GENETIC-ANALYSIS OF THE FLUORESCEIN ISOTHIOCYANATE BINDING-SITE OF THE YEAST PLASMA-MEMBRANE H-ATPASE()

The highly conserved motif of Saccharomyces cerevisiae H+-ATPase (474) KGAP has been proposed to participate in the formation of the phosphor ylated intermediate during the catalytic cycle (Portillo, F., and Serr ano, R. (1988) EMBO J. 7, 1793-1798), In addition, Lys-474 is the FITC binding site of the yeast enzyme (Portillo, F. and Serrano, R. (1989) Eur. J. Biochem. 186, 501-507). We have performed an intragenic suppr essor analysis of the K474R mutation to identify the interacting regio ns involved in these functions. Random in vitro mutagenesis of the K47 4R allele resulted in seven suppressor (second-site) mutations. One mu tation (V396I), located 18 residues away from the Asp-378 residue, whi ch is phosphorylated during catalysis, is allele-specific. This provid es genetic evidence of a direct interaction between the KGAP motif and the phosphorylation domain during the catalytic cycle. Three mutation s (V484I, V484I/E485K, and E485K/E486K) are located near Lys-474 and m ay compense the structural alteration introduced by the K474R mutation . Two substitutions at the end of the predicted transmembrane stretch 2 (A165V and V169I/D170N) and another in the predicted ATP binding dom ain (P536L) may act as allele-nonspecific suppressors, as they are als o able to suppress a mutation at the enzyme's carboxyl terminus.