Patricia M Kane
Upstate Medical University
Project start date: 1994-03-01
Project end date: 2016-01-31
Sponsored Links Excellgen http://Excellgen.com
Subunit Structure And Function In Vacuolar H+-ATPase
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 2R01GM050322-10A1 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Vacuolar proton-translocating ATPases (V-ATPases) couple hydrolysis of cytosolic ATP to proton transport into organelles of all eukaryotic cells and across the plasma membrane of some cell types. Organelle acidification, the major constitutive function of V-ATPases, is essntial for many physiological processes, but is also linked to a number of disease states. For example, acidification of phagosomes is essential for killing invading bacteria, but many viruses and toxins exploit the acidic environment generated by V-ATPases to facilitate their escape from organelles into the cytoplasm where they become biologically active. Plasma membrane V-ATPases are involved in renal acid secretion and osteoclast bone dissolution; mutations in tissue-specific V-ATPase subunit isoforms necessary for these processes result in genetic diseases characterized by metabolic acidosis and osteoporosis. The long-term goal of the lab is to understand the structure, function, assembly and regulation of V-ATPases by studying the yeast V-ATPase, which has proven to be an excellent model for all eukaryotic V-ATPases. All V-ATPases are composed of two multisubunit domains, a peripheral membrane complex involved in ATP hydrolysis and an integral membrane complex required for proton transport. In this proposal, we focus on the "stalk" subunits that structurally and functionally bridge these two domains. These subunits are responsible for transmission of conformational changes resulting from ATP hydrolysis to the proton pore, and are also key players in regulated disassembly of V-ATPases, an important regulatory mechanism. The aims of this proposal are 1) to position the stalk subunits in the yeast V-ATPase, by a combination of electron microscopy, hydrodynamic studies of subcomplexes, mutagenesis, and crosslinking experiments, 2) to elucidate the roles of the C and H subunits, particularly in the functionally important conformational change accompanying release of the peripheral sector from the membrane sector, 3) to examine protein-protein interactions with two isoforms of the "a" subunit and test their importance in regulated disassembly, and 4) to follow V-ATPase assembly and disassembly in vivo using GFP-tagged V-ATPase subunits.
Keywords: adenosinetriphosphatase, enzyme mechanism, enzyme structure, hydrogen transport, membrane transport protein, protein structure function, vesicle /vacuole, acidity /alkalinity, active site, conformation, cytoplasm, enzyme activity, isozyme, molecular assembly /self assembly, protein protein interaction, Saccharomyces cerevisiae, crosslink, electron microscopy, fluorescence resonance energy transfer, green fluorescent protein, mass spectrometry, site directed mutagenesis, yeast
Project start date: 1994-03-01
Project end date: 2007-05-31
2R01GM050322-10A1 (2003): $258400
SUBUNIT STRUCTURE/FUNCTION IN VACUOLAR H+ ATPASES
Patricia M Kane, Professor
Biochem And Molecular Biologyupstate Medical University
research Administration
syracuse, Ny 13210
Grant 2R01GM050322-06 from National Institute Of General Medical Sciences IRG: PB
Abstract: Vacuolar proton-translocating ATPases (V-ATPases) are found in all eukaryotic cells and appear to play both constitutive roles in all cells and more specialized roles in certain cell types. V-ATPases are multisubunit enzymes capable of coupling ATP hydrolysis to proton transport across membranes. The primary constitutive role of V-ATPases in all human cells appears to be acidification of certain intracellular compartments, and this acidification is critical for maintenance of the cell´s internal organization and ability to respond to extracellular stimuli. Organelle acidification mediated by V-ATPases is exploited by a variety of pathogens, including certain viruses and toxins, to allow these pathogens to enter the cell cytoplasm; other pathogens manipulate V- ATPase activity to allow them to exist in intracellular compartments. V-ATPases play specialized roles in regulated secretory granules of neural cells, where they are involved in sequestration of neurotransmitters, and at the plasma membrane of kidney intercalated cells, osteoclasts, macrophages and neutrophils, where they are involved in urinary acidification, bone resorption, and regulation of cytoplasmic pH, respectively. The V-ATPase of the yeast Saccharomyces cerevisiae has proven to be an excellent experimental model for V-ATPases of