An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca2+/Mn2+-ATPase

Pmr1, a novel member of the family of P-type ATPases, localizes to the Golg i compartment in yeast where it provides Ca2+ and Mn2+ for a variety of nor mal secretory processes. We have previously characterized Ca2+ transport in isolated Golgi vesicles; and described an expression system for the analys is of Pmr1 mutants in a yeast strain devoid of background Ca2+ pump activit y [Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895-9901]. Here we show, using recombinant bacterial fusions, that an N-terminal EF h and-like motif in Pmr1 binds Ca2+. Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51 A and D53A retained pump activity but with drastic reductions in the affini ty for Ca2+ transport, while the double mutant was largely unable to exit t he endoplasmic reticulum. In-frame deletions of the Ca2+-binding motif resu lted in complete loss of function. Interestingly, the single point mutation s conferred differential affinities for transport of Ca2+ and Mn2+ ions. Fu rther, the proteolytic stability of the catalytic ATP-binding domain is alt ered by the N-terminal mutations, suggesting an interaction between these t wo regions of polypeptide. These studies implicate the N-terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role o f N-terminal metal-binding sites of Cu2+-ATPases, defective in Wilson and M enkes disease.