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 elon gation factor 1-alpha (EF1 alpha) from the yeast Schizosaccharomyces pombe. The cDNA was cloned from an Schizosaccharomyces pombe expression library b y a two-hybrid selection for clones encoding calmodulin (CaM)-binding prote ins. The predicted protein is highly homologous to mammalian EF1 alpha, ind icating a strong tendency towards conservation of the primary amino acid se quence. The protein was expressed as a glutathione S-transferase fusion in both bacteria and in Schizosaccharomyces pombe. The bacterial protein was s hown by solution assay to compete with CaM kinase II for CaM. The CaM bindi ng domain was localized to the C-terminus of the protein by this method. Ex pression of full-length EF1 alpha in vivo caused an increase in cell cycle length and a decreased rate of growth as evidenced by a lack of elongated c ells in slowly dividing cultures. This effect appears to involve CaM bindin g because a truncation mutant version of EF1 alpha lacking the CaM binding domain did not cause cell cycle delay.