Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: Conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null

Raymond E Zielinski

Research output: Contribution to journalArticle

Abstract

Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

Original languageEnglish (US)
Pages (from-to)446-455
Number of pages10
JournalPlanta
Volume214
Issue number3
DOIs
StatePublished - Dec 1 2002

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calmodulin
Calmodulin
Arabidopsis
complement
Arabidopsis thaliana
Yeasts
yeasts
calcium
protein isoforms
Genes
Carrier Proteins
Protein Isoforms
genes
amino acid sequences
Gels
EF Hand Motifs
Peptides
Proteins
proteins
Expressed Sequence Tags

Keywords

  • Arabidopsis (calmodulin)
  • Calcium
  • Calmodulin
  • Protein-protein interaction
  • Signal transduction

ASJC Scopus subject areas

  • Plant Science

Cite this

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title = "Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: Conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null",
abstract = "Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49{\%} amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.",
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author = "Zielinski, {Raymond E}",
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N2 - Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

AB - Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

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