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

Teerapong Buaboocha, Birong Liao, Raymond E. Zielinski

Research output: Contribution to journalArticle

Abstract

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 Ca2+-regulated signals are transduced. An Arabidopsis thaliana L. flower cDNA expression library constructed in λZAPII 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-chainreaction (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 Ca2+-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.

Original languageEnglish (US)
Pages (from-to)774-781
Number of pages8
JournalPlanta
Volume212
Issue number5-6
DOIs
StatePublished - May 8 2001

Keywords

  • ATPase Calcium
  • Arabidopsis (ATPase, Ca)
  • Protein-protein interaction
  • Signal transduction

ASJC Scopus subject areas

  • Genetics
  • Plant Science

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