TY - JOUR
T1 - An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca2+/Mn2+-ATPase
AU - Wei, Ying
AU - Marchi, Valerie
AU - Wang, Runsheng
AU - Rao, Rajini
PY - 1999/11/2
Y1 - 1999/11/2
N2 - 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.
AB - 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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U2 - 10.1021/bi9911233
DO - 10.1021/bi9911233
M3 - Article
C2 - 10545175
AN - SCOPUS:0033517785
SN - 0006-2960
VL - 38
SP - 14534
EP - 14541
JO - Biochemistry
JF - Biochemistry
IS - 44
ER -