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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.

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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 (PADH1) or the CYC1 (PCYC1) 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.
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f3: 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 (PADH1) or the CYC1 (PCYC1) 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.

Mentions: To test whether TIPI is able to deplete S. cerevisiae proteins sufficiently to cause a phenotype similar to the corresponding gene-deletion, we fused the GFP–TDegF-tag to several soluble (nuclear and cytoplasmic) and membrane proteins, which are all essential for vegetative growth of S. cerevisiae. The amino terminus of all chosen proteins is either exposed to the cytoplasm or the nucleoplasm. The GFP–TDegX–tagged fusion proteins revealed localizations that were comparable to the corresponding C-terminally GFP-tagged proteins (Huh et al, 2003) (Supplementary Figure 2A). Expression of pTEV led to the cleavage of the fusion proteins (Supplementary Figure 2B) and to the inhibition of cell growth, which was either completely abolished (in 6/8 tested proteins) or reduced (Cdc15p and Nud1p) (Figure 3A). Growth was rescued by a UBR1 deletion or by using GFP–TDegM that contains a stabilizing amino acid (Figure 3A and data not shown). Importantly, expression of pTEV or pTEV+ protease alone did not affect growth of yeast cells (Figure 3A and B). Strong production of the target protein GFP–TDeg–Cdc14p using the ADH1 promoter (Janke et al, 2004) required the presence of the more active pTEV+ protease to result in a growth phenotype (Figure 3B). The use of the pTEV protease was sufficient to abrogate Cdc14 function, if the target protein was expressed from the weaker CYC1 promoter (Janke et al, 2004) (Figure 3A). Surprisingly, TIPI of the integral membrane proteins GFP–TDegF–Sec12p, GFP–TDegF–Pma1p, and GFP–TDegF–Alr1p resulted in non-viable cells. This indicates that these proteins are accessible to the degradation machinery, which may be the case prior or during their insertion into the membrane, or at their final localization. We analyzed whether pTEV is able to cut efficiently near the plasma membrane and found complete cutting within 3–4 h after induction of pTEV expression (Supplementary Figure 3). Up to now, there is no report of membrane proteins being degraded through the N-end rule pathway. It may be that degradation of these proteins is assisted by other ubiquitylation triggered degradation pathways, for example, through endocytosis and vacuolar degradation (Hicke, 1997; Hicke and Dunn, 2003).


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 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 (PADH1) or the CYC1 (PCYC1) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: 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 (PADH1) or the CYC1 (PCYC1) 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.
Mentions: To test whether TIPI is able to deplete S. cerevisiae proteins sufficiently to cause a phenotype similar to the corresponding gene-deletion, we fused the GFP–TDegF-tag to several soluble (nuclear and cytoplasmic) and membrane proteins, which are all essential for vegetative growth of S. cerevisiae. The amino terminus of all chosen proteins is either exposed to the cytoplasm or the nucleoplasm. The GFP–TDegX–tagged fusion proteins revealed localizations that were comparable to the corresponding C-terminally GFP-tagged proteins (Huh et al, 2003) (Supplementary Figure 2A). Expression of pTEV led to the cleavage of the fusion proteins (Supplementary Figure 2B) and to the inhibition of cell growth, which was either completely abolished (in 6/8 tested proteins) or reduced (Cdc15p and Nud1p) (Figure 3A). Growth was rescued by a UBR1 deletion or by using GFP–TDegM that contains a stabilizing amino acid (Figure 3A and data not shown). Importantly, expression of pTEV or pTEV+ protease alone did not affect growth of yeast cells (Figure 3A and B). Strong production of the target protein GFP–TDeg–Cdc14p using the ADH1 promoter (Janke et al, 2004) required the presence of the more active pTEV+ protease to result in a growth phenotype (Figure 3B). The use of the pTEV protease was sufficient to abrogate Cdc14 function, if the target protein was expressed from the weaker CYC1 promoter (Janke et al, 2004) (Figure 3A). Surprisingly, TIPI of the integral membrane proteins GFP–TDegF–Sec12p, GFP–TDegF–Pma1p, and GFP–TDegF–Alr1p resulted in non-viable cells. This indicates that these proteins are accessible to the degradation machinery, which may be the case prior or during their insertion into the membrane, or at their final localization. We analyzed whether pTEV is able to cut efficiently near the plasma membrane and found complete cutting within 3–4 h after induction of pTEV expression (Supplementary Figure 3). Up to now, there is no report of membrane proteins being degraded through the N-end rule pathway. It may be that degradation of these proteins is assisted by other ubiquitylation triggered degradation pathways, for example, through endocytosis and vacuolar degradation (Hicke, 1997; Hicke and Dunn, 2003).

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