Browsing by Author "Gottfried S"

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    A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (poster)
    (8/09/2022) Schiemann A; Gottfried S; Sattlegger E
    The General Amino Acid Control stress signalling pathway allows cells to sense and overcome starvation. One of the major players in this pathway is the protein kinase Gcn2 found in all eukaryotic cells. Activation of Gcn2 leads to phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF2α), which then leads to the re-programming of the cell’s gene transcription and translation profile, and ultimately allowing cells to cope with and overcome starvation. For sensing starvation, Gcn2 must directly bind to its effector protein Gcn1. This interaction is mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Overexpression of the RWDBD alone impairs Gcn2 activation through disrupting endogenous Gcn1-Gcn2 interaction, hampering eIF2α phosphorylation, and consequently cells cannot overcome starvation and fail to grow.  This dominant negative phenotype is dependent on Arg-2259 in RWDBD.  Taking advantage of this phenotype in yeast, we here found that the dominant negative phenotype was reverted by each of four amino acid substitutions, K2270A, R2289A, R2297A, and K2301A.  This correlated with increased eIF2α phosphorylation levels, suggesting their relevance for Gcn2 activation.  All but Lys-2270 are fully surface exposed, suggesting that these amino acids may directly contact Gcn2.  We also found amino acid substitutions that enhanced the dominant negative phenotype of the overexpressed RWDBD, and correlated with further reduction in eIF2α-P levels.  Our findings suggest that two helices in Gcn1 constitute at least one Gcn2 contact point.
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    A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (research article)
    (PLOS, 2022-11-28) Gottfried S; Koloamatangi SMBMJ; Daube C; Schiemann AH; Sattlegger E; Lustig AJ
    The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.
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    Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein
    (Elsevier Inc, 2023-03-17) Ghuge AA; Anderson RA; Gottfried S; Daube C; Koloamatangi SMBMJ; Schiemann AH; Sattlegger E
    Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.