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 multifunctional protein that functions by interacting with and modulating 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 Ca(2+)-regulated signals are transduced. An Arabidopsis thaliana L. flower cDNA expression library constructed in lambda ZAPII 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 nucleotide sequence was determined. The genomic DNA sequence was used to design oligonucleotide primers for polymerase-chain-reaction (PCR) 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 regulation, protein degradation, and vesicle-mediated protein transport. A truncated fusion protein encoded by the carboxy-terminal region of CIP111 was produced in Escherichia coli and shown to bind CaM in a Ca(2+)-dependent manner by protein gel blot and affinity chromatography binding assays. Reverse-transcription PCR analyses demonstrated that CIP111 mRNA is expressed in all organs examined including flowers, siliques, floral stalks, leaves, and roots. DNA blot hybridization analyses indicate that a single-copy gene in Arabidopsis is likely to encode CIP111.
Buaboocha
T
T
Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA.
Liao
B
B
Zielinski
R E
RE
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D002459:Cell Fusion; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015698:Genomic Library; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018514:Plant Structures / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000074405:Valosin Containing Protein
D029681:Arabidopsis Proteins; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D010940:Plant Proteins; D000251:Adenosine Triphosphatases; C435732:CIP111 protein, Arabidopsis; D000074405:Valosin Containing Protein; D002118:Calcium