title: Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis.
abstract: Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins--eIF5A(K56A) and eIF5A(Q22H,L93F)--and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.
authors.affiliation: Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rodovia Araraquara-Jaú km. 1, Araraquara, São Paulo, Brazil.
authors.forename: Camila A O
authors.initials: CA
authors.lastname: Dias
authors.forename: Veridiana S P
authors.initials: VS
authors.lastname: Cano
authors.forename: Suzana M
authors.initials: SM
authors.lastname: Rangel
authors.forename: Luciano H
authors.initials: LH
authors.lastname: Apponi
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Frigieri
authors.forename: João R C
authors.initials: JR
authors.lastname: Muniz
authors.forename: Wanius
authors.initials: W
authors.lastname: Garcia
authors.forename: Myung H
authors.initials: MH
authors.lastname: Park
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garratt
authors.forename: Cleslei F
authors.initials: CF
authors.lastname: Zanelli
authors.forename: Sandro R
authors.initials: SR
authors.lastname: Valentini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A