Identification of a calmodulin-regulated soybean Ca2+-ATPase (SCA1) that is located in the plasma membrane

Abstract

Ca2+-ATPases are key regulators of Ca2+ ion efflux in all eukaryotes. Animal cells have two distinct families of Ca2+ pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for (s) under bar oybean (C) under bar a(2+)-(A) under bar TPase (1) under bar), that encodes Ca2+-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca2+-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2)similar to N10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca2+ pumps. Our results indicate that sCA1p is structurally distinct from the plasma membrane-localized Ca2+ pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.