other eukaryotes, including humans. The long-term goals of this research are to understand the structure, function, assembly, and regulation of the yeast V-ATPase. The specific aims of this proposal are directed toward understanding the interaction between the peripheral V1 sector of the V-ATPase, which is responsible for ATP hydrolysis, and the integral membrane Vo sector, which is responsible for proton transport. The interaction between the V1 and Vo sectors is central to the catalytic activity of V-ATPases and is also a major site of enzyme regulation. Toward this goal, the functions of two subunits (Vma5p and Vma13p) that are known to be important for interaction between the V1 and Vo sectors will be studied in detail, both in wild-type yeast cells and in strains containing point mutations in each subunit gene. Reversible dissociation of V1-Vo complexes into cytosolic V1 sectors and membrane- bound Vo sectors has been shown to occur in vivo in response to nutrient deprivation in yeast and in insect cells, and is probably a general mechanism of regulation. Cytosolic V1 sectors will be isolated from yeast cells, and the biochemical and enzymatic properties of these sectors will be examined. Links between catalytic activity, nucleotide binding, changes in cytosolic pH, and assembly state of the V-ATPase will be explored in biochemical studies, and the physiological benefits of dissociation of the V-ATPase under conditions of nutrient deprivation will be examined by isolating yeast mutants defective in disassembly of the enzyme
Keywords: enzyme mechanism, enzyme structure, hydrogen transport, hydrogen transporting ATP synthase, vesicle /vacuole active site, adenosinetriphosphatase, conformation, cytoplasm, enzyme activity, membrane protein, molecular assembly /self assembly, mutant, point mutation, structural gene Saccharomyces cerevisiae, acidity /alkalinity, epitope mapping, site directed mutagenesis
Project start date: 1994-03-01
Project end date: 2003-02-28
2R01GM050322-06 (1999): $192705
Subunit Structure And Function In Vacuolar H+-ATPases
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 2R01GM050322-14 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: V-ATPases are ATP-driven proton pumps that are responsible for organelle acidification in all eukaryotic cells and are recruited to the plasma membrane for specialized functions in osteoclasts, kidney, and the male reproductive tract. V-ATPase activity is intimately linked to protein sorting in the endocytic and biosynthetic pathways, zymogen activation, and pH, calcium, and metal ion homeostasis. V-ATPase activity is also subverted to support metastasis of certain cancers and virus and toxin release from the endocytic pathway into the cytosol. V-ATPases are very highly conserved, and are comprised of a complex of peri- pheral membrane proteins containing the sites of ATP hydrolysis, V1, attached to a membrane complex containing the proton pore, Vo. V1 and Vo sectors must associate for proton pumping to occur, but they also reversibly dissociate under conditions of energy limitation. We propose to continue to use yeast as a model system to explore the structural basis of V-ATPase mechanism and regulation, along with the cellular context of V-ATPase activity, through the following three aims 1) We will define the interactions of the V1 C and H subunits with the V1 and Vo sectors and probe how these interactions change during glucose deprivation. Work in the current grant period indicated that the V1 sector has two stator stalks, and we hypothesize that the C and H bridge these stalks to different regions of the membrane sector, and from this position, regulate reversible disassembly of V1 from Vo. 2) Reversible disassembly in response to glucose suggests that V- ATPase activity is aligned to the varying needs of the cell through poorly understood metabolic signals. We will explore the cellular basis of this alignment by testing the functional roles of several proteins recently found to interact with the Vo sector and exploiting the sensitivity of diploid cells to H subunit haploin- sufficiency as a means to identify gene products important for V1-Vo reassembly. 3) We will determine the extent to which the V-ATPase is both a general pH regulator in the cell and a pH sensor. Using recently developed methods for flexible and robust vacuolar and cytosolic pH measurement in yeast, we will deter- mine the extent to which V-ATPase activity affects overall pH homeostasis. These experiments may help explain the far-reaching physiological defects of yeast mutants lacking V-ATPase activity. We will also examine the activity of the V-ATPase itself in response to measured cytosolic and vacuolar pH changes.
