Y. Wei et al., An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca2+/Mn2+-ATPase, BIOCHEM, 38(44), 1999, pp. 14534-14541
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.