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


RESULTS
In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot.


CONCLUSIONS
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.
Heidrich
Katja
K
Dept, of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston MA 02115, USA. kheid@server3.medizin.uni-leipzig.de
Fraenkel
Dan G
DG
D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D009154:Mutation; D010736:Phosphoglycerate Mutase / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics
D005656:Fungal Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D003513:Cycloheximide; D013762:Tetrahydrofolate Dehydrogenase; D010736:Phosphoglycerate Mutase; D005947:Glucose