Figure 3

Control of protein stability using combinations of stabilizing and destabilizing sequences. (A) Live-cell imaging of cODC1-mediated destabilization of GFP. Plasmid encoded GFP-TDegF-RFP and GFP-cODC1-TDegF-RFP were expressed constitutively under control of the ADH1 promoter in yeast cells (YCT1169). Images of the cells were taken at the indicated time points after induction of pTEV⁺ protease expression. Bar size, 5 μm. (B) Kinetics of cODC1-mediated destabilization of GFP. Fluorescence intensities of the fragments, which were generated by TEV protease cleavage, were plotted over time. Images recorded for the experiment shown in A were used for automated quantitative image analysis to measure intracellular GFP and RFP fluorescence in 1000 to 3000 cells per strain (error bars indicate the standard error of the mean). The fragment GFP-TDeg is marked by inverted triangles, F-RFP (derived from GFP-TDegF-RFP) by triangles, GFP-cODC1-TDeg by diamonds, and F-RFP (derived from GFP-cODC1-TDegF-RFP) by squares. (C) Processing of tester proteins by the pTEV⁺ protease. The plasmid encoded constructs GFP-cODC1-TDegM-RFP and GFP-TDegM-RFP were expressed in yeast cells (YCT1169) using the constitutive ADH1 promoter. Samples of logarithmically growing yeast cells were taken at the indicated time points after induction of pTEV⁺ protease expression and subjected to western blotting. For detection, anti-GFP, anti-tRFP, and anti-tub1 (loading control) antibodies were used. Positions of cleaved and uncleaved species are indicated in the figure.