Subunit and Nucleotide Interactions in the Chloroplast ATP Synthase

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Adenosine 5'-triphosphate (ATP) synthesis from adenosine 5'-diphosphate (ADP) and inorganic phosphate (Pi) is catalyzed by closely related enzymes in the energy transducing membranes of chloroplasts, mitochondria and some bacteria. Electron transport generates electrochemical proton gradients across the coupling membranes and the flow of protons down these gradients through the ATP synthase drives ATP synthesis. The chloroplast ATP synthase contains nine different polypeptides and a total of about twenty polypeptide chains. The enzyme may be separated into two parts: CF1 and CFo. CF1 contains the nucleotide binding sites of the enzyme and is catalytic. CFo anchors CF1 to the membrane and translocates protons. Interactions among the polypeptide subunits of CF1 and CFo will be studied, with emphasis on the smaller subunits of CF1 (gamma, delta and epsilon) and the regulation of activity. This project will examine roles of the binding of nucleotides in regulation. The location of the C-terminal domain of the e subunit within CF1 and its roles in ATP synthesis, regulation and proton gating will be evaluated making extensive use of site-directed mutagenesis. Whether the position of the C-terminal portion of the g subunit in CF1 differs from that in mitochondrial F1 will be investigated. The ease of preparation of large amounts of CF1, ability to manipulate its subunit composition and nucleotide content, and the high ATP synthesis activity of thylakoids, make the chloroplast ATP synthase an excellent subject for this research. The results of this work will be applicable to all ATP synthases and to protein-protein interactions, in general.