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 P TDH3 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.
Highlights
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
After 18 h in ethanol medium at 30 °C, cells were shifted to a glucose medium while the synthesis of both the fDHFRha reference protein and X-Mdh23f proteins was repressed by Tc, initiating a chase (Fig. 2)
Inasmuch as the Gid[4] Pro/N-recognin of the Pro/N-end rule pathway that targets P-Mdh23f for degradation (Fig. 2 and Fig. S1C) is known to be a short-lived protein (82), we asked whether results of a promoter reference technique (PRT)-based Tc chase with P-Mdh23f would significantly differ from those of an otherwise identical PRT-based CHX chase
Summary
Some proteins can be targeted through both of these routes concurrently, depending on the nature of a protein, its degron(s), and the physiological state of a cell (31) These and other complexities of intracellular protein degradation make it important to have the means for determining the in vivo decay curves of specific proteins with high accuracy and through methods that do not perturb the rates of proteolysis that these methods are designed to measure. 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). This technique is described in detail (Figs. 1– 4), was further optimized, and was 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)
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