title: Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2.
abstract: In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.
authors.affiliation: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Marton
authors.forename: C R
authors.initials: CR
authors.lastname: Vazquez de Aldana
authors.forename: H
authors.initials: H
authors.lastname: Qiu
authors.forename: K
authors.initials: K
authors.lastname: Chakraburtty
authors.forename: A G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D019892:eIF-2 Kinase
chemical_list: D018528:ATP-Binding Cassette Transporters; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D005656:Fungal Proteins; C501389:GCN1 protein, S cerevisiae; C094764:GCN20 protein, C elegans; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase