Efficient protein depletion by genetically controlled deprotection of a dormant N-degron

Abstract

Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.

Figure 1

TEV protease induced protein instability (TIPI). The principle of TIPI, a method to genetically control abundance of proteins with an N terminus exposed to the cytoplasm or nucleus. (A) The GFP–TDegX-tag is fused to the 5′-end of the target open reading frame (target ORF), directly in front of the ATG. The gene for pTEV expression is regulated by a controllable promoter; in this study, we used the galactose responsive GAL1-promoter in yeast. (B) Upon expression of pTEV, the pTEV protease binds to the GFP–TDegX-target protein. Binding is mediated by interaction of p14 with SF3B155^381−424. This interaction directs efficient cleavage of the GFP–TDegX-tag by the TEV protease at its consensus site (ENLYFQ-X). (C) Cutting of the GFP–TDeg-tag leads to deprotection of the dormant N-degron that is part of the GFP–TDegX-tag. The N-degron is constituted by the new N-terminal amino acid X and a sequence that promotes efficient poly-ubiquitylation by Ubr1p (Suzuki and Varshavsky, 1999). The exposed amino acid X determines the fate of the protein. In yeast, X=A, C, G, M, P, S, T, and V lead to stable proteins, whereas X = D, E, F, H, I, K, L, N, Q, R, W, and Y render proteins instable (half lives=2–30 min) (Bachmair et al, 1986). (D) The target protein is poly-ubiquitylated by Ubr1p and degraded by the proteasome.

Figure 2

TIPI mediates rapid degradation of proteins in yeast. (A) TIPI leads to rapid degradation of GFP–TDegD-tagged proteins. GFP–TDegD–DON1 was expressed chromosomally using the constitutive ADH1 promoter. Expression of pTEV or GFP–pTEV was induced by the addition of galactose (2% final concentration) to the culture. Samples of logarithmically growing yeast cells were removed from the culture at the indicated time points and subjected to western blotting. For detection of reporter constructs, anti-GFP and anti-Don1p antibodies were used. Detection of tubulin was used as a loading control. Positions of cleaved and uncleaved species are indicated in the figure. Strains used were either wild type or deleted for the gene UBR1 (ubr1Δ) as indicated. The strains we used in this experiment are described in Supplementary Table I, and their construction is indicated in Supplementary Table III. (B) Depletion of proteins by TIPI is reversible. GFP–TDegF–DON1 and GFP–TDegM–DON1 were expressed chromosomally using the constitutive ADH1 promoter. To induce pTEV expression galactose was added (at time point 0 h), repression of pTEV expression was done by adding glucose (at time point 3 h). Western blotting was performed as described in panel A. A # indicates the position of a non-specific band. (C) Modulation of protein abundance using different versions of GFP–TDegX. Protein levels of cleaved and uncleaved GFP–TDegF-, GFP–TDegM-, GFP–TDegK-, or GFP–TDegH–Don1p were assessed in crude extracts of yeast cells before and after 3 h of pTEV expression. GFP–TDegX constructs were expressed chromosomally from the ADH1-promoter. Western blotting was performed as described in Figure 2A. (D) C-terminal truncation of pTEV protease enhances proteolytic activity. Protein levels of cleaved and uncleaved GFP–TDeg–Don1p were assessed before and after 3 h of pTEV or C-terminally truncated pTEV⁺ expression. Strong overexpression of GFP–TDegD–DON1 constructs was achieved using the strong GPD-promoter. Western blotting was performed as described in panel A. (E) Protein depletion by TIPI can be followed by live cell imaging. Plasmid encoded CFP–TDegF–mKATE and CFP–TDegM–mKATE were expressed constitutively under control of the ADH1 promoter in wild-type cells and cells lacking UBR1 (ubr1Δ). Expression of pTEV⁺ (plasmid encoded) was induced by the addition of galactose (2% final concentration) to the cells. Images of the cells were taken at the indicated time points. (F) Quantification of the experiment shown in (E). Images from the cells used in (E) were recorded after induction of YFP–pTEV⁺. Automated quantitative image analysis was used to measure the cellular fluorescence of the different fluorescent protein reporters in 1000 to 3000 cells per strain (error bars represent the standard error of the mean). The yeast strains that were used to perform the experiments (A–F) are listed in Supplementary information. The genotypes are given in Supplementary Table I, the plasmids are described in Supplementary Table II.

Figure 3

TIPI of essential yeast proteins causes lethal phenotypes. (A) TIPI of essential proteins leads to impaired growth phenotypes. Serial dilutions (1:10) of yeast cultures (genotypes of yeast strains are indicated) were spotted on synthetic complete media containing either raffinose or galactose/raffinose and incubated at 30°C for 3 days. GFP–TDegF fusions were expressed either from the ADH1 (P_ADH1) or the CYC1 (P_CYC1) promoter (as indicated). (B) pTEV⁺ protease exhibits increased activity as compared with pTEV. Experimental conditions were the same as described in (A) using strains that express the indicated constructs. (C) TIPI of Cdc5p, Cdc14p or Cdc48p leads to cell-cycle defects. Cell-cycle phenotypes were assessed after 3 h of pTEV expression in GFP–TDegF–CDC5, GFP–TDegF–CDC14 and GFP–TDegF–CDC48 expressing strains. Wild-type cells with and without expression of pTEV were used as controls. Samples were fixed and cell-cycle stages assessed based on bud size, spindle morphology, and DNA segregation. (D) TIPI of Sec12p leads to impaired secretion. Samples of control cells and TDegF–Sec12p expressing cells were taken before (−) and after 3 h (+) of pTEV protease induction and subjected to western blotting. The secretory marker protein carboxypeptidase Y (CPY) was detected. mCPY, mature, vacuolar form of CPY; p1+p2CPY, ER and Golgi glycosylated forms of CPY. The yeast strains that were used to perform the experiments (A–D) are listed in Supplementary information. The genotypes of these strains are given in Supplementary Table I.