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
T. Buaboocha et al., 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, PLANTA, 212(5-6), 2001, pp. 774-781
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.