Modeling the Leigh syndrome nt8993 T-->C mutation in Escherichia coli F1F0 ATP synthase.
The mutations in human mitochondrial DNA at nt8993 are associated with a range of neuromuscular disorders. One mutation encodes a proline in place of a leucine conserved in all animal mitochondrial ATPase-6 subunits and bacterial a subunits of F1F0 ATP synthases. This conserved site is leu-156 and leu-207 in humans and Escherichia coli, respectively. An aleu-207-->pro substitution mutation has been constructed in the E. coli F1F0 ATP synthase in order to model the biochemical basis of the human disease mutation. The phenotype of the aleu-207-->pro substitution has been compared to that of the previously studied aleu-207-->arg substitution (Hartzog and Cain, 1993, Journal of Biological Chemistry 268, 12250-12252). The leu-207-->pro mutation resulted in approximately a 35% decrease in the number of intact enzyme complexes as determined by N, N'-dicyclohexylcarbodiimide-sensitive membrane associated ATP hydrolysis activity and western analysis using an anti-a subunit antibody. A 75% reduction in the efficiency of proton translocation through F1F0 ATP synthase was observed in ATP-driven proton pumping assays. Interestingly, the loss in F1F0 ATP synthase activity resulting from the leu-207-->pro substitution was markedly less dramatic than had been observed for the leu-207-->arg mutation studied earlier. By analogy, the human enzyme may also be affected by the leu-156-->pro substitution to a lesser extent than the leu-156-->arg substitution, and this would account for the milder clinical manifestations of the human leu-156-->pro disease mutations.
Hartzog
P E
PE
Department of Biochemistry and Molecular Biology, University of Florida Gainesville 32610, USA.
Gardner
J L
JL
Cain
B D
BD
D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D007888:Leigh Disease / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013577:Syndrome
D004272:DNA, Mitochondrial; D006180:Proton-Translocating ATPases