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High-throughput functional characterization of protein phosphorylation sites in yeast

Abstract

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

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Fig. 1: Phospho-mutant library construction, chemical genomics screen and quality control.
Fig. 2: Chemical genomics screen functional analysis.
Fig. 3: Molecular characterization of phospho-deficient mutants.
Fig. 4: Molecular characterisation of Vma2 and Sit4 phospho-deficient mutants.
Fig. 5: Yeast phospho-mutants with severe phenotypes can inform on the functional relevance of human phosphosites.

Data availability

The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017929. Yeast mutant strain growth measurements are provided in the Supplementary Data.

Code availability

Functional enrichment in the phosphorylation network was performed using SAFE (https://www.bioconductor.org/packages/release/bioc/html/safe.html). Image processing and S score calculations were performed using EMAP (https://www.bioconductor.org/packages/release/bioc/html/safe.html). GO enrichment analysis to assign annotations to yeast phosphosites was performed using the ClusterProfiler package (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html).

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Acknowledgements

We thank N. Krogan and J. Woolford Jr. for generously providing strains; M. Knop for generously providing plasmids; M. Gierlach and N. Nepke from the EMBL lab kitchen for their help making media and pouring screening plates; N. Gabrielli for providing nitrogen stress media; R. Gathungu and P. Phapale from the EMBL Metabolomics Core Facility; and K. Roy and J. Villen for critical reading of the manuscript. This study was funded by EMBL core funding and a Starting Grant Award from the European Research Council (ERC-2014-STG 638884 PhosFunc PB). L.M.S, S.C.V. and S.T.C. were supported by a European Research Council Advanced Investigator Grant under the European Union’s Horizon 2020 Research and Innovation Programme (AdG-742804-SystGeneEdit). S.C.V. was supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation (grant no. P300PA_177909). V.G.P. is supported by grants from the Swiss National Science Foundation, NCCR RNA & Disease, the Novartis Foundation and Olga Mayenfisch Stiftung. M.O.-O. was supported by a Boehringer Ingelheim Fonds PhD fellowship. K.-M.N. was supported by the DFG fund (SPP 1738) and Fondazione Cariplo (2018-0525 DEPRECAD).

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Authors

Contributions

C.V. and B.P.B. constructed the phospho-mutant library and performed the chemical genomics screen and spots assays, with assistance from A.J. and M.S. D.O., M.G. and D.M. performed computational analysis. C.V. and A.M. performed the 2D-TPP experiments, with assistance from C.M.P. F.S. analyzed the proteomics data. B.P.B. performed the lipidomics experiments. U.Y. and M.T. generated the human phospho-mutant cell lines. C.V., U.Y. and M.T. performed viability assays on human cell line clones. A.G.G. and M.O.-O. generated the humanized phospho-mutant yeast strains and performed the fluorescence in situ hybridization experiments. L.S., V.G.P., K.-M.N., M.M.S., A.T. and P.B. supervised the project. C.S.T. and S.V. performed genome sequencing. P.B. and C.V. wrote the manuscript, with assistance from all authors.

Corresponding authors

Correspondence to Mikhail M. Savitski, Athanasios Typas or Pedro Beltrao.

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The authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Stephen Tenzer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10

Reporting Summary

Supplementary Data 1

List of phosphorylation sites selected for mutation

Supplementary Data 2

Conditions used for growth measurments

Supplementary Data 3

Growth measurements for mutants in each condition reported as S scores and q values

Supplementary Data 4

Growth measurements for replicates

Supplementary Data 5

Spot assay growth measurements

Supplementary Data 6

Characterization of phospho-mutants, including degree of conservation, number of replicates and correlation with corresponding gene deletion

Supplementary Data 7

Predicted functional annotation for mutated phosphosite positions

Supplementary Data 8

Sequencing quality control information

Supplementary Data 9

Thermal proteome profile dataset

Supplementary Data 10

Lipidomics dataset on negative mode

Supplementary Data 11

Lipidomics dataset on positive mode

Supplementary Data 12

Lipidomics ID confirmations

Supplementary Data 13

GO enrichment for thermal proteome profile results

Supplementary Data 14

Lipid changes in VMA2 and ELO2 mutants

Supplementary Data 15

Annotations for human phosphosites at orthologous positions to the yeast phospho-mutants

Supplementary Data 16

Sample mapping for thermal proteome profiling dataset

Supplementary Data 17

List of plasmids used

Supplementary Data 18

List of generated humanized yeast strains

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Viéitez, C., Busby, B.P., Ochoa, D. et al. High-throughput functional characterization of protein phosphorylation sites in yeast. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-01051-x

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