The vacuolar type H+ATPase (V1Vo- or V-ATPase) is a fundamental component of all eukaryotic cells. The complex is found in the membranes of a wide variety of intracellular compartments like clathrin-coated vesicles, chromaffin granules, endosomes, lysosomes, synaptic vesicles, Golgi derived vesicles and the yeast vacuole. In higher eukaryotes, V-type ATPases are also found in the plasma membrane of polarized cells such as osteoclasts and renal epithelial cells. Structurally similar ATPases have also been identified in the plasma membrane of Archaea and bacteria, where they are called A-ATPases and bacterial A/V-ATPases, respectively. The proton pumping action of the vacuolar ATPase plays a vital role in a large number of intra- and inter- cellular processes. In eukaryotic cells, these processes include receptor mediated endocytosis, protein trafficking, pH maintenance, storage of metabolites and neurotransmitter release. In polarized cells of higher eukaryotes, a vacuolar type ATPase is pumping protons across the plasma membrane leading to an extra- cellular acidification. Acidification of the enclosed space between the ruffled membrane of osteoclasts and the bone surface plays an important role in bone resorption and remodeling. Defects in the human vacuolar ATPase have been associated with a number of diseases such as renal tubular acidosis, sensorineural deafness, osteoporosis, diabetes and cancer. Fighting these diseases on a molecular level will require a detailed understanding of the structure and mechanism of the eukaryotic V-ATPase complex, which is the long term goal of this project.
The Specific Aims of the now proposed work on the vacuolar ATPase are: (1) molecular structure and function of the vacuolar ATPase proton channel domain and (2) molecular structure and function of the V1 - Vo interface. In the first Aim, we plan to determine the atomic resolution x-ray crystal structure of the yeast vacuolar ATPase proton channel domain. In addition, we propose experiments to elucidate aspects of the mechanism of proton translocation across the isolated V-ATPase membrane domain. In the second Aim, we propose to determine the atomic resolution crystal structure of the subunit EGChead peripheral stalk complex and we will determine the molecular interactions that define the interface connecting V1-ATPase with the Vo proton channel domain. Results from the proposed studies will provide important molecular information on the mechanism of proton translocation and how the catalytic V1 ATPase sector and the membrane bound Vo proton channel domain interact to form a coupled enzyme complex. The proposed work will also shed light on the, as of yet poorly understood mechanism of V-ATPase activity regulation by regulated reversible enzyme dissociation and re-association, a mechanism now found to be involved in the development and maturation of cells in higher animals including human.
The vacuolar ATPase is a large, multi subunit enzyme complex that is involved in numerous fundamental cellular processes. A defective or hyper active vacuolar ATPase can be associated with devastating human diseases such as renal tubular acidosis, osteoporosis, diabetes and cancer. Understanding the molecular origin of these diseases requires detailed knowledge of the molecular structure of the disease causing bio macromolecules. This proposal requests funds for studying the structure and mechanism of the eukaryotic proton pumping vacuolar ATPase with the goal of gaining a molecular understanding of the enzyme's role in human disease.
|Stam, Nicholas J; Wilkens, Stephan (2017) Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION. J Biol Chem 292:1749-1761|
|Oot, Rebecca A; Kane, Patricia M; Berry, Edward A et al. (2016) Crystal structure of yeast V1-ATPase in the autoinhibited state. EMBO J 35:1694-706|
|Couoh-Cardel, Sergio; Hsueh, Yi-Ching; Wilkens, Stephan et al. (2016) Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Sci Rep 6:24774|
|Couoh-Cardel, Sergio; Milgrom, Elena; Wilkens, Stephan (2015) Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector. J Biol Chem 290:27959-71|
|Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy et al. (2014) Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models. Methods 66:168-79|
|Aggeli, Dimitra; Kish-Trier, Erik; Lin, Meng Chi et al. (2014) Coordination of the filament stabilizing versus destabilizing activities of cofilin through its secondary binding site on actin. Cytoskeleton (Hoboken) 71:361-79|
|Wen, Po-Chao; Verhalen, Brandy; Wilkens, Stephan et al. (2013) On the origin of large flexibility of P-glycoprotein in the inward-facing state. J Biol Chem 288:19211-20|
|Parsons, Lee S; Wilkens, Stephan (2012) Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays. PLoS One 7:e46960|
|Oot, Rebecca A; Wilkens, Stephan (2012) Subunit interactions at the V1-Vo interface in yeast vacuolar ATPase. J Biol Chem 287:13396-406|
|Verhalen, Brandy; Ernst, Stefan; Börsch, Michael et al. (2012) Dynamic ligand-induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy. J Biol Chem 287:1112-27|
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