Calmodulin/Ca²⁺‐ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform‐specific Ca²⁺ dependencies

Abstract

Arabidopsis thaliana plasma membrane (PM) Ca2+‐ATPase is a type IIB P‐type ATPase, which binds calmodulin (CaM) to an autoinhibitory N‐terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At‐ACA8, the first cloned A. thaliana PM Ca2+‐ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At‐ACA8 N‐terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide 41I‐T63 had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µM free Ca2+ about 25 nM], thus localizing At‐ACA8 CaM‐binding site within this sequence. The interaction of At‐ACA8 N‐terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca2+‐ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca2+‐ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca2+ concentration, suggesting that enzyme conformation is affected by Ca2+. Binding of CaM isoforms to At‐ACA8 N‐terminus was affected differently by free Ca2+ concentration, suggesting that plant CaMs may have different affinities for Ca2+.

Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

“…Thus, to determine the effect of the S/D mutations on ACA8 affinity for CaM, the first 116 amino acids of WT or mutant ACA8 fused to a 6His-tag were expressed in E. coli , purified, and used for CaM binding measurements by surface plasmon resonance (Bækgaard et al , 2006; Luoni et al , 2006), a technique which allows one to measure not only the affinity of the partners but also the kinetics of interaction.…”

Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps

“…Although the terminal regulatory domain of both animal and plant members of type 2B Ca 2ϩ -ATPases have been described in detail (2)(3)(4)(5)(11)(12)(13)(14), much less is known about how its autoinhibitory action is exerted. Cross-linking of PMCA with a peptide corresponding to the extended CaM-binding site allowed the identification of two putative sites of intramolecular interaction within the cytoplasmic head containing the catalytic domain: one is localized in the big cytoplasmic loop connecting transmembrane domains (TMs) 4 and 5 and the other in the small cytoplasmic loop connecting TM2 and TM3 (15,16).…”

Co-Immunoprecipitation of Membrane-Bound Receptors

“…For example, the kinase-deleted version of the receptor kinase ERECTA (ER: At2g26330) accumulates at much higher levels than the full-length, endogenous ERECTA protein (Shpak et al, 2003), while the removal of the kinase domain in the CLAVA-TA1 (CLV1: At1g75820) receptor improves its expression without affecting the ligand-binding affinity of its ectodomain (Ogawa et al, 2008). These truncated proteins have been successfully used in binding assays such as pull down, gel filtration, fluorescence anisotropy, and surface plasmon resonance (Pollard, 2010, Luoni et al, 2006, Lee et al 2012. However, interpretation of this binding data has to include a thorough analysis of the potential biological effects caused by removing the kinase domains and the changes in stoichiometry caused by the over-accumulation of stabilized receptors.…”