GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2.

Abstract

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...

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Inactivating mutations in Gcn1 or Gcn20, an ABC-type ATPase, prevent Gcn2 activation in response to amino acid starvation (Marton et al 1993;Vazquez de Aldana et al 1995). Both Gcn1 and Gcn20 bind elongating ribosomes (Marton et al 1997) via the Gcn1 NTD, and this binding is required for Gcn2 activation (Sattlegger and Hinnebusch 2005).…”

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response

“…This results in allosteric rearrangements in GCN2 that lead to its autophosphorylation at a threonine residue in the activation loop of the kinase domain, allowing GCN2 to efficiently bind and phosphorylate its substrate, eIF2α. At the GCN2 amino terminus, the RWD domain (from its presence in RING finger proteins, WD-repeat-containing proteins and DEAD-like helicases) binds directly to the effector protein GCN1, an interaction that is essential for GCN2 activation in vivo but not for the kinase activity per se (Marton et al, 1993;Sattlegger and Hinnebusch, 2000). GCN1 is believed to be involved in the transfer of uncharged tRNAs from the ribosomal A site to the HisRS domain of GCN2, when both proteins are bound to translating ribosomes (Marton et al, 1997;Sattlegger and Hinnebusch, 2000).…”

YIH1 Is an Actin-binding Protein That Inhibits Protein Kinase GCN2 and Impairs General Amino Acid Control When Overexpressed

“…The uncharged tRNA binds to a regulatory domain in GCN2 that resembles histidyl-tRNA synthetase, and this interaction is believed to induce a conformational change that overcomes an intrinsic defect in the adjacent kinase domain (7)(8)(9)(10). The products of GCN1 and GCN20 are also necessary for the activation of GCN2 by uncharged tRNA in starved cells (11,12). GCN1 and GCN20 form a protein complex (12) that binds to the N-terminal domain (NTD) of GCN2, which is highly conserved among all GCN2 orthologs.…”