regulation of V-ATPase.

The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It pumps protons into the vacuolar system of eukaryotic cells and provides the energy for numerous transport systems. Through our BARD grant we discovered a novel family of membrane chaperones that modulate the amount of membrane proteins. We also elucidated the mechanism by which assembly factors guide the membrane sector of V-ATPase from the endoplasmic reticulum to the Golgi apparatus. The major goal of the research was to understand the mechanism of action and biogenesis of V-ATPase in higher plants and fungi. The fundamental question of the extent of acidification in organelles of the vacuolar system was addressed by studying the V-ATPase of lemon fruit, constructing lemon cDNAs libraries and study their expression in mutant yeast cells. The biogenesis of the enzyme and its function in the Golgi apparatus was studied in yeast utilizing a gallery of secretory mutants available in our laboratories. One of the goals of this project is to determine biochemically and genetically how V-ATPase is assembled into the different membranes of a wide variety of organelles and what is the mechanism of its action.The results of this project advanced out knowledge along these lines.