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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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Related in: MedlinePlus

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 SF3B155381−424. 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.
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f1: 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 SF3B155381−424. 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.

Mentions: To create an N-degron that is activated only upon the conditional expression of a specific activator, we developed a degron that is protected at its N-terminus by an attached peptide that can be removed by proteolysis using the site-specific TEV protease (Parks et al, 1994). The TEV protease has been used in vivo in many different organisms (bacteria, yeast cells, drosophila, and mammalian cell culture) without negative side effects (Uhlmann et al, 2000; Kapust et al, 2002; Wehr et al, 2006; Pauli et al, 2008). Initially, we generated a fusion of a seven amino acid long TEV protease recognition site to the N terminus of an earlier developed N-degron (Suzuki and Varshavsky, 1999). The TEV protease cleaves between positions 6 and 7 of the recognition site. The enzymatic activity of TEV is somewhat flexible towards changes in the sequence, especially at position 7 (Kapust et al, 2002), which becomes the new N-terminal amino acid (in the following termed residue X) after proteolytic cleavage. Destabilizing amino-acid residues at the amino terminus (position X) target a protein for rapid destruction if the N-degron contains a sequence that allows the attachment of ubiquitin (Varshavsky, 1997). To monitor the cleavage, we fused a fluorescent protein to the N terminus of the TEV protease recognition site. To improve the processivity of the TEV protease, we enhanced the binding of the TEV protease to its substrate. We fused the N-degron construct with the TEV protease recognition site to the SF3b155381−424 protein domain. This domain is specifically recognized by the human spliceosome subunit p14 (Spadaccini et al, 2006), which we in turn fused to the TEV protease (named p14–TEV). Furthermore, we identified, by chance, a mutated allele of p14 (called p14*), which enhanced cleavage significantly. In summary, we have constructed a dormant N-degron that is constituted of a reporter, followed by a TEV protease recognition site (including residue X), an N-degron and SF3b155381−424 (in the following termed Reporter–TDegX-tag, e.g. GFP–TDegF-tag). This dormant N-degron can be deprotected by the expression of the p14*–TEV fusion protein (pTEV). An overview of the TEV protease induced protein inactivation (TIPI) system is shown in Figure 1. The mechanism underlying the enhanced activity of the p14*–TEV fusion versus p14–TEV is not clear, as the responsible mutation lies within a stretch of amino acids in p14 that is not involved in binding of p14 to SF3b155381−424 (data not shown; Schellenberg et al, 2006; Spadaccini et al, 2006). For details on the development of the TIPI system, see Supplementary information.


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

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

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 SF3B155381−424. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: 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 SF3B155381−424. 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.
Mentions: To create an N-degron that is activated only upon the conditional expression of a specific activator, we developed a degron that is protected at its N-terminus by an attached peptide that can be removed by proteolysis using the site-specific TEV protease (Parks et al, 1994). The TEV protease has been used in vivo in many different organisms (bacteria, yeast cells, drosophila, and mammalian cell culture) without negative side effects (Uhlmann et al, 2000; Kapust et al, 2002; Wehr et al, 2006; Pauli et al, 2008). Initially, we generated a fusion of a seven amino acid long TEV protease recognition site to the N terminus of an earlier developed N-degron (Suzuki and Varshavsky, 1999). The TEV protease cleaves between positions 6 and 7 of the recognition site. The enzymatic activity of TEV is somewhat flexible towards changes in the sequence, especially at position 7 (Kapust et al, 2002), which becomes the new N-terminal amino acid (in the following termed residue X) after proteolytic cleavage. Destabilizing amino-acid residues at the amino terminus (position X) target a protein for rapid destruction if the N-degron contains a sequence that allows the attachment of ubiquitin (Varshavsky, 1997). To monitor the cleavage, we fused a fluorescent protein to the N terminus of the TEV protease recognition site. To improve the processivity of the TEV protease, we enhanced the binding of the TEV protease to its substrate. We fused the N-degron construct with the TEV protease recognition site to the SF3b155381−424 protein domain. This domain is specifically recognized by the human spliceosome subunit p14 (Spadaccini et al, 2006), which we in turn fused to the TEV protease (named p14–TEV). Furthermore, we identified, by chance, a mutated allele of p14 (called p14*), which enhanced cleavage significantly. In summary, we have constructed a dormant N-degron that is constituted of a reporter, followed by a TEV protease recognition site (including residue X), an N-degron and SF3b155381−424 (in the following termed Reporter–TDegX-tag, e.g. GFP–TDegF-tag). This dormant N-degron can be deprotected by the expression of the p14*–TEV fusion protein (pTEV). An overview of the TEV protease induced protein inactivation (TIPI) system is shown in Figure 1. The mechanism underlying the enhanced activity of the p14*–TEV fusion versus p14–TEV is not clear, as the responsible mutation lies within a stretch of amino acids in p14 that is not involved in binding of p14 to SF3b155381−424 (data not shown; Schellenberg et al, 2006; Spadaccini et al, 2006). For details on the development of the TIPI system, see Supplementary information.

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