Cloning of a Schizosaccharomyces pombe homologue of elongation factor 1 alpha by two-hybrid selection of calmodulin-binding proteins.
This study reports the cloning and characterization of a cDNA encoding elongation factor 1-alpha (EF1alpha) from the yeast Schizosaccharomyces pombe. The cDNA was cloned from an Schizosaccharomyces pombe expression library by a two-hybrid selection for clones encoding calmodulin (CaM)-binding proteins. The predicted protein is highly homologous to mammalian EF1alpha, indicating a strong tendency towards conservation of the primary amino acid sequence. The protein was expressed as a glutathione S-transferase fusion in both bacteria and in Schizosaccharomyces pombe. The bacterial protein was shown by solution assay to compete with CaM kinase II for CaM. The CaM binding domain was localized to the C-terminus of the protein by this method. Expression of full-length EF1alpha in vivo caused an increase in cell cycle length and a decreased rate of growth as evidenced by a lack of elongated cells in slowly dividing cultures. This effect appears to involve CaM binding because a truncation mutant version of EF1alpha lacking the CaM binding domain did not cause cell cycle delay.
Rasmussen
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Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, Canada. colin@pombe.usask.ca
Wiebe
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D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D002455:Cell Division; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases