Mutational analysis of the first extracellular loop region of the H(+)-ATPase from Saccharomyces cerevisiae.
Transmembrane segments 1 and 2 of the yeast plasma membrane H(+)-ATPase are believed to form a helical hairpin structure that is joined by a short extracytoplasmic loop. The hairpin head region (Ala135-Phe144) was probed using site-directed mutagenesis. Scanning alanine mutagenesis produced functional H(+)-ATPase at all positions except Leu138, Asp143, and Phe144. D140A and V142A gave strong hygromycin B resistance and low pH sensitivity suggesting a major kinetic defect in these mutant enzymes. Other amino acid substitutions, such as L138Y, were highly perturbing, while mutations S139E and D140E produced minor effects on phenotype. Small uncharged residues Gly and Ala, which were inserted between Leu138 and Ser139 to examine the importance of loop length on H(+)-ATPase function, were well tolerated, while the insertion of a polar Ser residue was highly perturbing. Other additions were not tolerated by the enzyme. These results suggest that the turn region has limited structural flexibility. The conserved Phe144 residue could be changed to Trp with a minor effect on phenotype. However, neither Tyr, Arg, nor small hydrophobic residues could substitute, suggesting that this region is closely packed and hydrophobic. ATP hydrolysis measurements showed that Vmax was significantly reduced in nearly all mutant enzymes, except D140E; whereas, Km values were nearly normal. Vanadate-sensitivity and pH profiles for ATP hydrolysis were nearly normal for all mutant enzymes except insertion mutant S138+. Mutants with extreme phenotypes (S138+, Tyr138) showed significantly altered medium acidification profiles. These results support the notion that the hairpin head region linking transmembrane segments 1 and 2 forms a tightly packed conformationally sensitive domain that is coupled to the catalytic ATP hydrolysis domain.
Seto-Young
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Public Health Research Institute, New York, New York 10016.
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Monk
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Haber
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Perlin
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D004252:DNA Mutational Analysis; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011522:Protons; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000409:Alanine