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Published December 29, 2017 | Supplemental Material + Published
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A reference-based protein degradation assay without global translation inhibitors


Although it is widely appreciated that the use of global translation inhibitors, such as cycloheximide, in protein degradation assays may result in artefacts, these inhibitors continue to be employed, owing to the absence of robust alternatives. We describe here the promoter reference technique (PRT), an assay for protein degradation with two advantageous features: a reference protein and a gene-specific inhibition of translation. In PRT assays, one measures, during a chase, the ratio of a test protein to a long-lived reference protein, a dihydrofolate reductase (DHFR). The test protein and DHFR are coexpressed, in the yeast Saccharomyces cerevisiae, on a low-copy plasmid from two identical PTDH3 promoters containing additional, previously developed DNA elements. Once transcribed, these elements form 5′-RNA aptamers that bind to the added tetracycline, which represses translation of aptamer-containing mRNAs. The selectivity of repression avoids a global inhibition of translation. This selectivity is particularly important if a component of a relevant proteolytic pathway (e.g. a specific ubiquitin ligase) is itself short-lived. We applied PRT to the Pro/N-end rule pathway, whose substrates include the short-lived Mdh2 malate dehydrogenase. Mdh2 is targeted for degradation by the Gid4 subunit of the GID ubiquitin ligase. Gid4 is also a metabolically unstable protein. Through analyses of short-lived Mdh2 as a target of short-lived Gid4, we illustrate the advantages of PRT over degradation assays that lack a reference and/or involve cycloheximide. In sum, PRT avoids the use of global translation inhibitors during a chase and also provides a "built-in" reference protein.

Additional Information

© 2017 The American Society for Biochemistry and Molecular Biology. Received August 23, 2017; Accepted November 9, 2017; First Published on November 9, 2017. Author contributions: J.-H. O., S.-J. C., and A. V. designed the experiments. J.-H. O. and S.-J. C. performed the experiments. J.-H. O., S.-J. C., and A. V. wrote the paper. All authors discussed the results and commented on the manuscript. We thank the present and former members of the Varshavsky laboratory for helpful discussions during this study. We also thank A. Melnykov, I. Printsev, and B. Wadas for their comments on the manuscript. This work was supported by National Institutes of Health Grants 1R01GM031530 and 1R01DK039520 (to A. V.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Published - J._Biol._Chem.-2017-Oh-21457-65.pdf

Supplemental Material - jbc.M117.814236-1.docx


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