Identification and Characterization of a Calmodulin -Binding Protein Related to a Family of ATPases Involved in Cell Division and Membrane Fusion
Calmodulin (CaM) is a primary Ca2+-receptor, which interacts with and modulates the activities of a wide variety of target proteins. Identifying and characterizing these CaM-binding target proteins is essential to define the pathways by which Ca2+-regulated signals are transduced. An Arabidopsis thaliana L. flower cDNA expression library was screened for CaM-binding proteins with 35S-labeled CaM. A partial cDNA whose encoded protein shares a high level of similarity with yeast CDC48p was isolated. A genomic clone was isolated using the partial length cDNA clone as a probe, and its sequence was used to design oligonucleotide primers for polymerase chain reaction experiments that facilitated cloning and reconstructing a full length, 3.4-kb cDNA clone. The cDNA encodes a 111-kDa CaM-interacting protein (CIP111) containing motifs characteristic of a diverse family of ATPases, including proteins involved in cell cycle and membrane fusion. A truncated fusion protein encoding the carboxy-terminal 22-amino acids of CIP111 was produced in E. coli and shown to bind CaM in a Ca2+-dependent manner by affinity chromatography. RT-PCR analyses demonstrated that CIP111 mRNA is expressed in all organs examined including flowers, siliques, floral stalks, leaves, and roots. Furthermore, expression of the CIP111 promoter fused to a beta-glucuronidase reporter gene in transgenic plants demonstrated that CIP111 is expressed in young anthers, developing embryos, root tips, and lateral roots. An unusual characteristic of higher plants is that they express a variety of CaM isoforms. To explore the functional differences of plant CaM isoforms, their interaction with two peptide substrates was examined by fluorescence resonance energy transfer (FRET). FRET measurements between blue fluorescence protein fused with CaM-binding domains of CIP111 and green fluorescence protein fused with CaM isoforms indicate that they interact at very low level of free Ca2+. Apparent Kd values of CaM2, CaM8, and CaM9 complexes with CIP111 for Ca2+ are 38.9, 56.2, and 17.3 nM, respectively. GFP fused with CaM2 was shown to functionally substitute for yeast CMD1 to maintain viability and growth suggesting that GFP-CaM2 fusion proteins retain most, if not all, of the properties of unfused CaM.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign