Schmidt G, Rodgers A J, Howitt S M, Munn A L, Hudson G S, Holten T A, Whitfeld P R, Bottomley W, Gibson F, Cox G B
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra City.
Biochim Biophys Acta. 1990 Feb 2;1015(2):195-9. doi: 10.1016/0005-2728(90)90020-5.
The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.
叶绿体F0F1 - ATP酶的CF0I亚基的氨基酸序列与大肠杆菌F0F1 - ATP酶的b亚基的氨基酸序列仅有很低的相似性。然而，二级和三级结构预测以及疏水和亲水氨基酸的分布表明这两个亚基发挥相似的功能。该命题得到了直接研究。编码CF0I亚基的叶绿体atpF基因的cDNA克隆通过定点诱变进行了改造，使得翻译起始位点对应于成熟蛋白的N端。携带编码b亚基的uncF基因链终止突变的大肠杆菌突变菌株用携带改造后的atpF基因的质粒进行转化。所得转化体能够在琥珀酸盐上生长，并且生长产量与野生型对照相似。对转化体制备的膜进行的分析也清楚地表明，菠菜叶绿体的成熟CF0I亚基能够在大肠杆菌F0F1 - ATP酶中替代大肠杆菌的b亚基。
