The long term objective of this study is to understand the mechanism by which cells synthesize ATP, the basic unit of chemical energy in all organisms. ATP synthesis occurs at the plasma membrane of most bacteria, and for eukaryotes, in mitochondria and chloroplasts. The system of choice for this study is the F1F0 ATP synthase from Escherichia coli. The advantages of this system are the ea se of construction and analysis of mutants, and athe relatively simple subunit composition of the enzyme. The net synthesis of ATP is known to be coupled to the movement of protons across a membrane, but the details of this process remain to be determined. The mechanism of all members of the F1F0 ATP synthase family will be closely related to that of the well-studied, mammalian mitochondrial enzymes, and therefore, the results of our studies will be relevant both to human and bacterial metabolism. In particular, many mitochondrial myopathies are related to the inability to make sufficient ATP. This study will focus on two aspects of the structure and function of this enzyme. The F1F0 ATP synthase is a membrane-bound enzyme that is composed of 8 different types of subunits (alpha, beta, gamma, delta, epsilon, a, b, c) in the following stoichiometry, 3:3:1:1:1:1:2:10. Structurally, the enzyme is composed of 2 separable parts; F1, an ATP hydrolase composed of alpha, beta, gamma, delta, and epsilon subunits, and F0, an integral membrane complex composed of a, b, and c subunits. F1 must be attached to F0 (in a membrane) for ATP synthesis, Functionally, F1 is the site of ATP synthesis and hydrolysis, with the active site region composed of alpha, beta, and gamma subunits. Fo is the site of proton movement through the membrane, which is coordinated by a and c subunits. The third aspect of function is the linking of these 2 processes, which probably involves the remaining subunits: delta, epsilon, and b.. First,we propose to continue our studies of proton translocation by Fo. These studies will include (I) site-specific mutagenesis of the a subunit to test a recently developed model of proton translocation, (II) covalent linkage of the a subunit to b and c subunits via gene fusions, to test the possibility of F0 subunit rotation, and (III) structural analysis of the transmembrane segments, and of the loops connecting them, in the alpha subunit, by introduction of uniquely reactive cysteine residues. These cysteines can then by tested for disulfide formation, when introduced in pairs, or for labeling by surface or membrane-soluble reagents. Second, we propose to study the coupling of proton translocation to ATP synthesis by focusing on interactions of the gamma and epsilon subunits with the c subunit. We have recently mapped the binding surface of epsilon as it interacts with gamma and c subunit, by alanine-scanning mutagenesis. Future studies will continue to map functional regions of epsilon, and initiate analysis of residues of the gamma subunit that interact with epsilon and c subunits by mutagenesis.
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