Evidence that Yih1 resides in a complex with ribosomes

Area of Science:

• Molecular Biology
• Cellular Stress Response
• Protein Synthesis Regulation

Background:

• Protein synthesis regulation via eukaryotic translation initiation factor 2 (eIF2α) phosphorylation is crucial for eukaryotic stress adaptation.
• The Gcn2 kinase is essential for sensing and responding to amino acid starvation.
• The Gcn1-Gcn2-ribosome complex is implicated in starvation sensing.

Purpose of the Study:

• To investigate the role of yeast IMPACT homologue (Yih1) and its mammalian counterpart IMPACT in the Gcn2-mediated stress response.
• To determine if Yih1/IMPACT associates with ribosomes and how this interaction relates to Gcn1 and Gcn2 binding.

Main Methods:

• Velocity sedimentation assays to assess Yih1/IMPACT association with polyribosomes.
• Coprecipitation assays to identify binding partners of Yih1/IMPACT, including ribosomal proteins and Gcn1.
• Analysis of Yih1/IMPACT interaction under physiological conditions using myc-tagged Yih1.

Main Results:

• Overexpressed glutathione S-transferase (GST)-tagged Yih1 cosedimented with polyribosomes independently of Gcn1.
• GST-Yih1 coprecipitated large ribosomal protein Rpl39, and this interaction was Gcn1-independent.
• Yih1-ribosome interaction was confirmed under physiological conditions, and a similar interaction was observed for mammalian IMPACT with yeast ribosomes.
• GST-IMPACT also coprecipitated actin, similar to GST-Yih1.

Conclusions:

• IMPACT/Yih1 directly associates with ribosomes in a manner independent of Gcn1.
• This ribosome association is evolutionarily conserved between yeast and mammals.
• The findings suggest that IMPACT/Yih1 may act as a scaffold or regulator within the Gcn1-Gcn2-ribosome complex, facilitating rapid Gcn2 inhibition.