Project start date: 1994-03-01
Project end date: 2011-08-31
2R01GM050322-14 (2007): $282600
Grants awarded to Patricia M Kane
Subunit Structure And Function In Vacuolar H+-ATPase
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 5R01GM050322-13 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Vacuolar proton-translocating ATPases (V-ATPases) couple hydrolysis of cytosolic ATP to proton transport into organelles of all eukaryotic cells and across the plasma membrane of some cell types. Organelle acidification, the major constitutive function of V-ATPases, is essntial for many physiological processes, but is also linked to a number of disease states. For example, acidification of phagosomes is essential for killing invading bacteria, but many viruses and toxins exploit the acidic environment generated by V-ATPases to facilitate their escape from organelles into the cytoplasm where they become biologically active. Plasma membrane V-ATPases are involved in renal acid secretion and osteoclast bone dissolution; mutations in tissue-specific V-ATPase subunit isoforms necessary for these processes result in genetic diseases characterized by metabolic acidosis and osteoporosis. The long-term goal of the lab is to understand the structure, function, assembly and regulation of V-ATPases by studying the yeast V-ATPase, which has proven to be an excellent model for all eukaryotic V-ATPases. All V-ATPases are composed of two multisubunit domains, a peripheral membrane complex involved in ATP hydrolysis and an integral membrane complex required for proton transport. In this proposal, we focus on the "stalk" subunits that structurally and functionally bridge these two domains. These subunits are responsible for transmission of conformational changes resulting from ATP hydrolysis to the proton pore, and are also key players in regulated disassembly of V-ATPases, an important regulatory mechanism. The aims of this proposal are 1) to position the stalk subunits in the yeast V-ATPase, by a combination of electron microscopy, hydrodynamic studies of subcomplexes, mutagenesis, and crosslinking experiments, 2) to elucidate the roles of the C and H subunits, particularly in the functionally important conformational change accompanying release of the peripheral sector from the membrane sector, 3) to examine protein-protein interactions with two isoforms of the "a" subunit and test their importance in regulated disassembly, and 4) to follow V-ATPase assembly and disassembly in vivo using GFP-tagged V-ATPase subunits.
Keywords: adenosinetriphosphatase, enzyme mechanism, enzyme structure, hydrogen transport, membrane transport protein, protein structure function, vesicle /vacuole, acidity /alkalinity, active site, conformation, cytoplasm, enzyme activity, isozyme, molecular assembly /self assembly, protein protein interaction, Saccharomyces cerevisiae, crosslink, electron microscopy, fluorescence resonance energy transfer, green fluorescent protein, mass spectrometry, site directed mutagenesis, yeast
Project start date: 1994-03-01
Project end date: 2007-08-31
5R01GM050322-13 (2006): $252328
5R01GM050322-12 (2005): $258400
5R01GM050322-11 (2004): $258400
SUBUNIT STRUCTURE/FUNCTION IN VACUOLAR H+ ATPASES
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 5R01GM050322-08 from National Institute Of General Medical Sciences IRG: PB
Abstract: Vacuolar proton-translocating ATPases (V-ATPases) are found in all eukaryotic cells and appear to play both constitutive roles in all cells and more specialized roles in certain cell types. V-ATPases are multisubunit enzymes capable of coupling ATP hydrolysis to proton transport across membranes. The primary constitutive role of V-ATPases in all human cells appears to be acidification of certain intracellular compartments, and this acidification is critical for maintenance of the cell s internal organization and ability to respond to extracellular stimuli. Organelle acidification mediated by V-ATPases is exploited by a variety of pathogens, including certain viruses and toxins, to allow these pathogens to enter the cell cytoplasm; other pathogens manipulate V- ATPase activity to allow them to exist in intracellular compartments. V-ATPases play specialized roles in regulated secretory granules of neural cells, where they are involved in sequestration of neurotransmitters, and at the plasma membrane of kidney intercalated cells, osteoclasts, macrophages and neutrophils, where they are involved in urinary acidification, bone resorption, and regulation of cytoplasmic pH, respectively. The V-ATPase of the yeast