Soteropoulos P, Perlin D S
Public Health Research Institute, New York, New York 10016, USA.
J Biol Chem. 1998 Oct 9;273(41):26426-31. doi: 10.1074/jbc.273.41.26426.
The stalk region of the H+-ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport. Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively. Saturation mutagenesis was used to explore the role of side group character at position Ile183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438-25445). Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization. Substitution of glycine and proline at Ile183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position. Proline/glycine mutagenesis was used to study additional sites in S2 and S3. The substitution of proline at Gly186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly270 and proline or glycine at Thr287 resulted in viable mutants. Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively. The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport. The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth. Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore alpha-helical tendency. This study suggests that maintaining alpha-helical character is essential to S2 and may play an important role in S3.
酿酒酵母H⁺-ATP酶的柄部区域被认为在将ATP水解与质子运输偶联中发挥作用。采用基因探测来研究分别与M2和M3相关的柄部片段S2和S3的作用。利用饱和诱变来探究S2中Ile183位点侧链基团特性的作用，先前已表明该位点的丙氨酸取代会使酶部分解偶联（Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438 - 25445）。尽管I183N、I183R和I183Y这三个取代需要在酶的C末端进行第二位点突变以实现稳定，但该位点能耐受多种侧链基团取代。在Ile183处取代甘氨酸和脯氨酸会导致致死表型，这表明在此位置主链可能比侧链基团更重要。采用脯氨酸/甘氨酸诱变来研究S2和S3中的其他位点。在Gly186处取代脯氨酸会导致致死表型，而在S3中，Gly270处的脯氨酸或丝氨酸取代以及Thr287处的脯氨酸或甘氨酸取代会产生可存活的突变体。突变G270P和T287P产生的突变酶导致明显的生长缺陷，ATP水解速率分别比野生型酶低35%和60%。突变酶以与其ATP酶活性一致的速率运输质子，这表明观察到的生长缺陷是由于ATP水解速率降低而非质子运输解偶联所致。由突变G270P和T287P产生的显著生长表型使得能够分离出抑制突变，这些抑制突变恢复了野生型生长。大多数抑制突变要么用丙氨酸取代主要位点突变，要么通过与PMA2的异位重组恢复野生型残基，这两种方式都恢复了α-螺旋趋势。这项研究表明，维持α-螺旋特性对S2至关重要，并且可能在S3中发挥重要作用。
