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

Taxis C, Stier G, Spadaccini R, Knop M - Mol. Syst. Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: EMBL, Cell Biology and Biophysics Unit, Meyerhofstr. 1, Heidelberg, Germany.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2683728&req=5

f2: 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.

Mentions: We used Saccharomyces cerevisiae as a model organism to develop and test the TIPI system. As a target protein, we used the non-essential, soluble, and freely diffusible protein Don1p (Maeder et al, 2007). Don1p is a protein with a role only in yeast sporulation, and it is absent in vegetatively growing cells (Knop and Strasser, 2000). We monitored the processing and degradation of the GFP–TDegX–Don1p fusion proteins as a function of pTEV expression (driven by the inducible GAL1-promoter) using western blotting and antibodies specific for GFP or Don1p. The amino acid at position X of the GFP–TDegX-tag is predicted to influences both, the cleavage efficiency of pTEV and the half-life of the target protein. We found that X=Phe (F; GFP–TDegF) and X=Asp (D; GFP–TDegD) provide optimal combinations of both, excellent cleavage followed by rapid protein degradation resulting in very low Don1p protein amounts upon pTEV expression (Figure 2A–C). Degradation is dependent on the E3 protein, which is encoded by the ubiquitin-protein ligase gene UBR1 (Figure 2A), indicating proteasomal degradation by the N-end rule pathway (Bartel et al, 1990). Furthermore, repression of pTEV expression rapidly restores protein levels of the target protein (Figure 2B). The TEV protease cleaved target protein is not degraded in strains lacking Ubr1p or if the TDegM-tag is fused to the target protein (Figure 2A and B). This excludes that addition of the TDegF-tag or expression of the TEV protease caused side effects that act on target protein production. The use of different residues at position X enables specific modulation of the cleavage efficiency (e.g. TDegK) and the degradation rate (e.g. TDegH) (Figure 2C). In summary, TIPI is a new method suitable for the precise post-translational regulation of protein abundance.


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

Taxis C, Stier G, Spadaccini R, Knop M - Mol. Syst. Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683728&req=5

f2: 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.
Mentions: We used Saccharomyces cerevisiae as a model organism to develop and test the TIPI system. As a target protein, we used the non-essential, soluble, and freely diffusible protein Don1p (Maeder et al, 2007). Don1p is a protein with a role only in yeast sporulation, and it is absent in vegetatively growing cells (Knop and Strasser, 2000). We monitored the processing and degradation of the GFP–TDegX–Don1p fusion proteins as a function of pTEV expression (driven by the inducible GAL1-promoter) using western blotting and antibodies specific for GFP or Don1p. The amino acid at position X of the GFP–TDegX-tag is predicted to influences both, the cleavage efficiency of pTEV and the half-life of the target protein. We found that X=Phe (F; GFP–TDegF) and X=Asp (D; GFP–TDegD) provide optimal combinations of both, excellent cleavage followed by rapid protein degradation resulting in very low Don1p protein amounts upon pTEV expression (Figure 2A–C). Degradation is dependent on the E3 protein, which is encoded by the ubiquitin-protein ligase gene UBR1 (Figure 2A), indicating proteasomal degradation by the N-end rule pathway (Bartel et al, 1990). Furthermore, repression of pTEV expression rapidly restores protein levels of the target protein (Figure 2B). The TEV protease cleaved target protein is not degraded in strains lacking Ubr1p or if the TDegM-tag is fused to the target protein (Figure 2A and B). This excludes that addition of the TDegF-tag or expression of the TEV protease caused side effects that act on target protein production. The use of different residues at position X enables specific modulation of the cleavage efficiency (e.g. TDegK) and the degradation rate (e.g. TDegH) (Figure 2C). In summary, TIPI is a new method suitable for the precise post-translational regulation of protein abundance.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: EMBL, Cell Biology and Biophysics Unit, Meyerhofstr. 1, Heidelberg, Germany.

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.

Show MeSH
Related in: MedlinePlus