Identification of a high-affinity Ca2+ pump associated with endocytotic vesicles in Dictyostelium discoideum.
In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca2+ are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca2+ levels in this organism, we previously isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump which appeared to be a component of "inside-out" plasma membrane vesicles [J. L. Milne and M. B. Coukell (1988) Biochem. J. 249. 223-230]. In this report, we demonstrate that a high-affinity Ca2+ pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca2+-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca2+ transporter is associated with a high-capacity non-IP3-releasable Ca2+ store which is generated by endocytosis. A possible role for this store in maintaining Ca2+ homeostasis in Dictyostelium is discussed.
Milne
J L
JL
Department of Biology, York University, Ontario, Canada.
Coukell
M B
MB
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002463:Cell Membrane Permeability; D002499:Centrifugation, Density Gradient; D004023:Dictyostelium / Q000378:metabolism*; D004705:Endocytosis; D005372:Filipin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D011189:Potassium Chloride; D011205:Povidone; D012822:Silicon Dioxide
C016039:Percoll; D011189:Potassium Chloride; D012822:Silicon Dioxide; D005372:Filipin; D011205:Povidone; D002118:Calcium