title: Features of vacuolar H(+)-ATPase revealed by yeast suppressor mutants.
abstract: The yeast Saccharomyces cerevisiae serves as an excellent model for the study of the structure and function of proteins. Numerous amino acid substitutions in the proteolipid subunit of yeast vacuolar H(+)-ATPase have been reported. Suppressed variants for several of the inactive mutants were selected after subjecting them to chemical or polymerase chain reaction mutagenesis and screening for second site suppressors. Suppressors for the mutation Gln90 to Lys change were intragenic and resulted from the changes: Ala14 to Val, Val74 to Ile, Ile89 to Leu, and Ile89 to Met. These residues are found on three different transmembrane segments but presumably at the same surface of the membrane. A new inactive proteolipid mutation was constructed by changing Val138 to Leu. This residue is situated in the middle of the fourth transmembrane segment, neighboring Glu137 which is the potential dicyclohexylcarbodiimide-binding site. The intragenic suppressor mutations for the above amino acid replacement resulted in changes of Val55 to Ala, Met59 to Val, or Ile130 to Thr. These residues are found in the second and fourth transmembrane segments, presumably on the same interface. It seems as if all those internal suppressor mutations compensate for the volume changes caused by the original displacement of the given amino acid. Five glycine residues, situated on the same face of the third transmembrane helix, were changed to valine and all these mutants were inactive. A suppressor mutation to one of those mutants (Gly101 to Val) was identified as substitution of Ile134 to Val. The structural and functional implications of these findings are discussed.
authors.affiliation: Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
authors.forename: F
authors.initials: F
authors.lastname: Supek
authors.forename: L
authors.initials: L
authors.lastname: Supekova
authors.forename: N
authors.initials: N
authors.lastname: Nelson
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry* / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D011510:Proteolipids; D006180:Proton-Translocating ATPases