Phosphorylation of the a subunit of eukaryotic translation initiation factor 2 (eIF-2a) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCNI. Inactivation of GCNI did not affect the level of eIF-2ot phosphorylation when mammalian eIF-2a kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2a. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2a by GCN2, cell extracts from gcnlA strains contained wild-type levels of GCN2 eIF-2a-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.Starvation for an amino acid or the presence of a defective aminoacyl-tRNA synthetase in the yeast Saccharomyces cerevisiae leads to increased transcription of more than 30 genes encoding amino acid biosynthetic enzymes in 10 different pathways. This response, called general amino acid control (reviewed in reference 35), is mediated by the transcriptional activator GCN4 that binds to promoter regions of the coregulated structural genes subject to the general control. Expression of GCN4 itself is regulated by the availability of amino acids, but at the level of translation initiation. When amino acids are abundant, four short upstream open reading frames (uORFs) in the leader of GCN4 mRNA restrict the flow of scanning ribosomes to the GCN4 initiation codon. Under starvation conditions, trans-acting positive factors encoded by GCN2 and GCN3 allow ribosomes to bypass the most inhibitory of the uORFs and initiate translation at the GCN4 AUG codon, thus increasing the level of GCN4 protein in the cell (1, 35).GCN2 is a protein kinase that stimulates GCN4 translation by phosphorylating the a subunit of eukaryotic translation initiation factor 2 (eIF-2a; encoded by SUI2) on the serine residue at position 51 (20,75). In mammalian cells, phosphorylation of eIF-2a on serine 51 inhibits general translation initiation by impairing the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. GDP-GTP exchange on eIF-2 catalyzed by eIF-2B is essential for translation because only the GTP-bound form of eIF-2 can deliver initiator tRNAMet to small ribosomal subunits (32). By analogy with mammalian systems, it was proposed that eIF-2a phosphorylation by GCN2 in S. cerevisiae would de...

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

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Two homogeneous protein phosphatases, termed ‘smooth muscle phosphatase‐I’ and ‘smooth muscle phosphatase‐III’, isolated from turkey gizzard as enzymes active against the 20‐kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purfied as an enzyme active against protein synthesis initiation factor eIF‐2, were classified using the criteria defined by Ingebritsen and Cohen [Eur J. Biochem. (1983) 132, 255–261]. All three enzymes were type‐2 protein phopsphatases bawsed on their specificity for the α subunit of phosphorylase kinase and insensitivity to inhibitor‐1 and inhibitor‐2. The substrate specificities of smoth muscle phosphatase‐I and the eIF‐2 phosphatase were similar to the catalytic subunit of protein phosphatase‐2A. Smooth muscle phosphatase‐I could be designated as protein phosphatase‐2A1 and eIF‐2 phosphatase as protein phosphatase‐2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase‐II allowed its assignment as protein phosphatase‐2C.