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 indica te that significant interactions occur between the region near the sit e of phosphorylation, stalk segment 3 (S3), and the N-terminal transme mbrane 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, stab le substitutions with Arg, Tyr or Asn were often accompanied by second -site mutations at the extreme C-terminus, suggesting a close interact ion between these regions. Several mutations in the putative stalk dom ain are known to alter coupling, and scanning glycine and proline muta genesis was used to probe the predicted a-helical character of the sta lk segments. The results indicate that the introduction of proline or glycine in S2, S4 or S5, was highly disruptive to enzyme function ofte n resulting in cell death. Similar substitutions in stalk 3 yielded vi able 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 .