Probing conserved regions of the cytoplasmic LOOP1 segment linking transmembrane segments 2 and 3 of the Saccharomyces cerevisiae plasma membrane H+-ATPase.
Genetic probing was used to examine conserved amino acid clusters in the first cytoplasmic loop domain (LOOP1) linking transmembrane segments 2 and 3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae. Deletion of the LOOP1 region in PMA1 resulted in a defective enzyme. Scanning alanine mutagenesis of conserved residues produced lethal cell phenotypes in 14 of 26 amino acids, suggesting major enzyme defects. Most viable mutants showed growth characteristics that were comparable to wild type. Two mutations, I183A and D185A, produced reduced growth rates, hygromycin B resistance, and low pH sensitivity, which are phenotypes associated with defects in the H+-ATPase. However, both mutant enzymes displayed near-normal kinetics for ATP hydrolysis in vitro. Localized random mutagenesis was also performed at sites Glu195, Val196, and Ile210, which all showed lethal phenotypes upon conversion to alanine. Amino acids with polar side groups could substitute for Glu195, while Val196 could not tolerate polar side group moieties. Nine mutations at Ile210 proved lethal, including K, R, E, P, H, N, V, G, and A, while functional enzyme was obtained with S, C, M, and L. Normal rates and extents of pH gradient formation were observed for all mutant enzymes, except I183A and D185A. Detailed analysis of the I183A enzyme indicated that it hydrolyzed ATP like wild type, but it appeared to inefficiently couple ATP hydrolysis to proton transport. In total, these results affirm that conserved amino acids in LOOP1 are important to H+-ATPase function, and purturbations in this region can alter the efficiency of energy coupling.
Wang
G
G
The Public Health Research Institute, New York, New York 10016, USA.
Tamás
M J
MJ
Hall
M J
MJ
Pascual-Ahuir
A
A
Perlin
D S
DS
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D006863:Hydrogen-Ion Concentration; D006921:Hygromycin B / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D017384:Sequence Deletion
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006921:Hygromycin B; D006180:Proton-Translocating ATPases; D000409:Alanine