Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca2+ and Mn2+ for a variety of normal secretory processes. We have previously characterized Ca2+ transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca2+ pump activity [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 hand-like motif in Pmr1 binds Ca2+. Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51A and D53A retained pump activity but with drastic reductions in the affinity for Ca2+ transport, while the double mutant was largely unable to exit the endoplasmic reticulum. In-frame deletions of the Ca2+-binding motif resulted in complete loss of function. Interestingly, the single point mutations conferred differential affinities for transport of Ca2+ and Mn2+ ions. Further, the proteolytic stability of the catalytic ATP-binding domain is altered by the N-terminal mutations, suggesting an interaction between these two regions of polypeptide. These studies implicate the N- terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role of N-terminal metal-binding sites of Cu2+-ATPases, defective in Wilson and Menkes disease.
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