Genetic and biochemical evidence for yeast GCN2 protein kinase polymerization.
The GCN2 (general control kinase 2) protein is an eIF2-alpha (eukaryotic initiation factor alpha) kinase which mediates translational derepression of the yeast general control transcriptional activator, GCN4, upon amino-acid starvation. We isolated and characterized GCN2 mutations differentially affecting GCN2 function. Mutations mapping in, or close to, the ATP-binding site of the kinase moiety result in constitutively activated GCN2 molecules. A C-terminal regulatory mutation dramatically affects translation initiation rates resulting in pleiotropic phenotypes. The effect of mutations in both regions were found to depend on eIF2-alpha phosphorylation. We have demonstrated that GCN2 mutants have altered autophosphorylation activities in vitro, depending on the presence or absence of a wild-type GCN2 gene and that GCN2 elutes in gel-filtration chromatography fractions with high apparent molecular mass. Both these genetic and biochemical findings suggest that GCN2 functioning might involve polymerization to form dimers or tetramers.
Diallinas
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Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece.
Thireos
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D000596:Amino Acids / Q000378:metabolism; D002850:Chromatography, Gel; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D009154:Mutation; D010448:Peptide Initiation Factors / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics*
D000596:Amino Acids; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D005656:Fungal Proteins; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases