Molecular structure of H^+ transporting ATPase and its rotation mechanisms in the catalysis

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Concentrations of various ions including H^+ and Na^+ are strictly regulated within living cells. These regulations are important to keep internal environment of cells and also their growth , which are related to several diseases like cancer, when they are deregulated. Ion transporting proteins in various biological membranes have very important roles in such regulations. Further, some of them have essential roles in energy transduction such as ATP synthesis. In the present study, we have studied structure, function, and regulation for two of these proteins, ATP synthase with techniques of genetic engineering and biochemistry.ATP synthase of Escherichia coli has 8 independent subunits and the genes of subunits were cloned and sequenced. Electrochemical proton gradient across biological membranes and H^+ flow through this membrane bound enzyme is essential for catalysis of ATP synthesis. However, relationship bewteen such complex subunit structure and the catalytic mechanisms of the enzyme is not well understood. Topological arrangement of the subunits should be understood to realize the function of subunits. Here, we developed a new approach with a combination of genetics and biochemistry. We have shown that a new genetic approach, the yeast two-hybrid system, could be applicable to reveal subunit interactions within the enzyme. We have shown tight interactions of alpha-beta gamma-epsilon and b-delta by this procedure. These interactions were further. evidenced by in vitro reconstitution of the subunit complex. Functional importance of the interactions was analyzed by introducing various functionally defective mutations for the interactions. One of the most important findings was b-delta interaction which contributed for the interaction of membrane integral and peripheral portions.