Abstract
Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead. In a yeast t-degron (ubiquitin-argDHFRts)- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37°C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23°C in vivo by glucose metabolism and confirmed by immunoblot. An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.
Highlights
Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead
Rather than a temperature sensitive enzyme, which is not suited for graded levels of activity, we here report application of the td-mutant technique of Dohmer et al [4], where the temperature-inactivated degron, a ts-version of mouse-dihydrofolate reductase carrying an N-terminal arginine residue, is placed between ubiquitin and a protein of interest (POI), i.e., a Ub-td-POI gene construction, giving a td-POI protein fusion which is stable at 23°C but degraded at 37°C
Lower panels, the 37°C-pretreated mutant suspension was considerably lowered in its rate of glucose metabolism and glycerate-3-P was accumulated to high level, as in a phosphoglycerate mutase mutant [3]; the control cells not exposed to 37°C showed slightly elevated levels of this metabolite
Summary
Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead. In an earlier report we have assessed glucose metabolism in yeast gcr mutants where levels of several glycolytic enzymes are decreased [3] and a logical step was to arrange their individual decreases. The usual way to decrease an enzyme level is to change expression of the gene by promoter alteration or regulation. Rather than a temperature sensitive enzyme, which is not suited for graded levels of activity, we here report application of the td-mutant technique of Dohmer et al [4], where the temperature-inactivated degron, a ts-version of mouse-dihydrofolate reductase carrying an N-terminal arginine residue, is placed between ubiquitin and a protein of interest (POI), i.e., a Ub-td-POI gene construction, giving a td-POI protein fusion (the ubiquitin is postranslationally cleaved) which is stable at 23°C but degraded at 37°C
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