Multiprotein bridging factor 1 is required for robust activation of the integrated stress response on collided ribosomes
The multiprotein bridging factor 1 (Mbf1) proteins are a family of conserved eukaryotic proteins thought to mediate the activity of stress-induced transcription factors. In yeast, Mbf1 has been proposed to function in the integrated stress response (ISR) by mediating an interaction between the basal transcription machinery and the process's key effector Gcn4. Curiously, mounting recent evidence has strongly suggested that the factor and its human homologue to be recruited to collided ribosomes, the very signal newly recognized to activate the ISR. Here we provide data that connect these otherwise supposedly disparate functions of the factor. Our findings revealed that Mbf1 functions as a core factor in the ISR process, responding early to stress and mediating the activation of Gcn2. We further show that the factor serves no role as a transcriptional coactivator of Gcn4; instead, it is required for optimal stress-induced eIF2a phosphorylation and downstream de-repression of GCN4 translation. Detailed mutational and biochemical analysis, together with a cryo-EM structure of the factor bound to collided ribosomes, showed that Mbf1's activity in the ISR is enabled through its interaction with ribosomes. Collectively, our data resolve an important question about how Mbf1 factors function in signaling by acting as sensors of stress-induced ribosomes collisions, and not as coactivators of the effector proteins as widely accepted in the field. Overall design: RNA-seq analysis of wild type, gcn2?, gcn4?, and mbf1? strains treated with 0.1% methyl methanesulfonate (MMS) for one hour, as well as their matched no treatment controls. Each condition had three biological replicates and differential gene expression analysis was done using a Salmon-DESeq2 pipeline to investigate the interaction effect of treatment with genotype.