ARCHIVES
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 376-381, 1992
Escherichia co/l’ H+-ATPase: Role of the 6 Subunit in Binding F, to the F, Sector’ Masayoshi Yoshinori
Jounouchi, Moriyama,
Michiyasu Masatomo
Takeyama, Pawinee Chaiprasert, Takato Maeda, and Masamitsu Futai’ and Biochemistry, The Institute of Scientific and Industrial
Noumi,
Department of Organic Chemistry Osaka University, Ibaraki, Osaka 567, Japan
Research,
Received September 3, 1991, and in revised form September 26, 1991
The roles of the Escherichia coli H+-ATPase (F,F,) 6 subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23 + end, Gln-29 + end, Gln-74 + end) and missense (Gly150 + Asp) mutants had very low ATPase activities, indicating that the 6 subunit is essential for the binding of the F, portion to F,. The Gln- 176 + end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174 -+ end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu- 175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150 + Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the 6 subunit is necessary, but that individual residues 0 1992 Academic may not have strict functional roles. Press,
Inc.
The H+-translocating ATPase (F,F,) of Escherichia cd catalyzes ATP synthesis utilizing an electrochemical gradient of protons across the cytoplasmic membrane gen1 This research was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, grants for a research program on the “Creation of New Materials through Intelligent Design” (ISIR, Osaka University), and grants from the Human Frontier Science Program. ’ To whom correspondence should be addressed.
erated by the electron transfer chain (for review, see Refs. (l-4)). The ATPase consists of two portions, F1 and F,. The catalytic sector F, extrinsic to the membrane has five subunits, 01,p, y, 6, and 6, while the sector F, in the membrane has three subunits, a, b, and c, and functions as a proton pathway. The 6 subunit (177 amino acid residues), coded by the uncH gene, is required together with the t subunit for binding of the ~$37 complex to the F, sector, as shown by in vitro reconstitution experiments (5). Thus the 6 subunit, a unique polypeptide with a high o( helical content (6), seems to be located in the part connecting FI and F,. A close interaction of the 6 subunit with the (Y subunit was suggested by cross-linking between the two subunits (7, 8). The corresponding subunits from mitochondria (9) and chloroplasts (10, 11) seem to stabilize the linkage between FI and F,: chloroplast F1 lacking the 6 subunit can bind to F, but the resulting F,F
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 376-381, 1992
Escherichia co/l’ H+-ATPase: Role of the 6 Subunit in Binding F, to the F, Sector’ Masayoshi Yoshinori
Jounouchi, Moriyama,
Michiyasu Masatomo
Takeyama, Pawinee Chaiprasert, Takato Maeda, and Masamitsu Futai’ and Biochemistry, The Institute of Scientific and Industrial
Noumi,
Department of Organic Chemistry Osaka University, Ibaraki, Osaka 567, Japan
Research,
Received September 3, 1991, and in revised form September 26, 1991
The roles of the Escherichia coli H+-ATPase (F,F,) 6 subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23 + end, Gln-29 + end, Gln-74 + end) and missense (Gly150 + Asp) mutants had very low ATPase activities, indicating that the 6 subunit is essential for the binding of the F, portion to F,. The Gln- 176 + end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174 -+ end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu- 175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150 + Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the 6 subunit is necessary, but that individual residues 0 1992 Academic may not have strict functional roles. Press,
Inc.
The H+-translocating ATPase (F,F,) of Escherichia cd catalyzes ATP synthesis utilizing an electrochemical gradient of protons across the cytoplasmic membrane gen1 This research was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, grants for a research program on the “Creation of New Materials through Intelligent Design” (ISIR, Osaka University), and grants from the Human Frontier Science Program. ’ To whom correspondence should be addressed.
erated by the electron transfer chain (for review, see Refs. (l-4)). The ATPase consists of two portions, F1 and F,. The catalytic sector F, extrinsic to the membrane has five subunits, 01,p, y, 6, and 6, while the sector F, in the membrane has three subunits, a, b, and c, and functions as a proton pathway. The 6 subunit (177 amino acid residues), coded by the uncH gene, is required together with the t subunit for binding of the ~$37 complex to the F, sector, as shown by in vitro reconstitution experiments (5). Thus the 6 subunit, a unique polypeptide with a high o( helical content (6), seems to be located in the part connecting FI and F,. A close interaction of the 6 subunit with the (Y subunit was suggested by cross-linking between the two subunits (7, 8). The corresponding subunits from mitochondria (9) and chloroplasts (10, 11) seem to stabilize the linkage between FI and F,: chloroplast F1 lacking the 6 subunit can bind to F, but the resulting F,F