Saccharomyces cerevisiae has proven to be an excellent experimental model for V-ATPases of other eukaryotes, including humans. The long-term goals of this research are to understand the structure, function, assembly, and regulation of the yeast V-ATPase. The specific aims of this proposal are directed toward understanding the interaction between the peripheral V1 sector of the V-ATPase, which is responsible for ATP hydrolysis, and the integral membrane Vo sector, which is responsible for proton transport. The interaction between the V1 and Vo sectors is central to the catalytic activity of V-ATPases and is also a major site of enzyme regulation. Toward this goal, the functions of two subunits (Vma5p and Vma13p) that are known to be important for interaction between the V1 and Vo sectors will be studied in detail, both in wild-type yeast cells and in strains containing point mutations in each subunit gene. Reversible dissociation of V1-Vo complexes into cytosolic V1 sectors and membrane- bound Vo sectors has been shown to occur in vivo in response to nutrient deprivation in yeast and in insect cells, and is probably a general mechanism of regulation. Cytosolic V1 sectors will be isolated from yeast cells, and the biochemical and enzymatic properties of these sectors will be examined. Links between catalytic activity, nucleotide binding, changes in cytosolic pH, and assembly state of the V-ATPase will be explored in biochemical studies, and the physiological benefits of dissociation of the V-ATPase under conditions of nutrient deprivation will be examined by isolating yeast mutants defective in disassembly of the enzyme.
Keywords: enzyme mechanism, enzyme structure, hydrogen transport, hydrogen transporting ATP synthase, vesicle /vacuole, active site, adenosinetriphosphatase, conformation, cytoplasm, enzyme activity, membrane protein, molecular assembly /self assembly, mutant, point mutation, structural gene, Saccharomyces cerevisiae, acidity /alkalinity, epitope mapping, site directed mutagenesis
Project start date: 1994-03-01
Project end date: 2003-02-28
5R01GM050322-08 (2001): $204255
5R01GM050322-09 (2002): $210293
5R01GM050322-07 (2000): $198395
A Skpl-containing Complex Regulating V-ATPase Activity
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 5R01GM063742-04 from National Institute Of General Medical Sciences IRG: PB
Abstract: Vacuolar H-+ translocating ATPases (V-ATPases) are highly conserved enzymes that play a central role in cell physiology stemming from their ability to acidify intracellular compartments, regulate cytosolic pH and calcium concentrations, and establish proton gradients that drive other transporters. These functions can be adapted to many different cellular contexts, and as a result, V-ATPase activity is linked to disease states as diverse as viral infection, metabolic acidosis due to impaired kidney function, and osteoporosis. Skp1 is a highly conserved protein that plays a central role in ubiquitin-dependent proteolysis as an essential member of the family of SCF E3 ubiquitin ligases in addition to a growing number of non-proteolytic roles. SCF complexes show specificity for a wide array of phosphorylated substrates and thus interact with many signal transductional pathways. Recently a new Skp1-containing complex, RAVE, was identified in yeast and shown to contain two other uncharacterized proteins. The RAVE complex does not appear to be an SCF ubiquitin ligase; instead, it appears to post-translationally regulate V-ATPase activity in response to extracellular conditions by modulating the extent of assembly of the V-ATPase complex. In the project proposed here, we will use the yeast Saccharomyces cerevisiae as a model system to examine the structural and functional basis for regulation of V-ATPase activity and assembly by the RAVE complex. We will examine how the three subunits of the RAVE complex interact with each other and with the peripheral V1 sector of the V-ATPase. We will determine whether protein components of RAVE are directly affected by changes in extracellular conditions, and how these changes might affect interaction with the V1 sector. We will develop a system for monitoring interactions between RAVE and the V-ATPase in vivo, using fluorescent derivatives of the two complexes. Finally, we will probe the molecular basis of RAVE action on the V-ATPase by developing an in vitro model for RAVE-dependent assembly of the peripheral and integral membrane sectors of the ATPase and using this model system to test the hypothesis that RAVE assists functional attachment of one of the V1 subunits.
