The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional. However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions. Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together. Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type. The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Bakhtiari N, Lai-Zhang J, Yao B, Mueller DM

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