Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2

In eukaryotes, amino acid deprivation leads to the accumulation of uncharged tRNAs that are detected by Gcn2, which in turn phosphorylates eIF2α, an essential process for overcoming starvation. In Saccharomyces cerevisiae, sensing amino acid shortages requires that Gcn2 binds directly to its effector protein Gcn1, and both must associate with the ribosome. Our hypothesis is that uncharged tRNAs occur in the ribosomal A-site, and that Gcn1 is directly involved in transfer of this starvation signal to Gcn2. Here we provide evidence that Gcn1 directly contacts the small ribosomal protein Rps10. Gcn1 residues 1060-1777 showed a yeast 2-hybrid interaction with Rps10A. In vitro, Rps10A or Rps10B co-precipitated Gcn1[1060-1777] in an RNA-independent manner. rps10AΔ or rps10BΔ strains showed reduced eIF2α phosphorylation under replete conditions and shortly after onset of starvation, suggesting that Gcn1-mediated Gcn2 activation was impaired. Overexpression of GST-tagged Rps10 reduced growth under amino acid starvation, and this was exacerbated by the Gcn1-M7A mutation known to impair Gcn1-ribosome interaction and Gcn2 activity. Under amino acid starvation, eEF3 overexpression known to weaken Gcn1 function on the ribosome, exacerbated the growth defect of rps10AΔ or rps10BΔ strains. Taken together, these data support the idea that Gcn1 contacts ribosome-bound Rps10 to efficiently mediate Gcn2 activation.

Source: BJ Signal - December 1, 2014 Category: Biochemistry Authors: S Lee, M J Swanson, E Sattlegger Tags: BJ Signal Source Type: research