Proton Pumping of the Yeast Plasma Membrane H+-ATPase

Abstract : This proposal was to study the structure and function of the yeast plasma membrane H(+)-ATPase. We proposed to study (1) the kinetics of proton pumping, (2) the transmembrane distribution of the protein and (3) the interaction between the ATPase and an associated protein kinase. Our studies of the proton pumping were unsuccessful due to an inability to reconstitute high activity enzyme into non-leaky vesicles at a high protein to lipid ratio so that proton flux could be measured. The transmembrane distribution project was progressing well until the same project was published by an other group. We have shown a potential link between the level of phosphorylation and the specific activity of the ATPase. We have also shown that the site of kinase mediated phosphorylation is in the C-terminal 5,000 Da of the protein. This domain has previously been shown to be involved in environmentally mediated changes in the ATPase activity. In an effort to produce enzyme with a high protein concentration and high protein/lipid ratio we have begun construction of a Baculovirus expression vector that we will transfect into insect cells. We hope that this system will not suffer from the same low level expression seen in E. coli expression systems. ATPase, proton, protein structure