An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca(2+)/Mn(2+)-ATPase

Biochemistry. 1999 Nov 2;38(44):14534-41. doi: 10.1021/bi9911233.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Base Sequence
  • Calcium / metabolism
  • Calcium-Transporting ATPases / chemistry*
  • Calcium-Transporting ATPases / genetics
  • Calcium-Transporting ATPases / metabolism*
  • Catalytic Domain / genetics
  • DNA Primers / genetics
  • Golgi Apparatus / enzymology
  • Ion Transport
  • Kinetics
  • Molecular Sequence Data
  • Point Mutation
  • Protein Conformation
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Deletion

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • Calcium-Transporting ATPases
  • Calcium