Genetic characterization of the (534)DPPR motif of the yeast plasma membrane H(+)-ATPase.
The highly conserved motif +(534)DPPR of Saccharomyces cerevisiae H(+)-ATPase, located in the putative ATP binding site, has been mutagenized and the resulting 23 mutant genes conditionally expressed in secretory vesicles. Fourteen mutant ATPases (D534A, D534V, D534L, D534N, D534G, D534T, P535A, P535V, P535L, P535G, P535T, P535E, P535K and R537T) failed to reach the secretory vesicles. Of these mutants, nine (D534N, D534T, P535A, P535V, P535L, P535G, P535T, P535E and P535K) were not detected in total cellular membranes, and five (D534A, D534V, D534G, D534L and R537T) were retained at the endoplasmic reticulum and exhibited a dominant lethal phenotype. The remaining mutants (D534E, R537A, R537V, R537L, R537N, R537G, R537E, R537K and R537H) reached the secretory vesicles at levels similar to that of the wild type. Of these, six (R537A, R537V, R537L, R537N, R537G, and R537E) showed severely decreased ATPase activity compared to the wild type enzyme, and three (D534E, R537K and R537H) rendered an enzyme with an altered K(m) for ATP.
Portillo
F
F
Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain. fportillo@iib.uam.es
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D022142:Secretory Vesicles / Q000201:enzymology
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases