Molecular genetic probing of energy coupling by the yeast plasma membrane proton pump.
Genetic probing of PMA1, which encodes the plasma membrane H(+)-ATPase, has highlighted the putative role of the N-terminal half of the enzyme in the coupling process. Recent second-site suppressor studies indicate that significant interactions occur between the region near the site of phosphorylation, stalk segment 3 (S3), and the N-terminal transmembrane segments. Saturation mutagenesis was used to explore I183 in S2, which partially uncouples proton transport when converted to alanine. Numerous substitutions could be made at this position. However, stable substitutions with Arg, Tyr or Asn were often accompanied by second-site mutations at the extreme C-terminus, suggesting a close interaction between these regions. Several mutations in the putative stalk domain are known to alter coupling, and scanning glycine and proline mutagenesis was used to probe the predicted alpha-helical character of the stalk segments. The results indicate that the introduction of proline or glycine in S2, S4 or S5, was highly disruptive to enzyme function often resulting in cell death. Similar substitutions in stalk 3 yielded viable but significantly altered enzymes. These results suggest that the helical properties of these segments may be important for catalysis. Finally, the stalk region has been modeled as a helical bundle, which helps account for the effects of specific perturbations in this region.
Soteropoulos
P
P
Public Health Research Institute, New York, NY 10016, USA.
Wang
G
G
Perlin
D S
DS
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004734:Energy Metabolism / Q000502:physiology*; D006868:Hydrolysis; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000166:cytology / Q000201:enzymology*
D017494:Proton Pumps; D000255:Adenosine Triphosphate