Characterization of eukaryotic initiation factor 5A (eIF-5A) in the dinoflagellate crypthecodinium cohnii biecheler

Eukaryotic Initiation Factor 5A (eIF-5A) is a highly conserved ubiquitous protein present in all eukaryotes and archaeas analyzed. Previous studies in yeast and mammalian cells show the requirement of eIF-5A in G1/S transition and more recent ones reveal the involvement of this gene in cytoplasmic-nuclear transportation and metabolism of RNAs. eIF-5A is the only known protein that contains the unusual amino acid hypusine, which is essential for eIF-5A's function. Hypusine is synthesized post-translationally by modifying a specific lysine residue with a 4-aminobutyl moiety from spermidine. Spermidine and other polyamines were found to be required in eukaryotic cell proliferation, and were also suggested to stimulate the blooming of certain dinoflagellate species. Although the exact function of polyamines is not known, one of the growth-stimulating mechanisms might be the hypusine modification. A cDNA encoding eIF-5A homolog in heterotrophic dinoflagellate Crypthecodinium cohnii was isolated from a C. cohnii cDNA library. The deduced amino acids sequence contains the 12 amino acids around the hypusination site that are strictly conserved among eukaryotes. A single 1.2 kb transcript was detected in C. cohnii total RNA using this cDNA as a probe. The transcript level peaks at early Gl and drops continuously till the end of the cell cycle. Expression of the cDNA in E. coli yielded a 23 kDa protein in the soluble fraction, which is comparable with other species. Phylogenetic trees constructed using parsimony and UPGMA method show C. cohnii eIF-5A emerges as an independent lineage within the eukaryotes, while neighbor-joining tree shows C. cohnii eIF-5A might have a close relationship with the plants. Polyamines synthesis inhibitor DFMO was found to retard growth of C. cohnii, but no growth arrest was observed. Mimosine, which was claimed to specifically inhibit hypusine synthesis in all eukaryotic cells treated so far, was found to stimulate growth of C. cohnii. Our findings show the highly conserved nature of C. cohnii eIF-5A gene and suggest the possible involvement of eIF-5A in dinoflagellate cell cycle control