Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Filters

2015 - 201912020 - 20241

Settings

Has files: YesSubject: search.filters.subject.Gcn1

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    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.