An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca(2+)/Mn(2+)-ATPase.
Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca(2+) and Mn(2+) for a variety of normal secretory processes. We have previously characterized Ca(2+) transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca(2+) 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 Ca(2+). 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 Ca(2+) transport, while the double mutant was largely unable to exit the endoplasmic reticulum. In-frame deletions of the Ca(2+)-binding motif resulted in complete loss of function. Interestingly, the single point mutations conferred differential affinities for transport of Ca(2+) and Mn(2+) 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 Cu(2+)-ATPases, defective in Wilson and Menkes disease.
Wei
Y
Y
Department of Physiology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Marchi
V
V
Wang
R
R
Rao
R
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006056:Golgi Apparatus / Q000201:enzymology; D017136:Ion Transport; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium