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  1. Author Response

    Reviewer #1 (Public Review):

    In this study Crawford et al., studied the protein composition of translating ribosomes in yeast under unstressed or stress conditions. To achieve this, the authors employed a combination of polysome profiling, which is a method that separates translating from non-translating ribosomes based on their sedimentation in sucrose gradients, and mass spectrometry. They identified aspartate aminotransferase, Aat2 as one of the proteins that is enriched in translating ribosomes in stressed cells. Crawford et al., went further to show that deletion of Aat2 impairs adaptation of yeast to osmotic stress and provided some evidence that Aat2 may play a role in integrated stress response. Finally, the authors show that the aminotransferase activity is not required for its function in translation and stress responses. Altogether, this study was found to be of high interest as it provides further potential insights in the molecular underpinnings of stress adaptation and further emphasizes potential unconventional roles of metabolic enzymes in regulation of translation. It was therefore thought that this study is likely to be of broad interest to the fields of biochemistry, molecular and cellular biology and beyond.

    Strengths: This study is based on an elegant combination of biochemical and genetic approaches. Evidence implicating Aat2 in oxidative stress response was found to be strong. In addition, it was appreciated that authors demonstrate that a potential role of Aat2 in regulation of protein synthesis under stress is independent of its aminotransferase activity.

    Weaknesses: The major weaknesses were thought to be related to the relative lack of the mechanistic evidence of how Aat2 is recruited to the ribosomes. In addition, factor(s) that transduce signals from stressors to entice Aat2:ribosome association remain(s) elusive.

    We thank the reviewer for their positive comments about our manuscript. To address the weaknesses: as outlined below a fraction of Aat2 is constitutively associated with ribosomes under the conditions we have investigated, rather than appearing during stress.

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  2. Evaluation Summary:

    Using mass spectrometry, Crawford et al. identify aspartate aminotransferase 2 (Aat2) as a protein whose polysome-association is increased under oxidative stress in yeast. Aat2 deletion sensitizes yeast to oxidative stress, which is paralleled by an aberrantly elevated integrated stress response, although polysome-association of Aat2 and its effect on oxidative stress response are independent of its aminotransferase activity. This provides evidence that metabolic enzymes may "moonlight" as post-transcriptional regulators. The study will appeal to experts in the fields of biochemistry, genetics, cellular and molecular biology. The presented data mostly support the authors' conclusions, but there are a few technical issues that should be addressed. These include corroborating Aat2:ribosome association and characterizing the effects of non-catalytic Aat2 mutants on the integrated stress response.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer 3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    In this study Crawford et al., studied the protein composition of translating ribosomes in yeast under unstressed or stress conditions. To achieve this, the authors employed a combination of polysome profiling, which is a method that separates translating from non-translating ribosomes based on their sedimentation in sucrose gradients, and mass spectrometry. They identified aspartate aminotransferase, Aat2 as one of the proteins that is enriched in translating ribosomes in stressed cells. Crawford et al., went further to show that deletion of Aat2 impairs adaptation of yeast to osmotic stress and provided some evidence that Aat2 may play a role in integrated stress response. Finally, the authors show that the aminotransferase activity is not required for its function in translation and stress responses. Altogether, this study was found to be of high interest as it provides further potential insights in the molecular underpinnings of stress adaptation and further emphasizes potential unconventional roles of metabolic enzymes in regulation of translation. It was therefore thought that this study is likely to be of broad interest to the fields of biochemistry, molecular and cellular biology and beyond.

    Strengths: This study is based on an elegant combination of biochemical and genetic approaches. Evidence implicating Aat2 in oxidative stress response was found to be strong. In addition, it was appreciated that authors demonstrate that a potential role of Aat2 in regulation of protein synthesis under stress is independent of its aminotransferase activity.

    Weaknesses: The major weaknesses were thought to be related to the relative lack of the mechanistic evidence of how Aat2 is recruited to the ribosomes. In addition, factor(s) that transduce signals from stressors to entice Aat2:ribosome association remain(s) elusive.

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  4. Reviewer #2 (Public Review):

    Translational control is critical for adaptation to environmental stresses, such as those inflicting oxidative damage and these regulatory mechanisms are suggested to involve the interplay of proteins associating with ribosomes or translating mRNAs. This manuscript uses the yeast model system and an unbiased proteomics approach to measure proteins associating with monosomes and heavy ribosomes (measures of robust mRNA translation) to measure this interplay in response to oxidative stress (acute hydrogen peroxide exposure). The manuscript identifies clusters of proteins that are enriched with polysomes, some differentially bound, in response to the oxidative stress. The study provides a thoughtful analysis of the clusters of proteins that that differentiall engage with the ribosomes and their regulatory meaning. One protein aspartate amino transferase (Aat2) was determined with be associated with heavy polysome duyring oxidative stress and this association was genetically determined to be independent of its metabolic activity and show to moderate Gcn2 activation and the integrated stress response (aka yeast general amino acid control). Deletion of AAT2 conferred sensitivity to H2O2 and had an accompanying enhanced Gcn2 phosphorylation of eIF2 and translational control, showing its in vivo significance in translational control. Overall, this is a significant manuscript that provides insights into the dynamics of changing protein associations with ribosomes and their implications for translational control. The manuscript is well organized, generally clearly presented, and experiments are appropriately interpreted. The manuscript would be of broad interest. There are some concerns involving clarification of some text and figures.

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  5. Reviewer #3 (Public Review):

    In the presented paper, Crawford et al. used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in yeast cells under unstressed vs. ISR-inducing stressed conditions (oxidative stress and amino acid starvation). Numerous proteins, including expected ribosomal proteins and translation factors, as well as proteins with less known translation-related roles such as metabolic enzymes were identified and clustered based on their polysome enrichment (PE) during tested conditions. One such a polysome-enriched protein, the cytosolic aspartate aminotransferase, Aat2, was characterized further. It was shown that its deletion conferred growth sensitivity to oxidative stress, causing aberrantly high activation of the ISR via enhanced eIF2a phosphorylation and GCN4 activation. Since non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity, the authors proposed that Aat2 has a separate ribosome-associated translational regulatory / moonlighting function that modulates the ISR. Despite some reservations listed below, overall I conclude that the authors achieved their aims and stress that for the most part the results perfectly support the outlined conclusions.

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