Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.
The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.
Lill
H
H
Abteilung Biophysik, Universität Osnabrück, Germany.
Burkovski
A
A
Altendorf
K
K
Junge
W
W
Engelbrecht
S
S
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases