Mutation of alanine 24 to serine in subunit c of the Escherichia coli F1F0-ATP synthase reduces reactivity of aspartyl 61 with dicyclohexylcarbodiimide.
Dicyclohexylcarbodiimide (DCCD) inhibits the activity of the F1F0-H+ ATP synthase of Escherichia coli by reacting with aspartyl 61 in subunit c of the FO sector to form a stable N-acylurea. The segment of chromosomal DNA which codes the subunits of the FO was cloned from four independently isolated DCCD-resistant mutants, and the sequence of the subunit c gene (uncE) was determined. An Ala24 to serine (A24S) substitution was found in the subunit c gene of each mutant. The A24S uncE gene was cloned into the BamHI site of a mutant derivative of plasmid pBR322. The A24S subunit c conferred DCCD resistance to a variety of recipient E. coli strains when it was overexpressed from this plasmid. A 7-base pair deletion beginning at position 132 of the plasmid vector was responsible for the observed overexpression. Hoppe et al. (Hoppe, J., Schairer, H. U., and Sebald, W. (1980) Eur. J. Biochem. 112, 17-24) had previously shown that mutation of subunit c Ile28 to threonine or valine resulted in DCCD resistance. The DCCD sensitivities of the membrane ATPase of these mutants and the A24S mutant were compared. DCCD sensitivity decreased in the order: wild-type much greater than I27V greater than I28T = A24S. The venturicidin sensitivities of wild-type and mutant membranes were also examined. The membrane ATPase of the I28T and I28V mutants was venturicidin resistant whereas the A24S substitution resulted in a hypersensitivity to inhibition by venturicidin. These results support a model in which subunit c folds in the membrane like a hairpin, where the region of residues 24-28 in transmembrane helix-1 is close to that of aspartyl 61 in transmembrane helix-2.
Fillingame
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RH
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison 53706.
Oldenburg
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M
Fraga
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D000409:Alanine / Q000502:physiology; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine / Q000502:physiology; D013329:Structure-Activity Relationship; D014698:Venturicidins / Q000494:pharmacology
D004269:DNA, Bacterial; D014698:Venturicidins; D012694:Serine; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases; D000409:Alanine