Identification of a Calmodulin-Regulated Soybean Ca²⁺-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 ‫ف‬ 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. INTRODUCTIONCa 2 ϩ plays a central role as a second messenger in signal transduction of all eukaryotes (Bootman and Berrige, 1995;Clapham, 1995). The transient increase of cytosolic free Ca 2 ϩ concentration, [Ca 2 ϩ ] cyt , is correlated with a variety of external signals such as touch, temperature shift, abscisic acid, auxin, red light, fungal elicitors, salinity/drought, anoxia, gravity, gibberellic acid, cytokinin, oxidative stress, and hypoxia (Bush, 1995; Sanders et al., 1999). In turn, the increased [Ca 2 ϩ ] cyt triggers many signal transduction pathways, including the regulation of enzyme activity, ion channel activity, and gene expression, which results in diverse cellular responses (Bush, 1995). In addition to its role as a second messenger, Ca 2 ϩ also is important in regulating the processing of proteins in secretory pathways (Rudolph et al., 1989). Thus, cells require carefully regulated transport systems to control [Ca 2 ϩ ] in the cytoplasm and endomembrane compartments. Influx of Ca 2 ϩ to the cytosol occurs as a "downhill" transport through Ca 2 ϩ channels (Sanders et al., 1999). In contrast, the efflux of Ca 2 ϩ from the cytosol is mediated by two active Ca 2 ϩ transporters-Ca 2 ϩ pumps and Ca 2 ϩ /H ϩ antiporters-which are powered by ATP hydrolysis and proton motive force, respectively (Bush, 1995). In plants, Ca 2 ϩ -ATPases are believed to be a major Ca 2 ϩ transporter for the endoplasmic reticulum (ER), Golgi apparatus, vacuole, plastid inner membrane, and plasma membrane (Sander...

Genome‐wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice

“…This is quite coherent also as fluctuations in the cytosolic calcium levels are one of the primary responses to various stimuli and Ca 2+ transport elements actively participate in maintaining this flux and homeostasis. Previously, it was reported that NaCl treatment leads to upregulation of the Ca 2+ ‐ATPase transcript level in different plant species such as tomato, tobacco, soybean and Arabidopsis and it was speculated that increased capacity of Ca 2+ ‐ATPase may help in lowering the cytosolic calcium level, which was elevated due to NaCl stress and might be involved in maintenance of Ca 2+ homeostasis. Also, overexpression of N‐terminal modified ACA4 in Arabidopsis seedlings resulted in increased salt tolerance in comparison with wild type plants .…”

ACA12 is a deregulated isoform of plasma membrane Ca2+-ATPase of Arabidopsis thaliana

“…Moreover, despite its low expression level, ACA12 was able to complement the K616 phenotype, making it able to grow in the presence of 5 mM EGTA (Fig. 4), as only deregulated mutants of other ACA isoforms are able to do (Harper et al 1998; Chung et al 2000; Geisler et al 2000b; Hwang et al 2000; Sze et al 2000; Bonza et al 2004; Kabala and Klobus 2005; Schiøtt and Palmgren 2005; Baekgaard et al 2006; Lee et al 2007). Taken together, these results clearly indicate that ACA12 is a deregulated isoform of Arabidopsis type 2B Ca 2+ -ATPase.…”

Structure and function of CrACA1, the major PM‐type Ca2+‐ATPase, expressed at the peak of the gravity‐directed trans‐cell calcium current in spores of the fern Ceratopteris richardii

“…Based on the success of this method with the soybean Ca 2+ ‐ATPase SCA1 (Chung et al . ), an analogous region of CrACA1 was chosen for this expression. Primers were designed to amplify the A504‐V596 region of the gene (Table S1) for expression using a pETBlue bacterial expression vector to allow for His‐tag/Ni affinity purification.…”