Dennis K. Stone
Our laboratory is focused on a molecular analysis of a novel protontranslocating ATPase. Members of this new class of proton pumpshave been localized to a wide variety of intracellular organelles,including clathrin-coated vesicles, endosomes, lysosomes, synaptosomes, chromaffin granules, golgi membranes and endoplasmic reticulum.The functions of these proton pumps are diverse, commiserate withtheir distribution and include processing of endocytosed receptor-ligand complexes and viruses, degradation of metabolic waste products,and storage of catecholamines. In addition, this proton pump isresponsible for osteoporosis, and is a potential pharmacotherapeutictarget for treatment of cancer and malaria.
We have solubilized and purified the clathrin-coated vesicle protonpump, and have reconstituted the enzyme into artificial, purelipid membranes. The proton pump is a large (530 kDa), multimeric,integral membrane protein composed of eight subunits.
We are in the process of assembling a functional proton pump fromentirely recombinant components. Toward this end, seven of thesubunits have been cloned and expressed, and we have used thesecomponents to reconstitute the ATP-hydrolytic sector of the enzyme.Resolution of subunit function at this level will not only provide a critical insight into the structural properties of the enzyme,but also will ultimately allow for an analysis of the biogenesisof the pump.