Browsing by Author "Bolech, Michael"

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    Identification of novel proteins that potentially are in complex with Yih1 and that are required for promoting Gcn2 function : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Philosophy in Biochemistry at Massey University, Auckland, New Zealand
    (Massey University, 2015) Bolech, Michael
    The ability of organisms to respond to various stress conditions is important for life. Under amino acid starvation conditions the protein Gcn2 is activated and phosphorylates the translation initiation factor eIF2α. This leads to a downregulation of general protein synthesis and an upregulation of the synthesis of proteins that are involved in helping the cell overcome starvation, a process called General Amino Acid Control (GAAC). It is important that the GAAC is only switched on when necessary and for this Gcn2 needs to be regulated. For instance, the protein Gcn1 is needed for Gcn2 activation. Another protein, Yih1, inhibits Gcn2 activity by competing with Gcn2 for Gcn1 binding. However, the balance between Gcn2 activation by Gcn1 and Gcn2 inhibition by Yih1 is not well understood. Actin was already identified as a Yih1-binding protein and modelling exercises strongly suggests that additional proteins bind Yih1. The aim of this project was to identify novel proteins that are in a complex with Yih1 (Yih1-binding proteins, YBP) and to then discover which are required for Gcn2 activation. For the first aim, YBP were ascertained from published large-scale protein-protein interactions studies and from data generated in-house. 19 different strains deleted for one putative YBP exhibited an impaired growth under starvation conditions. Of those, four deletion mutants showed a reduced Gcn2 activity. One protein was Spc72 which is involved in mitochondrial organisation. Another protein was Idh2, an enzyme of the citric acid cycle. The growth defect of strains deleted for SPC72 or IDH2 was complemented with a plasmid containing SPC72 or IDH2, respectively, and other genes. This suggested their involvement in Gcn2 activation. Elongation factor eEF1A was found as a putative YBP and as a co-precipitator of Yih1, supporting previous unpublished observations. eEF1A was found to bind Gcn2 in previous studies and this suggested that Yih1-eEF1A interaction may regulate Gcn2 activation. Another putative YBP, the heat shock protein Hsc82, is needed for Gcn2 maturation. Strains deleted for HSC82 showed an impaired growth under starvation conditions and this was reversed by deleting YIH1. This suggested that Yih1 may regulate Hsc82-Gcn2 interaction and thus Gcn2 activity. This study was a step to further advance our understanding of Yih1-binding proteins and Gcn2 activity. In addition, this further emphasised the idea of Yih1 as an important regulator inside the cell.