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    Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation
    (Portland Press on behalf of the Biochemical Society, 2024-03-05) Shanmugam R; Anderson R; Schiemann AH; Sattlegger E
    The protein kinase Gcn2 and its effector protein Gcn1 are part of the General Amino Acid Control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis.  Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P).  This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation.  Xrn1 is involved in RNA decay, quality control and processing.  We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex.  Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels.  Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion doesn't enhance eIF2α de-phosphorylation.  Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly.  Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.
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    Identification of Gcn1 binding proteins and characterization of their effect on Gcn2 function : a thesis submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry, Massey University, Albany, New Zealand
    (Massey University, 2015) Shanmugam, Renuka
    All cells must have the ability to deal with a variety of environmental stresses. Failure to adapt and protect against adverse stress conditions can lead to cell death. One important stress that affects all cells is amino acid limitation. Amino acids are building blocks of proteins. Gcn2 is a protein kinase, activated under conditions of amino acid limitation and the active Gcn2 reduces the general protein synthesis and specifically increases the synthesis of a protein called Gcn4, a transcription factor of stress response genes. Gcn2 is found in virtually all eukaryotes. In addition to the amino acid limitation it protects cells to a large array of stress conditions such as glucose and purine limitation, high salt, reactive oxygen species and UV irradiation. Interestingly, Gcn2 has been found to have acquired additional functions in higher eukaryotes such as cell cycle regulation, viral defense and memory formation. Not surprisingly, Gcn2 has been implicated in diseases and disorders such as abnormal feeding behaviour, cancer, Alzheimer’s disease, impaired immune response, congestive heart failure, and susceptibility to viruses including HIV. Despite of its medical relevance, so far it is unknown how the cell ensures proper Gcn2 function. Yeast studies have uncovered that for almost all Gcn2 functions Gcn2 must bind to its positive effector protein Gcn1. Gcn1 is proposed to be a scaffold protein, strongly suggesting that it serves as a platform for recruiting other proteins close to Gcn2 to fine-tune its activity. For this reason, in this study, we set out to comprehensively identify all proteins binding to Gcn1, i.e. generate the Gcn1 interactome, using a procedure that allowed us to also identify proteins that only weakly or transiently contact Gcn1 (a typical property of regulatory proteins). We have identified several potential Gcn1 binding proteins from published and in house data. Sixty six of these were further analyzed using the respective deletion strains. Ten of these deletion strains were unable to grow under amino acid starvation conditions. Five of these showed reduced eIF2! phosphorylation, strongly suggesting that they are positive effectors of Gcn2. Using plasmids from the Yeast Genome Tiling Collection, we were able to rescue the Gcn2 function of three deletion strains (kem1", msn5" and sin3"), indicating that the defect was due to the deletion of the respective gene. In addition, some of these proteins were confirmed to reciprocally bind to Gcn1. Finally, we show that Kem1 partially facilitates activation of Gcn2 via Gcn1 and it may play a role as a positive regulator of Gcn2. Furhther the interactions were validated by reciprocal immunoprecipitation. Taken together, this study sheds light on novel Gcn1 binding proteins regulating Gcn2.
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    Identification of ribosomal proteins that are necessary for fully activating the protein kinase Gcn2 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Albany, New Zealand
    (Massey University, 2014) Jochmann, Viviane Aleta
    The environment in which cells grow often changes rapidly and in order to survive, cells need to adjust their metabolic pathway to these changes. Vitally important for all organisms is the constant availability of amino acids as they are building blocks for proteins. Proteins are essential molecules involved in most biological processes in a cell. Yeast and mammals overcome amino acid limitation by switching on a signalling pathway named General Amino Acid Control (GAAC), which triggers a decrease in general protein synthesis by inhibiting translation initiation while upregulating the transcription of stress-response genes. For sensing starvation in yeast, the GAAC requires the kinase Gcn2 and its effector protein Gcn1. Gcn2 phosphorylates the α-subunit of the eukaryotic initiation factor 2 (eIF2α), which ultimately induces the selective expression of stress-response genes, leading to the de novo synthesis of all amino acids. In order to recognize the deacylated tRNA as an immediate signal for starvation, Gcn1 and Gcn2 need to be in direct contact and associated with the translating ribosome. The current model for sensing starvation by Gcn2 suggests that deacylated tRNA enters the ribosomal Asite and Gcn1 concomitantly transfers the starvation signal to Gcn2. However, the molecular details of this process are still unclear. Deletion analysis of GCN1, suggested that Gcn1 has multiple contact points with the ribosome. We therefore aim to uncover ribosomal proteins that are required to fully activate Gcn2 in order to better understand the starvation recognition process. The fact that Gcn1 has many ribosomal contact points implies that the deletion of one contact point will not remove Gcn1 from the ribosome and therefore maintains Gcn2 activation. This allows us to identify Gcn1-ribosome interaction points which are not only required to position Gcn1on the ribosome but also facilitate in Gcn1 mediated Gcn2 activation per se. Genetic studies conducted in this thesis reveal that ribosomal proteins rps18, rps26, rps28, rpl21 and rpl34 are necessary for full Gcn2 activation. The deletion of their genes resulted in an impaired growth on starvation media and in a reduction in eIF2α phosphorylation. With these results we are able to create a first map of Gcn1 contact points of the ribosome that are necessary to promote Gcn2 activation. Two ribosomal proteins that are necessary for fully activated Gcn2 are located on the large ribosomal subunit. Three others are located on the ribosomal head region of the small ribosomal subunit in proximity to the A-site region. Considering that Gcn1 is a large protein, our results support the idea that Gcn1 has multiple contact points with the ribosome and that some important contact points for Gcn2 activation are located near the ribosomal A-site.