Figure 2

Control of protein abundance using the bidirectional degron. (A) The conditional C-degron included in cODC1-TDegF induces rapid protein depletion. The plasmid encoded constructs GFP-TDegF-RFP, GFP-cODC1-TDegF-RFP, and GFP-cODC2-TDegF-RFP were expressed in yeast cells (YCT1169) using the constitutive ADH1 promoter. Cells without a construct (lane C) served as control of antibody specificity. Expression of the pTEV⁺ protease was induced by addition of galactose (2% final concentration). Samples of logarithmically growing yeast cells were taken at the indicated time points 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. (B) Observation of Cap2 depletion by live-cell imaging. CAP2-GFP-cODC1-TDegF-RFP or CAP2-GFP-cODC2-TDegF-RFP (chromosomally encoded) was expressed in strains containing or lacking the gene encoding for the pTEV⁺ protease. Images (maximum intensity projections shown) were recorded before and after 4 hours of pTEV⁺ protease production. Bar size, 2.5 μm. (C) Translation shut-off experiment to measure the destabilizing properties of the dormant degrons in GFP-TDegF-RFP, GFP-cODC1-TDegF-RFP, GFP-cODC2-TDegF-RFP, and GFP-cODC1-TDegF (plasmid encoded). The same conditions were used as described in A. Galactose and the translation elongation inhibitor cycloheximide were added at time point 0 hours. (D) Proteasomal activity is necessary for protein depletion mediated by the bidirectional degron. Plasmid encoded CFP-cODC1-TDegF-RFP was expressed in wild type and proteasomal mutant cells. Expression of the pTEV⁺ protease (plasmid encoded) was induced by the addition of galactose. Cells were kept at 30°C during the experiment (semi-permissive conditions for the proteasomal mutants). Samples were prepared as described in A. (E) Destabilizing activity of the conditional C-degron in cODC1-TDegF depends on the cysteine-alanine motif. The plasmid encoded constructs GFP-cODC1-TDegF-RFP and GFP-cODC1^C243A-TDegF-RFP were expressed in yeast cells using the constitutive ADH1 promoter. Experimental procedure as described in A.