Keywords: adenosinetriphosphatase, enzyme activity, protein binding, protein protein interaction, protein structure function, ubiquitin, biological signal transduction, biological transport, hydrogen transport, membrane transport protein, oxidative phosphorylation, SDS polyacrylamide gel electrophoresis, fluorescence resonance energy transfer, gene deletion mutation, immunoprecipitation, point mutation, western blotting, yeast two hybrid system
Project start date: 2002-08-01
Project end date: 2007-07-31
5R01GM063742-04 (2005): $202160
5R01GM063742-03 (2004): $212800
5R01GM063742-02 (2003): $212800
SUBUNIT STRUCTURE AND FUNCTION IN VACUOLAR H+-ATPASES
Patricia M Kane
Upstate Medical University, Research Administration, Syracuse, Ny 13210
Grant 5R01GM050322-17 from National Institute Of General Medical Sciences
Abstract: V-ATPases are ATP-driven proton pumps that are responsible for organelle acidification in all eukaryotic cells and are recruited to the plasma membrane for specialized functions in osteoclasts, kidney, and the male reproductive tract. V-ATPase activity is intimately linked to protein sorting in the endocytic and biosynthetic pathways, zymogen activation, and pH, calcium, and metal ion homeostasis. V-ATPase activity is also subverted to support metastasis of certain cancers and virus and toxin release from the endocytic pathway into the cytosol. V-ATPases are very highly conserved, and are comprised of a complex of peri- pheral membrane proteins containing the sites of ATP hydrolysis, V1, attached to a membrane complex containing the proton pore, Vo. V1 and Vo sectors must associate for proton pumping to occur, but they also reversibly dissociate under conditions of energy limitation. We propose to continue to use yeast as a model system to explore the structural basis of V-ATPase mechanism and regulation, along with the cellular context of V-ATPase activity, through the following three aims 1) We will define the interactions of the V1 C and H subunits with the V1 and Vo sectors and probe how these interactions change during glucose deprivation. Work in the current grant period indicated that the V1 sector has two stator stalks, and we hypothesize that the C and H bridge these stalks to different regions of the membrane sector, and from this position, regulate reversible disassembly of V1 from Vo. 2) Reversible disassembly in response to glucose suggests that V- ATPase activity is aligned to the varying needs of the cell through poorly understood metabolic signals. We will explore the cellular basis of this alignment by testing the functional roles of several proteins recently found to interact with the Vo sector and exploiting the sensitivity of diploid cells to H subunit haploin- sufficiency as a means to identify gene products important for V1-Vo reassembly. 3) We will determine the extent to which the V-ATPase is both a general pH regulator in the cell and a pH sensor. Using recently developed methods for flexible and robust vacuolar and cytosolic pH measurement in yeast, we will deter- mine the extent to which V-ATPase activity affects overall pH homeostasis. These experiments may help explain the far-reaching physiological defects of yeast mutants lacking V-ATPase activity. We will also examine the activity of the V-ATPase itself in response to measured cytosolic and vacuolar pH changes
