Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system.
Subunit interactions among the F1-ATPase subunits were studied by the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Escherichia coli H+-ATPase fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of the alpha and beta subunit genes, and of the epsilon and gamma subunit genes showed high levels of reporter gene expression, while those of alpha and delta, beta and delta, gamma and delta, and delta and epsilon demonstrated weak but significant reporter gene expression. However, combinations of alpha and gamma, beta and gamma, alpha and epsilon, and beta and epsilon did not induce reporter expression. None of the fused genes alone induced reporter gene expression. These results suggested that specific and strong interactions between the alpha and beta, gamma and epsilon, and weak interactions between the alpha and delta, beta and delta, and gamma and delta subunits occurred in yeast cells in the two-hybrid system. Effects of previously identified mutant beta subunits with Leu-40 to Pro. Glu-41 to Lys or Pro-332 to Gln substitutions which caused defects in molecular assembly of F1-ATPase were analyzed with regard to alpha-beta interactions. No interaction of the alpha and beta subunits was observed in this system using the beta subunit with mutation of Pro-332 to Gln. However, for the other two mutations, alpha-beta interactions were observed. This system may be useful for isolating mutants which have defects in interaction of F1-ATPase subunits.
Moritani
C
C
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Japan.
Sawada
K
K
Takemoto
K
K
Shin
Y
Y
Nemoto
S
S
Noumi
T
T
Kanazawa
H
H
D004926:Escherichia coli / Q000201:enzymology*; D017930:Genes, Reporter; D006824:Hybridization, Genetic; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D015003:Yeasts / Q000235:genetics
D006180:Proton-Translocating ATPases