SITE-DIRECTED MUTAGENESIS OF THE YEAST PMA1 H-ATPASE - STRUCTURAL ANDFUNCTIONAL-ROLE OF CYSTEINE RESIDUES()

The yeast plasma-membrane H+-ATPase contains nine cysteines, three in presumed transmembrane segments (Cys-148, Cys-312, and Cys-867) and th e rest in hydrophilic regions thought to be exposed at the cytoplasmic surface (Cys-221, Cys-376, Cys-409, Cys-472, Cys-532, and Cys-569). T o gather new functional and structural information, we have studied th e yeast ATPase by cysteine mutagenesis. It proved possible to replace seven of the nine cysteines by alanine, one at a time, without any sig nificant decrease in ATP hydrolysis or ATP-dependent proton pumping. I n the remaining two cases (Cys-409 and Cys-472), there were small but reproducible effects; the results clearly indicated, however, that no single Cys is required for activity and that, if a disulfide bridge is formed in the yeast ATPase, it does not play an obligatory structural or functional role. Next, multiple mutants were constructed to ask ho w many Cys residues could be replaced simultaneously while leaving a f ully functional enzyme. After substitution of all ''membrane'' Cys (Cy s-148, Cys-312, and Cys-867) together with two non-conserved Cys locat ed in hydrophilic regions (Cys-221 and Cys-569), there were no signifi cant abnormalities in expression (87%) or activity (89% ATP hydrolysis /93% H+ pumping) of the mutant protein. Replacement of two additional cysteines (Cys-376 near the phosphorylation site and Cys-532, in or ne ar the ATP-binding site) caused a drop in expression (to 54%), althoug h the corrected hydrolytic and H+ pumping activities were still normal . When Cys-472 was also mutated, the corrected activity fell to 44% hy drolysis/47% pumping; finally, substitution of Cys-409 to give a ''cys teine-free'' ATPase led to a very poorly expressed and poorly active e nzyme. Brief exposure of the ''one-cysteine'' and ''two-cysteine'' ATP ases to trypsin revealed a normal pattern of degradation, but there wa s a slight impairment in the ability of vanadate to protect against pr oteolysis. Thus, although single Cys replacements are tolerated well b y the yeast ATPase, multiple replacements are progressively more harmf ul, suggesting that they cause small but additive perturbations of pro tein folding.