Phenotypic Characterization of Yeasts Aiming at Bioethanol Production

Worldwide, the production of bioethanol is derived through first-generation technology, where plants, vegetables, and cereals, that have high levels of sucrose, are fermented by yeast. Brazil, for the production of bioethanol from sugarcane, is among the world’s leading producers. The process for bioethanol production is a complex that involves a variety of environmental factors, resulting in different phenotypic profiles of strain used. It has been evidenced that the interaction between environmental factors and microorganism can influence in the identification of different characteristics of Saccharomyces cerevisiae. Also, the bioethanol is developed by the second and third generations, and new yeast strains may also contribute to the feasibility of production. Successful performance of fermentation depends on the ability of the yeast to deal with a number of factors that occur during the fermentation, such as concentration of sugar, ethanol, nitrogen, pH, resistance to contaminants, stress protein, temperature change, and osmotic pressure

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.Bill & Melinda Gates Foundation FAPES

Is there a role for eIF5A in translation?

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archaebacteria to mammals. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. This review focuses on the functional characterization of eIF5A. Although this protein was originally identified as a translation initiation factor, subsequent studies did not support a role for eIF5A in general translation initiation. eIF5A has also been implicated in nuclear export of HIV-1 Rev and mRNA decay, but these findings are controversial in the literature and may reflect secondary effects of eIF-5A function. Next, the involvement of eIF5A and hypusination in the control of the cell cycle and proliferation in various organisms is reviewed. Finally, recent evidence in favor of reconsidering the role of eIF5A as a translation factor is discussed. Future studies may reveal the specific mechanism by which eIF5A affects protein synthesis

eIF5A and EF-P: two unique translation factors are now traveling the same road

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation. (C) 2014 John Wiley & Sons, Ltd.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Functional significance of eIF5A and its hypusine modification in eukaryotes

The unusual basic amino acid, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Synthetic lethality between eIF5A and Ypt1 reveals a connection between translation and the secretory pathway in yeast

The putative translation initiation factor 5A (eIF5A) is a small protein, highly conserved and essential in all organisms from archaea to mammals. Although the involvement of eIF5A in translation initiation has been questioned, new evidence reestablished the connection between eIF5A and this cellular process. In order to better understand the function of elF5A, a screen for synthetic lethal gene using the tif51A-3 mutant was carried out and a new mutation (G80D) was found in the essential gene YPT1, encoding a protein involved in vesicular trafficking. The precursor form of the vacuolar protein CPY is accumulated in the ypt1-G80D mutant at the nonpermissive temperature, but this defect in vesicular trafficking did not occur in the tif51A mutants tested. Overexpression of eIF5A suppresses the growth defect of a series of ypt1 mutants, but this suppression does not restore correct CPY sorting. on the other hand, overexpression of YPT1 does not suppress the growth defect of tif51A mutants. Further, it was revealed that eIF-5A is present in both soluble and membrane fractions, and its membrane association is ribosome-dependent. Finally, we demonstrated that the ypt1 and other secretion pathway mutants are sensitive to paromomycin. These results confirm the link between translation and vesicular trafficking and reinforce the implication of eIF5A in protein synthesis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq