FEATURES OF VACUOLAR H-ATPASE REVEALED BY YEAST SUPPRESSOR MUTANTS()

The yeast Saccharomyces cerevisiae serves as an excellent model for th e 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 mu tants were selected after subjecting them to chemical or polymerase ch ain reaction mutagenesis and screening for second site suppressors. Su ppressors for the mutation GLn(90) to Lys change were intragenic and r esulted from the changes: Ala(14) to Val, Val(74) to Ile, Ile(89) to L eu, and Ile(89) to Met. These residues are found on three different tr ansmembrane segments but presumably at the same surface of the membran e. A new inactive proteolipid mutation was constructed by changing Val (138) to, Leu. This residue is situated in the middle of the fourth tr ansmembrane segment, neighboring Glu(137) which is the potential dicyc lohexylcarbodiimide-binding site. The intragenic suppressor mutations for the above amino acid replacement resulted in changes of Val(55) to Ala, Met(59) to Val, or Ile(130) to Thr. These residues are found in the second and fourth transmembrane segments, presumably on the same i nterface. It seems as if all those internal suppressor mutations compe nsate for the volume changes caused by the original displacement of th e given amino acid. Five glycine residues, situated on the same face o f the third transmembrane helix, were changed to valine and all these mutants were inactive. A suppressor mutation to one of those mutants ( Gly(101) to Val) was identified as substitution of Ile(134) to Val. Th e structural and functional implications of these findings are discuss ed.