Genetic analysis of the fluorescein isothiocyanate binding site of the yeast plasma membrane H(+)-ATPase.
The highly conserved motif of Saccharomyces cerevisiae H(+)-ATPase 474KGAP has been proposed to participate in the formation of the phosphorylated intermediate during the catalytic cycle (Portillo, F., and Serrano, 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 suppressor analysis of the K474R mutation to identify the interacting regions involved in these functions. Random in vitro mutagenesis of the K474R allele resulted in seven suppressor (second-site) mutations. One mutation (V396I), located 18 residues away from the Asp-378 residue, which is phosphorylated during catalysis, is allele-specific. This provides genetic evidence of a direct interaction between the KGAP motif and the phosphorylation domain during the catalytic cycle. Three mutations (V484I, V484I/E485K, and E485K/E486K) are located near Lys-474 and may 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 domain (P536L) may act as allele-nonspecific suppressors, as they are also able to suppress a mutation at the enzyme's carboxyl terminus.
Maldonado
A M
AM
Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Portillo
F
F
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate