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• W2767800575 abstract "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. 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. In vivo half-lives of intracellular proteins range from less than a minute to many days (1Varshavsky A. Regulated protein degradation.Trends Biochem. Sci. 2005; 30: 283-286Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar2Inobe T. Matouschek A. 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Graciet E. Varshavsky A. Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes.PLoS One. 2013; 8: e67952Crossref PubMed Scopus (7) Google Scholar). One class of widely employed approaches, historically among the first to be introduced, are assays for in vivo protein degradation that involve the use of a global translation inhibitor to halt protein synthesis, followed by measurements of decreasing levels of a protein of interest (a “chase”) by SDS-PAGE and immunoblotting or by other means. The resulting decay curves are usually calibrated by measuring, in parallel, the levels of an abundant endogenous protein (e.g. tubulin or actin) that is presumed to be both long-lived and expressed at approximately equal levels under different physiological conditions and in varying genetic backgrounds. A shortcoming, in the context of these assays, of a global translation inhibitor such as, for example, cycloheximide (CHX), 3The abbreviations used are: CHXcycloheximideDHFRdihydrofolate reductasePRTpromoter reference techniqueTctetracyclineUbubiquitinSCsynthetic complete. is not only the inhibitor’s cytotoxicity but also the fact that a proteolytic pathway under study may itself involve a short-lived protein(s). Naturally unstable components of specific proteolytic systems continue to be identified (49Li X.S. Trojer P. Matsumura T. Treisman J.E. Tanese N. Mammalian SWI/SNF—a subunit BAF250/ARID1 is an E3 ubiquitin ligase that targets histone H2B.Mol. Cell. Biol. 2010; 30: 1673-1688Crossref PubMed Scopus (90) Google Scholar50Wu X. Yen L. Irwin L. Sweeney C. Carraway 3rd, K.L. Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8.Mol. Cell. Biol. 2004; 24: 7748-7757Crossref PubMed Scopus (129) Google Scholar, 51Galan J.M. Peter M. 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In all such cases, a global halt to the synthesis of cellular proteins would perturb the very process that a degradation assay is meant to measure, because the activity of a relevant proteolytic pathway would start to decrease as soon as its short-lived component is no longer produced. In addition and independently, it can be problematic to calibrate chase-based degradation assays by measuring the relative amounts of endogenous “marker” proteins, inasmuch as their levels often change in response to altered physiological conditions or genetic backgrounds. cycloheximide dihydrofolate reductase promoter reference technique tetracycline ubiquitin synthetic complete. Two of our 2017 studies described and employed a degradation assay termed the promoter reference technique (PRT) (54Chen S.J. Wu X. Wadas B. Oh J.-H. Varshavsky A. An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes.Science. 2017; 355: 366Crossref Scopus (89) Google Scholar, 55Oh J.H. Hyun J.Y. Varshavsky A. Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway.Proc. Natl. Acad. Sci. U.S.A. 2017; 114: E4370-E4379Crossref PubMed Scopus (36) Google Scholar). Key features of PRT are (i) a coexpression, from identical transcriptional promoters, of a test protein and a long-lived reference protein; and (ii) a gene-specific (i.e. not global) inhibition of translation during a chase (Fig. 1). In the present work, this technique is described in detail (Figs. 1–Figure 3, Figure 4), was further optimized, and was also used, in specific PRT-based degradation assays, to demonstrate a significant downside of using a global translation inhibitor, in comparison with a gene-specific inhibition of translation (Fig. 4).Figure 2PRT-based chase assays with Mdh2. A, S. cerevisiae Mdh2 is the cytosolic malate dehydrogenase and a substrate of the GID-mediated Pro/N-end rule pathway (54Chen S.J. Wu X. Wadas B. Oh J.-H. Varshavsky A. An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes.Science. 