Genetic Probing of the Stalk Segments Associated with M2 and M3 of the Plasma Membrane H+-ATPase fromSaccharomyces cerevisiae

Abstract

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 Ile 183 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 Ile 183 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 Gly 186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly 270 and proline or glycine at Thr 287 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 ␣-helical tendency. This study suggests that maintaining ␣-helical character is essential to S2 and may play an important role in S3.The plasma membrane H ϩ -ATPase from Saccharomyces cerevisiae is an electrogenic proton pump that is essential to cell survival. It plays a critical role in fungal cell physiology by helping to regulate intracellular pH and by generating the large electrochemical proton gradient necessary for nutrient uptake (1). The enzyme is a member of the P-type family of ion translocating ATPases that includes diverse members from plants, animals, and bacteria (2, 3). P-type ATPases generically consist of a membrane transport domain with 10 transmembrane segments, a large cytoplasmic ATP hydrolysis domain, and a narrow stalk domain that links the two larger domains. These enzymes couple ATP hydrolysis in the cytoplasmic domain to ion transport in the membrane-embedded domain forming an acylphosphate intermediate during catalysis (4, 5). The yeast H ϩ -ATPases couples energy with one proton transported per ATP hydrolyzed (6). There is ample evidence that dynamic changes in protein structure occur during ...

Helical Stalk Segments S4 and S5 of the Plasma Membrane H+-ATPase from Saccharomyces cerevisiae Are Optimized to Impact Catalytic Site Environment

“…We previously demonstrated that the helical properties of S2 and S3 are important to enzyme function (15). In this report, we have extended that analysis to examine the importance of helical character in S4 and S5, which are more directly linked to coupling of ion transport.…”

“…Targeted Proline Mutagenesis of S4 -Scanning proline mutagenesis has been used to probe the helical properties of stalk segments S2 and S3 (15). This approach was extended to assess the importance of helical backbone structure in S4 and S5 by making strategic substitutions in largely non-conserved residues near the ends of the two stalk segments.…”

Catalytic Phosphorylation of Na,K-ATPase Drives the Outward Movement of Its Cation-Binding H5−H6 Hairpin

“…It was earlier proposed that key conformational changes are transmitted between the phosphorylation and the cation-binding sites via the stalk region of the enzyme constituted by extensions of TMs H2, H3, H4, and H5 (Figure ) (). Several laboratories have explored the putative role of this region in energy transduction in different P-type ATPases ( − ). However, analysis of the Ca-ATPase structure and the Na,K-ATPase model suggests a quite simple link between the phosphorylation and cation-binding sites via a long central helical structure.…”

“…Numbering of this sequence does not include five residues in the leader sequence. P-type ATPases (14)(15)(16)(17). However, analysis of the Ca-ATPase structure and the Na,K-ATPase model suggests a quite simple link between the phosphorylation and cationbinding sites via a long central helical structure.…”

Different Flour Microbial Communities Drive to Sourdoughs Characterized by Diverse Bacterial Strains and Free Amino Acid Profiles

“…The DNA was used as template in PCR using primers LacbF/LacbR (Corsetti et al, 2004). DNA was extracted from yeasts using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI), according to the manufacturer's instructions (Soteropoulos and Perlin, 1998). The DNA was used as template in PCR using primers NL-1/NL-4, targeting the D1/D2 domain of the 26S rRNA gene (Kurtzman and Robnett, 1998).…”