Identification Of Four Putative Calmodulin-binding Proteins In Arabidopsis Cell Membrane

In plant systems, a series of work in our and other laboratorys has provided evidences that Calmodulin (CaM), well-known as an intracellular calcium sensor, has also been found extracellularly and play several important biological roles. Based on these data, our laboratory suggested that extracellular CaM might act as a polypeptide signal and there was a possible transmembrane signaling mechanism. In this model, the extracellular CaM binds to its receptor (or receptors) in cell membrane and then the receptor (or receptors) activates downstream signal pathway to regulate physiological reactions as well as gene expression. Cui et al. firstly reported the presence of apoplastic CaM receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells. However, we have not indentified the candidate receptor (or receptors) on cell surface for apoplast CaM. So we conducted the experiments below to study the CaM-binding proteomics in Arabidopsis cell membranes.We previously purified the microsome fractions of Arabidopsis suspension-cultured cells, and the proteins purified from CaM affinity chromatography were identified by LC-MS/MS or MALDI-TOF-MS. The characterization of the total 35 matched proteins was analyzed by bioinformatic prediction. Under laser confocal microscopy, five proteins specifically or mostly locate in onion epidermal cell plasma membrane by transient expression.And the interaction between these proteins and CaM in vivio need to be further confirmed with other methods. Mating-based Split ubiquitin system (mbSUS) and Fluorescence Resonance Energy Transfer (FRET) were used to identify the interaction between CaM and its putative binding proteins. Using mbSUS system, four proteins (PCaMBP1,PCaMBP2,PCaMBP3,PCaMBP4) could interact with CaM. But only PCaMBP4 could interact with CaM in transient co-expression onion epidermal cell by FRET assay.In this paper, one T-DNA insertion mutant was identified in DNA level. Identification of other insertion mutants was on going.