Mapping eIF5A binding sites for Dys1 and Lia1: in vivo evidence for regulation of eIF5A hypusination.
The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal alpha-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required.
Thompson
Gloria M
GM
Department of Biological Sciences, School of Pharmacy, São Paulo State University - UNESP, Rodovia Araraquara-Jaú, Km01, Araraquara, SP, 14801-902, Brazil.
Cano
Veridiana S P
VS
Valentini
Sandro R
SR
D001665:Binding Sites; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques; D000097574:Eukaryotic Translation Initiation Factor 5A
D018076:DNA, Complementary; D005656:Fungal Proteins; D005982:Glutathione Transferase; D008024:Ligands; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C063507:deoxyhypusine synthase