2017; 355: 366Crossref Scopus (89) Google Scholar). Lane 1, kDa markers. Tc-based chases were performed at 30 °C during the transition from ethanol to glucose medium (see “Experimental procedures”) with wild-type (lanes 2–5 and 10–13) or gid2Δ S. cerevisiae (lanes 6–9 and 14–17) expressing the fDHFRha reference and either wild-type P-Mdh23f (bearing N-terminal Pro; lanes 2–9) or S-Mdh23f (bearing N-terminal Ser; lanes 10–17). At the indicated times of a chase, proteins in cell extracts were fractionated by SDS-PAGE, followed by immunoblotting with anti-flag antibody. The bands of X-Mdh23f test proteins and the fDHFRha protein are indicated on the right. B, quantification of the data in A. The time 0 (before-chase) level of S-Mdh23f in gid2Δ cells was taken as 100%. Note the much lower time 0 level (40%) of the short-lived, wild-type P-Mdh23f in wild-type cells. Its half-life (the time it took for the level of P-Mdh23f to decrease from the initial 40% to 20%) was ∼20 min. All chases in this study were performed at least twice and yielded results that differed by <10%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Optimizing the level of tetracycline during a chase. ubr1Δ S. cerevisiae carried the previously described (55Oh J.H. Hyun J.Y. Varshavsky A. Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway.Proc. Natl. Acad. Sci. U.S.A. 2017; 114: E4370-E4379Crossref PubMed Scopus (36) Google Scholar), PRT-based plasmid pJO630 that expressed the Chk13f protein and the fDHFRha reference protein (see “Experimental procedures” and “Results and discussion” for a brief introduction of the mitotic checkpoint kinase Chk1, a physiological substrate of the Arg/N-end rule pathway). Cells were labeled for 3 min at 30 °C with 35S-EXPRESS Met/Cys at the specified times after the addition of Tc to the indicated varying final concentrations. Lanes 1–9, total 35S-labeling patterns, before immunoprecipitation. Lanes 10–18, same as lanes 1–9 but after immunoprecipitation of labeled cell extracts with anti-flag antibody (Ab). Lane 1, no addition of Tc before a 3-min pulse with 35S-EXPRESS Met/Cys. Lanes 2–6 and 10–15, Tc was added to cells to a final concentration of 0.1 mm, followed by incubation for 2, 4, 8, 15, and 20 min, respectively, before a 3-min pulse with 35S-EXPRESS Met/Cys. Lanes 7–9 and 16–18, Tc was added to cells to the final concentrations of 0.2, 0.3, and 0.5 mm, respectively, followed by incubation for 20 min and a 3-min pulse with 35S-EXPRESS Met/Cys. See “Results and discussion” for additional details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Comparing a PRT-based tetracycline chase with a cycloheximide chase. PRT-based Tc chases and CHX chases were carried out with wild-type S. cerevisiae growing in SC medium and expressing either P-Mdh23f (bearing N-terminal Pro) or S-Mdh23f (bearing N-terminal Ser). A, lane 1, kDa markers. Lanes 2–5, CHX chase, for 0, 0.5, 1, and 2 h, with P-Mdh23f. Lanes 6–9, same as lanes 2–5 but with S-Mdh23f. Lanes 10–13, same as lanes 2–5 but a Tc chase. Lanes 14–17, same as lanes 6–9, but a Tc chase. The bands of X-Mdh23f test proteins and the fDHFRha protein are indicated on the right. B, quantification of the data in A. The time 0 (before-chase) level of S-Mdh23f in the Tc chase was taken as 100%. Note the time 0, before-chase degradation of ∼85% of P-Mdh23f, relative to S-Mdh23f, and the similar time 0 levels (in contrast to subsequent levels) of P-Mdh23f in the Tc chase versus the CHX chase. Note, also, the lower rate of P-Mdh23f decrease (and the subsequent flattening of the decay curve) in the CHX chase, in comparison with the Tc chase. All chases in this study were performed at least twice and yielded results that differed by <10%. Also see “Results and discussion.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) PRT is described below through its applications to one of the N-end rule pathways (Fig. 1 and Fig. S1). These pathways are a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing the degradation of these proteins, largely by the proteasome (and also by autophagy) in eukaryotes and by the proteasome-like ClpAP protease in bacteria (4Varshavsky A. The N-end rule pathway and regulation by proteolysis.Prot. Sci. 2011; 20: 1298-1345Crossref PubMed Scopus (475) Google Scholar, 54Chen S.J. Wu X. Wadas B. Oh J.-H. Varshavsky A. An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes.Science. 2017; 355: 366Crossref Scopus (89) Google Scholar, 55Oh J.H. Hyun J.Y. 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