Identification of a Calmodulin-Regulated Soybean Ca 2+ -ATPase (SCA1) That Is Located in the Plasma Membrane

Abstract

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) 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 soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-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 Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 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 Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) 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.

Mlo, a Modulator of Plant Defense and Cell Death, Is a Novel Calmodulin-binding Protein

“…The polyclonal antibody against the OsMloAg peptide was raised in a New Zealand White rabbit, and the polyclonal antiserum was purified by antigen affinity chromatography on an OsMloAg peptide-Sepharose column as described (35). The column was prepared by conjugating the OsMloAg peptide to CNBr-activated Sepharose 4B according to the procedure recommended by the manufacturer (Amersham Biosciences).…”

“…Preparation of Rice Membrane Fractions-A microsomal membrane fraction was prepared from 10-day-old rice leaves according to the procedure described by Chung et al (35). Rice leaves were ground on ice in prechilled homogenization buffer of 50 mM Tris-HCl (pH 8.0), 20% (w/w) sucrose, 2 mM EDTA, 4 mM DTT, 2 mM PMSF, and complete protease inhibitor mixture (1 tablet per 10 ml of lysis buffer) (Roche Molecular Biochemicals).…”

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

“…4A). In the soybean Ca 2ϩ -ATPase, SCA1, two CaMBDs have been identified in the N-terminal domain (31). We therefore tested the possibility that ACA8 could contain an additional CaMBD downstream of Ala-143 and therefore not present in the (His) 6 -tagged N termini of ACA8.…”

Molecular Responses to Osmotic Stresses in Soybean

“…In soybean, SCA1 is a plasma membrane-localized Ca 2+ -ATPase (encoded by GmSCA1) that belongs to a novel family of plant type IIB Ca 2+ pump. It is stimulated by CaM (Chung et al, 2000) and can be rapidly and dramatically induced by NaCl stress and fungal elicitor (Chung et al, 2000) although its role in shaping Ca 2+ signatures awaits further studies. On the other hand, the soybean gene GmSTL encodes a calcineurin-like protein which shares 63.6% protein sequence identity with its homologue AtSTO in A. thaliana.…”

“…Upon perception, stress specific Ca 2+ signature may be triggered. GmSCA1, a salt responsive plasma membrane-localized Ca 2+ -ATPase (Chung et al, 2000), may play roles in shaping the Ca 2+ signatures. SPK1 and/or SPK2 are activated by upstream osmosensors.…”