Keywords: ATP Hydrolysis; ATP Synthesis; ATP Synthesis Pathway; ATP phosphohydrolase; ATP-protein phosphotransferase; ATPase; Adenosine Triphosphatase; Adenosinetriphosphatase; Affect; Autoregulation; Biological Models; Blood Coagulation Factor IV; Ca++ element; Calcium; Cancers; Cell Communication and Signaling; Cell Signaling; Cell membrane; Cells; Chronic; Coagulation Factor IV; Complex; Cytoplasmic Membrane; Cytosol; D-Glucose; Defect; Dextrose; Diploid; Diploid Cells; Diploidy; EC 2.7; Elements; Energy Supply; Enzyme Precursors; Enzymes; Eukaryote; Eukaryotic Cell; Factor IV; Genetic; Genetic Screening; Glucose; Grant; H(+) Pump; H+ element; Heavy Metals; Homeostasis; Hydrogen Ions; Intracellular Communication and Signaling; Investigators; Ions; Kidney; Kinases; Link; Malignant Neoplasms; Malignant Tumor; Measurement; Measures; Membrane; Membrane Proteins; Membrane-Associated Proteins; Metabolic; Metals; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Methods; Mining; Minings; Model System; Models, Biologic; Neoplasm Metastasis; Organelles; Osteoclasts; Oxidative Stress; Pathway interactions; Peripheral; Phosphotransferases; Physiologic; Physiological; Physiological Homeostasis; Plasma Membrane; Play; Position; Positioning Attribute; Proenzymes; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Kinase; Proteins; Proton Pump; Protons; Recruitment Activity; Regulation; Research Personnel; Researchers; Role; Secondary Neoplasm; Secondary Tumor; Signal Transduction; Signal Transduction Systems; Signaling; Site; Sorting - Cell Movement; Structure; Surface Proteins; Testing; Time; Toxin; Transphosphorylases; Tumor Cell Migration; Urinary System, Kidney; V-ATPase; V-type ATPase; Vacuole; Virus; Viruses, General; Work; Yeasts; Zymogens; base; biological signal transduction; cancer metastasis; deprivation; enzyme activity; eukaryotida; experiment; experimental research; experimental study; extracellular; flexibility; fungus; gene product; glycogen synthase a kinase; hydroxyalkyl protein kinase; in vivo; interest; male; malignancy; membrane structure; mutant; neoplasm/cancer; novel; pH Homeostasis; pathway; phosphorylase b kinase kinase; plasmalemma; programs; recruit; renal; reproductive; research study; response; sensor; social role; sorting; trafficking; uptake; vacuolar ATPase; vacuolar H+-ATPase; vacuolar membrane H(+)-ATPase
Project start date: 1994-03-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
5R01GM050322-17 (2010): $279774
Sponsored Links Excellgen http://Excellgen.com
5R01GM050322-16 (2009): $282600
5R01GM050322-15 (2008): $282600
Molecular & Cellular Bioenergetics Gordon Conf. 2005
Patricia M Kane, Professor
Gordon Research Conferences West Kingston, Ri 02892
Grant 1R13GM074281-01 from National Institute Of General Medical Sciences IRG: ZGM1
Abstract: The Molecular and Cellular Bioenergetics Gordon Conference will be held June 26-July 1, 2005 at Kimball Union Academy in Meriden, New Hampshire. Although the traditional focus of this meeting has been on mechanisms of electron transport and oxidative phosphorylation, the conference now includes greater content of cellular bioenergetics. This change recognizes substantial recent data highlighting the contributions of high resolution structural information to understanding cell biological processes and the importance of bioenergetic processes in aging and disease. The proposed conference will continue the tradition of presenting high resolution structures of membrane proteins and state-of-the art biochemical approaches to bioenergetic problems, but will also incorporate a number of sessions with a focus on integrating cellular and molecular bioenergetics. Sessions on high-resolution structures of bioenergetic complexes, V- and F-type ATPases, regulation of pH gradients in vivo, bioenergetics and disease, genomic and proteomic approaches to bioenergetics, assembly of respiratory chain complexes, structure and function of electron transport complexes, and new frontiers in mitochondrial biology are proposed. The Molecular and Cellular Bioenergetics Gordon Conference has an attendance of 100-120, and it attracts leaders in the field, drawn from academics, government, and industry, as well as younger scientists (graduate students, postdoctoral fellows, and assistant professors). The format of the Gordon Conference is designed to maximize opportunities for discussion and to encourage presentation of the latest results in the field.
