The putative eukaryotic translation initiation factor 5A (eIF5A) is a very abundant and highly conserved protein in all eukaryotes and archaebacteria. It undergoes a unique post-translational modification called hypusination, in which deoxyhypusine synthase (Dys1) catalyzes the transfer of the butylamine moiety from the spermidine to the amino group of a specific lysine residue of eIF5A. Mutation of the target lysine (K51R) or DYS1 deletion in yeast does not allow cell growth, demonstrating that hypusination of eIF5A is an essential event. Although eIF5A function is not clear, it has been implicated in translation initiation, nucleocytoplasmic transport and mRNA decay. Our laboratory has previously performed a high-copy suppression screen using a temperature-sensitive eIF5A mutant and the gene encoding Pkc1 was isolated. Both yeast and mammalian deoxyhypusine synthases were found to be phosphorylated by different mammalian protein kinase C (PKC isoforms). Yeast and human deoxyhypusine synthases are 58% identical and have, at least, three conserved PKC consensus phosphorylation sites. Then, we have been studying whether Pkc1 can influence Dys1 activity in vivo and how this possible interaction interferes with eIF5A function in yeast. In a previous work, we presented the dys1^T118A mutant, which was only able to complement a dys1::HIS3 strain in the presence of 1M sorbitol. Here, we demonstrate by western blot analysis that dys1^T118A is unstable. Moreover, sequencing of this allele revealed two additional mutations (W75R and A147T), naturally present in a wild-type DYS1 from W3O3. Site-directed mutagenesis was conducted again to modify possible PKC phosphorylation sites in Dys1 and we obtained a novel dys1^T118A mutant as well as the mutants dys1^S160A and dys1^T268A, using another DYS1 wild-type template. Surprisingly, none of these DYS1 mutants are inviable and western blot analysis shows that the proteins encoded by these three new alleles are stable. Experiments are being carried out to evaluate whether these mutated alleles might affect the high-copy suppression of an eIF5A mutant by PKC1, thereby contributing to elucidate the mechanism of this phenotypic suppression by Pkc1.

Supported by: FAPESP and CNPq / PIBIC