Isolation of cDNA and genomic DNA clones encoding a calmodulin-binding protein related to a family of ATPases involved in cell division and vesicle fusion

Calmodulin (CaM), a primary Ca2+ receptor in all eukaryotic cells, is a mul tifunctional protein that functions by interacting with and modulating the activities of a wide variety of target proteins. Identifying and characteri zing 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 constructed in lambda ZAPII was screened for CaM-binding proteins with S-35-labeled CaM. A partial cDNA whose encoded p rotein 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 nucleotide sequence was determined. The genomic DNA sequence was us ed to design oligonucleotide primers for polymerase-chain-reaction (PCR) ex periments that facilitated cloning and reconstructing a full-length, 3.4-kb cDNA clone. The cDNA encodes a 111-kDa CaM-interacting protein (CIP111) co ntaining motifs characteristic of a diverse family of ATPases, including pr oteins involved in cell cycle regulation, protein degradation, and vesicle- mediated protein transport. A truncated fusion protein encoded by the carbo xy-terminal region of CIP111 was produced in Escherichia coli and shown to bind CaM in a Ca2+-dependent manner by protein gel blot and affinity chroma tography binding assays. Reverse-transcription PCR analyses demonstrated th at CIP111 mRNA is expressed In all organs examined including Hewers, siliqu es, floral stalks, leaves, and roots. DNA blot hybridization analyses indic ate that a single-copy gene in Arabidopsis is likely to encode CIP111.