Molecular Cloning of an Intracellular P-type ATPase from Dictyostelium That Is Up-regulated in Calcium-adapted Cells

doi:10.1074/jbc.270.47.28276

Molecular Cloning of an Intracellular P-type ATPase from Dictyostelium That Is Up-regulated in Calcium-adapted Cells (*)

From the Department of Biology, York University, North York, Ontario M3J 1P3, Canada

^¶ To whom all correspondence should be addressed:
Dept. of Biology, York University, 4700 Keele St., North York, Ontario M3J 1P3, Canada.
Tel.: 416-736-2100 (ext. 33554); Fax: 416-736-5698; FS300047{at}SOL.YORKU.CA.

Abstract

Results from a number of laboratories suggest that intracellular Ca is involved in the regulation of Dictyostelium discoideum growth and development. To learn more about the regulation and function of intracellular Ca in this organism, we have cloned and sequenced cDNAs that encode a putative P-type Ca ATPase designated patA. The deduced protein product of this gene (PAT1) has a calculated molecular mass of 120,718 daltons. It exhibits about 46% amino acid identity with Ca ATPases of the plasma membrane Ca ATPase family and lower identity with sarco(endo)plasmic reticulum Ca ATPase family members and monovalent cation pumps. However, PAT1 lacks the highly conserved calmodulin-binding domain present in the C-terminal region of most plasma membrane Ca ATPase-type enzymes. When Dictyostelium amoebae are adapted to grow in the presence of 80 mM CaCl, both the patA message and protein product are up-regulated substantially. These cells also exhibit an increase in the rate and magnitude of intracellular P-type Ca uptake activity. Immunofluorescence analysis indicates that PAT1 colocalizes with bound calmodulin to intracellular membranes, probably components of the contractile vacuole complex. The presence of PAT1 on the contractile vacuole suggests that in Dictyostelium this organelle might function in Ca homeostasis as well as in water regulation.

Footnotes

↵^§ Recipient of an Ontario Graduate Scholarship.
↵* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to M. B. C. and A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89369[GenBank].
↵⁽⁾ The abbreviations used are:

PMCA

plasma membrane Ca ATPase

SERCA

sarco(endo)plasmic Ca ATPase

PCR

polymerase chain reaction

kb

kilobase

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

vatP

vacuolar-type H ATPase proteolipid

MES

2-(N-morpholino)ethanesulfonic acid

PBS

phosphate-buffered salts

patA

intracellular P-type ATPase A.
↵⁽⁾J. Moniakis and Y. Xie, unpublished observation.
↵⁽⁾Y. Xie, M. B. Coukell, and Z. Gombos, manuscript in preparation.
↵⁽⁾C.-H. Siu, personal communication.
- Received July 20, 1995.
- Revision received September 19, 1995.