PROBING CONSERVED REGIONS OF THE CYTOPLASMIC LOOP1 SEGMENT LINKING TRANSMEMBRANE SEGMENT-2 AND SEGMENT-3 OF THE SACCHAROMYCES-CEREVISIAE PLASMA-MEMBRANE H-ATPASE()

Genetic probing was used to examine conserved amino acid clusters in t he first cytoplasmic loop domain (LOOP1) linking transmembrane segment s 2 and 3 of the plasma membrane H+-ATPase from Saccharomyces cerevisi ae. Deletion of the LOOP1 region in PMA1 resulted in a defective enzym e. Scanning alanine mutagenesis of conserved residues produced lethal cell phenotypes in 14 of 26 amino acids, suggesting major enzyme defec ts. Most viable mutants showed growth characteristics that were compar able to wild type. Two mutations, I183A and D185A, produced reduced gr owth rates, hygromycin B resistance, and low pH sensitivity, which are phenotypes associated with defects in the H+-ATPase. However, both mu tant enzymes displayed near-normal kinetics for ATP hydrolysis in vitr o. Localized random mutagenesis was also performed at sites Glu(195), Val(196) and Ile(210), which all showed lethal phenotypes upon convers ion to alanine. Amino acids with polar side groups could substitute fo r Glu(195), while Val(196) could not tolerate polar side group moietie s, Nine mutations at Ile(210) 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 fo r 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. I n 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.