Keywords: bioenergetics, cell biology, meeting /conference /symposium, molecular biology, electron transport, oxidative phosphorylation, travel
Project start date: 2005-05-01
Project end date: 2006-04-30
1R13GM074281-01 (2005): $7000
SUBUNIT STRUCTURE AND FUNCTION IN VACUOLAR H+/ATPASES
Patricia M Kane, Professor
Upstate Medical University Research Administration Syracuse, Ny 13210
Grant 5R01GM050322-05 from National Institute Of General Medical Sciences IRG: PB
Abstract: The aim of the proposed research is to define the subunit structure and function relationships in vacuolar proton-translocating ATPases (H+- ATPases). Vacuolar H+-ATPases are found in all eukaryotic cells, where they may play both constitutive and specialized roles. Constitutive organelle acidification is involved in receptor-mediated endocytosis, targeting of proteins in the biosynthetic pathway, activation of zymogens, protein degradation, ion homeostasis, and uptake of basic compounds into regulated secretory granules. Specialized cells of the kidney and bone contain vacuolar H+-ATPases at the plasma membrane, where they pump protons out of the cell, resulting in urinary acidification and bone resorption. Constitutive organelle acidification is also exploited by viruses, such as influenza, toxins, including diphtheria toxin, and cellular pathogens, such as Salmonella,in invasion of the host cell, suggesting that the ability to regulate compartment acidification could have implications for a wide variety of human disease states. Although the cellular regulation of acidification is quite complex, it is clear that the central player is the vacuolar H+ATPase. Vacuolar H+-ATPases are remarkably similar in fungi, plants, and animals, both in overall structure and in the primary amino acid sequences of the subunits. This proposal focuses on the vacuolar H+-ATPase of the yeast Saccharomyces cerevisiae, with the goal of bridging the genetic information available about the enzyme in yeast and the biochemical characterization derived from work on other cell types. The experiments proposed will also extend the characterization of several of the subunits that have not been well- studied. A collection of subunit-specific antibodies will be generated and used as probes in biochemical studies aimed at defining subunit interactions in the native enzyme. In order to address structure- function relationships in the peripheral sector of the enzyme, which contains the catalytic sites for ATP hydrolysis, the structural genes for three of the peripheral subunits will be randomly mutagenized and mutations affecting assembly and function of the enzyme will be identified. The functional relationships in the membrane sector, containing the proton pore, will be elucidated by biochemicaIly defining interactions between the membrane subunits and the regions of these subunits important for binding of the peripheral sector and coupling of ATP hydrolysis and proton transport. Structural models generated by both the biochemical and genetic approaches will be genetically tested by directed mutagenesis of regions of the subunit genes believed to be functionally important.
Keywords: adenosinetriphosphatase, enzyme mechanism, enzyme structure, hydrogen transport, vesicle /vacuole, chemical binding, conformation, crosslink, eukaryote, hydrolysis, membrane protein, mutant, proteolysis, structural gene, Saccharomyces cerevisiae, laboratory mouse, laboratory rabbit, monoclonal antibody, site directed mutagenesis
Project start date: 1994-03-01
Project end date: 1999-02-28
5R01GM050322-05 (1998): $159068
5R01GM050322-02 (1995): $112094
1R01GM050322-01 (1994): $99215
5R01GM050322-04 (1997): $152984
5R01GM050322-03 (1996): $137181