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Induction of GCN4translation in amino acid-starved cells involves the inhibition of initiator tRNAMetbinding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal(tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded byPUS4also leads to translational derepression of GCN4(Gcd−phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd−phenotype of high-copy-number PUS4(hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate thatPUS4overexpression did not diminish functional initiator tRNAMetlevels. The presence of hcPUS4or hcNME1led to the accumulation of certain tRNA precursors, and their Gcd−phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5′-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyrthat cannot be processed by RNase P had a Gcd−phenotype. Interestingly, the Gcd−phenotype of hcPUS4also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Δ cells have a Gcd−phenotype. Overproduced PUS4 appears to impede 5′-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal*showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMetbinding to the ribosome.

ABSTRACTInduction of GCN4translation in amino acid-starved cells involves the inhibition of initiator tRNAMetbinding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4translation could be induced independently of GCN2 by overexpressing a mutant tRNAAACVal(tRNAVal*) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded byPUS4also leads to translational derepression ofGCN4(Gcd-phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd-phenotype of high-copy-number PUS4(hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate thatPUS4overexpression did not diminish functional initiator tRNAMetlevels. The presence of hcPUS4or hcNME1led to the accumulation of certain tRNA precursors, and their Gcd-phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNATyrthat cannot be processed by RNase P had a Gcd-phenotype. Interestingly, the Gcd-phenotype of hcPUS4also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1? cells have a Gcd-phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNAVal*showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNAMetbinding to the ribosome.

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.