Study of the eIF5A impact on cellular proteomic profile using Saccharomyces cerevisiae as model

Author(s)

Natália Moreira Barbosa

• Document type: Doctoral Thesis
• Press: Araraquara. 2019-09-23.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2019-09-13
• Advisor: Cleslei Fernando Zanelli; Sandro Roberto Valentini

Abstract

The translation factor 5A (eIF5A) is conserved and essential for cell viability. This is the only protein known to contain the amino acid residue hypusine, essential for eIF5A function, generated by a post-translational modification. Although it was initially suggested a function for eIF5A in the translation initiation, eIF5A has been demonstrated to have a role in translation elongation. More recent studies have established that eIF5A is necessary for the elongation of specific sequences, which are able to induce a ribosome stalling. Still, there are few studies with proteomic profile in the absence of eIF5A function and the proteins which syntheses are dependent on eIF5A are not well known. Thus, the present study aims to search for the proteins which syntheses are dependent on eIF5A by proteomic profile comparison between wild-type strains and eIF5A mutants in Saccharomyces cerevisiae. We present a proteomic profile for GFP fluorescence using a 4156 collection of strains, each one containing a different ORF fused to the C-terminal GFP and a protein RFP constitutively produced as normalizing, both in the wild and eIF5A mutant background. This thesis presents GFP / RFP data analysis and data validation using western blot. Our data using an in vivo proteome profile of the ORFs-GFP collection in a hyp2-3 mutant background demonstrating that yeast eIF5A shows several mitochondrial proteins downregulated in the eIF5A mutant. To confirm eIF5A involvement with mitochondrial function, we characterized mitochondrial physiology in eIF5A mutants. We revealed the inability of the eIF5A mutant to grow in non-fermentable carbon sources, where they depend on mitochondria for its oxidation. eIF5A mutant also showed reduced respiratory activity, increased membrane potential and mitochondrial DNA amount, although morphology does not appear to be affected. Mostly, mitochondrial proteins are either coded into the nucleus, translated into the cytoplasm and imported into mitochondria as an unfolded polypeptide, or are translocated in a conventional manner to mitochondria, and somehow eIF5A may be regulating the translation of these proteins specifically. Interestingly, the slowdown of translation elongation rates by rare codons or other translation stalling motifs can facilitate binding of the Signal Recognition Particle (SRP) to the ribosome, which may interfere with ER targeting. Thus, there are different ways by which eIF5A could interfere with translation of proteins targeted to the mitochondria or the ER and further work are necessary to address the mechanisms underlying this role of eIF5A in the cell. (AU)

• FAPESP's process: 15/23203-0 - Study of the eIF5A impact on cellular proteomic profile using Saccharomyces cerevisiae as model
• Grantee: Natália Moreira Barbosa
• Support Opportunities: Scholarships in Brazil - Doctorate