Cloning of aSchizosaccharomyces pombehomologue of elongation factor 1 alpha by two-hybrid selection of calmodulin-binding proteins

Abstract

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.