Title: Gap1 amino acid signaling and translation initiation in Saccharomyces cerevisiae.
Other Titles: Gap1 aminozuursignalering en initiatie van translatie in Saccharomyces cerevisiae.
Authors: Kimpe, Marlies
Issue Date: 7-Jun-2012
Abstract: Saccharomyces cerevisiae serves as one of the main species of yeast for the production of beer, bread and wine, but is also used as a model organism for the study of the eukaryotic cell. In nature, yeast cells are exposed to rapid and extreme changes in the environment, ranging from temperature stress to nutrient deprivation and osmotic stress. Therefore, the ability of yeast to cope with these adverse conditions is crucial for its survival. In particular, regulation of cell growth in response to nutrients is not only crucial for yeast, but for all organisms. Besides their role as building blocks and energy providers, nutrients function as signaling molecules that transmit environmental information to cells. These nutrients induce signaling cascades that control growth, several of which are highly conserved from yeast to human. Importantly, during the process of brewing, wine or bioethanol production, yeast cells are exposed to similar stresses which adversely affect growth, fermentative capacity and cell viability. Therefore, the discovery of novel molecular mechanisms that underlie nutrient-dependent control of growth in yeast will not only reveal equivalent mechanisms in higher eukaryotes but is also of great value for optimizing the use of yeast in the fermentation industry.When starved for an essential nutrient, such as carbon, nitrogen, phosphate or sulfate, yeast cells arrest growth and enter stationary phase. During this quiescent state, the cells adopt a number of properties characteristic for low protein kinase A (PKA) activity such as decreased expression of ribosomal protein genes, accumulation of reserve carbohydrates, increased stress resistance andgrowth arrest. Re-addition of the missing nutrient triggers rapid activation of PKA, reversing these effects and allowing a fast start-up of growth. In case of nitrogen starvation, regulation of PKA activity by amino acids is mediated by the general amino acid permease Gap1. This transporter protein has an additional function as a receptor for activation of PKA in response to amino acids via the so-called fermentable growth medium-induced (FGM) pathway. Exactly how Gap1 activates PKA is not known, but evidence has been provided that the yeast S6K/PKB ortholog Sch9 and the yeast PDK1 orthologs Pkh1-3 are implicated. One approach to elucidate the molecular mechanisms behind FGM signaling is the identificationof proteins physically interacting with the transceptor Gap1. Previous work in our lab has identified the alfa-subunit of the translation initiation factor eIF2 as a potential direct interaction partner of Gap1. Translation initiation is the first step in the process of protein synthesis, and its proper regulation in response to nutrient availability is crucial for eukaryotic cell growth. Throughout this research the possible direct connection between amino acid-induced FGM signaling and components of the translation initiation machinery in yeast has been investigated.We show that Gap1 physically interacts with different subunits of eIF2 but also the guaninenucleotide exchange factor eIF2B, using several protein-protein interaction assays. Importantly, in the absence of the eIF2alfa kinase Gcn2, the activity of the downstream PKA target trehalase is increased and also mutations in eIF2 and eIF2B affect amino acid-induced activation of the FGM pathway. These results suggest that eIF2, eIF2B but also the regulatory eIF2alfa kinase Gcn2 play a role in amino acid-induced activation of the FGM pathway. Moreover, several direct interactions were observed of Tpk1, Sch9 and Pkh1 with eIF2, eIF2B and Gcn2, both in vitro and in vivo. We also show for the first time that Pkh1 is able to phosphorylate Gcn2 in vitro, suggesting a possible role for PDK1 signaling in the regulation of GCN2 activity and translation initiation in mammalian cells. In addition, we observed that Gcn2, but also Gcn4, a transcription factor and key regulator of amino acid biosynthesis, are required for start-up of growth after addition of certain, but not all, amino acids to nitrogen-starved yeast cells. This indicates that under conditions of nitrogen starvation, Gcn4 has an important role in supporting the utilization of certain amino acids as sole nitrogen source, rather than a general role for start-up of amino acid biosynthesis. We also provide evidence for a rapid, Gap1-dependent, increase of eIF2alfa phosphorylation in response to addition of amino acids to nitrogen starved yeast cells. This suggests a possible role for Gap1 in regulation of translation initiation. Last but not least, we provide evidence that the use of auxotrophic yeast strainsunder conditions of nitrogen starvation can give rise to results that may not be comparable with their prototrophic counterparts. These results also suggest a possible role for intracellular amino acid levels in the regulation of FGM signaling. Although this study has improved our insight in the possible direct connection between translation initiation and Gap1-dependent activation of the FGM pathway in yeast, many questions remain and several new questions have been raised. Additional research is required to fully elucidate the molecular mechanisms that underlie activation of PKA by amino acids and the role of Gap1 amino acid signaling in the regulation of translation initiation.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Microbiology and Biotechnology Section - miscellaneous (-)
Laboratory for Molecular Cell Biology (-)

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