Evidence that Yih1 resides in a complex with ribosomes

Adjusting protein synthesis by phosphorylating eukaryotic translation initiation factor 2 (eIF2α) is a major mechanism by which eukaryotes adapt to and overcome stress. The eIF2α kinase Gcn2 is essential for overcoming amino acid starvation in all eukaryotes. We have shown that to sense starvation, the Gcn2 RWD domain must directly contact its effector protein, Gcn1, and both must bind to the ribosome, suggesting that starvation is sensed within a Gcn1–Gcn2–ribosome complex. The mammalian protein IMPACT, highly expressed in neurons, and its yeast orthologue yeast IMPACT homologue (Yih1) harbour an RWD domain with Gcn1-binding activity. We have shown that Yih1 downregulates Gcn2 by competing with Gcn2 for Gcn1-binding. Here, we provide evidence that Yih1 forms a complex with ribosomes. In velocity sedimentation assays, overexpressed glutathione S-transferase (GST)-tagged Yih1 cosedimented with polyribosomes independently of Gcn1. Reduction of polyribosomes to monosomes concomitantly decreased GST–Yih1 sedimentation in the heavy fractions where polyribosomes are normally found. Furthermore, GST–Yih1 coprecipitated large ribosomal protein Rpl39 independently of Gcn1. GST–Yih1 overexpression did not significantly affect Gcn1–ribosome or Gcn2–ribosome cosedimentation. myc-tagged Yih1 expressed from its own promoter cosedimented with polyribosomes independently of Gcn1, indicating that Yih1–ribosome interaction occurs under physiological conditions. GST–IMPACT cosedimented with yeast ribosomes and coprecipitated Rpl39 in a Gcn1-independent fashion, suggesting that Yih1/IMPACT–ribosome association is evolutionarily conserved. Moreover, GST–IMPACT coprecipitated actin as found for GST–Yih1. Taken together, our findings strongly suggest that IMPACT/Yih1 associates with ribosomes and that these ribosomes may simultaneously carry Gcn1 and Gcn2. Close physical proximity of Yih1 to the Gcn1–Gcn2–ribosome complex would allow cells to quickly inhibit Gcn2 whenever or wherever necessary.

Structured digital abstract

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   Polyribosomes physically interact with YIH1, GCN1, GCN2, eEF3, eEF1A, GIR2, RPL39, RPS22, by co-sedimentationassays (Viewinteraction)
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   YIH1 physicallyinteracts with RPL39 by cross-linkingstudy (Viewinteraction)
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   IMPACT physicallyinteracts with GCN1, actin, RPS22 and RPL39 by pulldown (Viewinteraction)