Ichikawa Naoki, Chisuwa Naomi, Tanase Mamiko, Nakamura Mai
Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sumiyoshi.
J Biochem. 2005 Aug;138(2):201-7. doi: 10.1093/jb/mvi116.
Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.
线粒体ATP合酶（F(1)F(0)-ATP酶）受内在的ATP酶抑制蛋白IF(1)调控。我们之前发现酵母IF(1)的六个残基（苯丙氨酸17、精氨酸20、谷氨酸21、精氨酸22、谷氨酸25和苯丙氨酸28）形成一个ATP酶抑制位点[市川，N.和小仓，C.（2003年）《生物能量学与生物膜杂志》35卷，399 - 407页]。在F(1)/IF(1)复合物的晶体结构中[卡韦松，E.等人（2003年）《自然结构生物学》10卷，744 - 750页]，抑制位点的核心残基与F(1)β亚基的精氨酸408、精氨酸412和谷氨酸454相互作用。在本研究中，我们通过定点诱变研究了β亚基上这三个残基的作用。总共构建了六个酵母突变体：R408I、R408T、R412I、R412T、E454Q和E454V。β精氨酸412和β谷氨酸454突变体（R412I、R412T、E454Q和E454V）能够在不可发酵的乳酸培养基上生长，但β精氨酸408突变体（R408I和R408T）不能。在R412I、R412T、E454Q和E454V突变体细胞中，分离的线粒体的ATP酶活性降低，而在R408I和R408T细胞中未检测到。通过免疫印迹在每个突变体的线粒体中检测到了F(1)的亚基（α、β和γ），并从它们中分离出了F(1)F(0)复合物。这些结果表明，β精氨酸408对于F(1)F(0)复合物的组装不是必需的，但对于该酶的催化活性是必需的。在F(1)的晶体结构中，β精氨酸408与α(DP)/β(DP)对中的α谷氨酸399结合，似乎对于形成封闭的α(DP)/β(DP)构象很重要。IF(1)似乎通过与β(DP)精氨酸408结合来破坏这种α(DP)谷氨酸399/β(DP)精氨酸408相互作用，并干扰F(1)F(0)-ATP酶催化所需的从开放的α(DP)/β(DP)构象到封闭构象的转变。
