Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase

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.Modeling of metabolism – the matching of enzyme and physiology – is a familiar theme in biochemistry and can be an exacting exercise even for the long studied glycolytic pathway [1,2]. A contribution of genetics would be to supply strains with altered levels of a particular enzyme as desired. Knock-outs are inappropriate for quantitative study of the reaction itself, and although increases are readily got with cloned genes on multicopy vectors, with many enzymes in apparent excess lowered levels are of more interest. 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 next 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. Apart from the practicalities, such methods have the limitation that since the adjustment is a process that must occur during growth, compensating enzymes, known or not, can also change in level. So for some purposes conditional activity is preferred over conditional expression. 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 carr