EVIDENCE THAT GCN1 AND GCN20, TRANSLATIONAL REGULATORS OF GCN4, FUNCTION ON ELONGATING RIBOSOMES IN ACTIVATION OF EIF2-ALPHA KINASE GCN2

In the yeast Saccharomyces cerevisae, phosphorylation of translation i nitiation factor eIF2 by protein kinase GCN2 leads to increased transl ation of the transcriptional activator GCN4 in amino acid-starved cell s. The GCN1 and GCN20 proteins are components of a protein complex req uired for the stimulation of GCN2 kinase activity under starvation con ditions, GCN20 is a member of the ATP-binding cassette (ABC) family, m ost of the members of which function as membrane-bound transporters, r aising the possibility that the GCN1/GCN20 complex regulates GCN2 indi rectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no ob vious association with plasma or vacuolar membranes. In addition, a fr action 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 dispe nsable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism f or 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, w as found to interact with an internal segment of GCN1 similar in seque nce to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EM-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 co uld allow for modulation of GCN1 activity, and the ABC domains in GCN2 0 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly cons erved with yeast GCN1 has sequence similarity to EF3. Thus, similar me chanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.