Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Aug 14, 2012The Journal of biological chemistry

Too much elongation factor 3 reduces activation of Gcn2 protein

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Abstract

Overexpression of eEF3 in Saccharomyces cerevisiae diminished growth on amino acid starvation medium.

Gcn2 activation by uncharged tRNA involves direct interaction with Gcn1 and the ribosome.
Gcn1 and Gcn2 may not function simultaneously on the same ribosome due to overlapping binding sites with eEF3.
eEF3 overexpression decreased phosphorylation of eIF2α, indicating reduced activation of the amino acid starvation response.
The growth defect from constitutively active Gcn2 was alleviated by eEF3 overexpression.
eEF3 overexpression did not significantly alter Gcn1's association with the ribosome but worsened the Gcn(-) phenotype associated with a Gcn1 variant with lower ribosome affinity.

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In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn(-) phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn(-) phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn(-) phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.

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Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

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