Abstract
This chapter discusses genetic probing that has been a valuable asset in developing mechanistic information about specific functional domains in the yeast H+–ATPase. The chapter discusses the dynamic interactions that provide information about coupling between ATP hydrolysis and proton transport domains and provides an overview of how genetic probing can aid in structure predictions by focusing on transmembrane segments 1 (Ml) and 2 (M2). The chapter also discusses the catalytic center and the nature of the phosphate binding domain by analyzing mutations within the first cytoplasmic loop domain linking M2 and M3 and the large central cytoplasmic domain linking M4 and M5 that confer relative vanadate insensitivity on the H+–ATPase. Mutations mapping within close proximity to the site of phosphorylation (D378) is described and the interesting consequences of lethal mutations at this site is presented. The role of the C-terminus as a regulatory domain of catalytic activity is discussed.