Figure 4

Depletion of essential yeast proteins using the GFP-cODC1-TDegF-RFP and GFP-cODC1-TDegF tag causes phenotypes close to deletion mutants. (A) The GFP-cODC1-TDegF-RFP tag was fused to the 3'-end of CDC14 and CDC48 in strains with or without the gene encoding for the pTEV+ protease. Serial dilutions (1:5) of cells were spotted on agar plates supplemented with either raffinose or galactose and raffinose and incubated at 30°C for 3 days. (B) The GFP-cODC1-TDegF tag or the GFP-cODC1-TDegF-RFP tag were fused to the 3'-end of CDC14, CDC48, CYR1, KOG1, CDC20, MCM1, and CDC5 in strains with the gene encoding for the pTEV⁺ protease. Serial dilutions (1:5) of cells were spotted on agar plates supplemented with either glucose or galactose and incubated at 30°C for 3 days. (C) Cdc14-GFP-cODC1-TDegF-RFP and Cdc48-GFP-cODC1-TDegF-RFP are depleted quickly after pTEV⁺ protease expression. The GFP-cODC1-TDegF-RFP tag was inserted at the 3'-end of CDC14 and CDC48 in yeast strains ESM356-1 and YCT1169. Maximum intensity projection images are shown. The Images were recorded at the indicated time points after induction of pTEV⁺ protease production. Bar size, 2 μm. (D) Kinetics of Cdc14-GFP-cODC1-TDegF-RFP and Cdc48-GFP-cODC1-TDegF-RFP depletion. Automated quantitative image analysis was used to measure the cellular fluorescence of the green fluorescent protein in 1000 to 3000 cells per strain (error bars represent the standard error of the mean). Images recorded for the experiment shown in C were used for quantification. (E) Depletion of Cdc48-, Mcm1-, Cdc14-, Cdc5-, and Cyr1-GFP-cODC1-TDegF leads to cell-cycle defects. Cell-cycle stages were assessed after 4 hours of pTEV⁺ protease expression. The bud size and spindle morphology were taken into account to classify the cells. Wild-type cells with and without expression of pTEV⁺ protease were used as controls. Bar size, 2 μm.