Coupling auxin‐inducible degradation with quantitative proteomics for in‐vivoprotein functional characterization
Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting fro...
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Veröffentlicht in: | The FASEB journal 2022-05, Vol.36, p.n/a |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long‐term changes to the target protein expression/function. In contrast, the auxin‐inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long‐term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well‐suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10‐15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long‐term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms. |
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ISSN: | 0892-6638 1530-6860 |
DOI: | 10.1096/fasebj.2022.36.S1.R2524 |