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Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

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Comparison of different depletion systems. (A) Schematic representation of the depletion cassettes used. The promoter is indicated by a broken arrow; DAA, destabilizing amino acid; tc, tetracycline aptamer. (B) Growth of strains with the standard TetO7 promoter and the new TetO7-Ubi-Leu cassette during depletion. Doxycycline or ethanol (solvent) was added to exponentially growing cultures to a final concentration of 2 μg/ml, and optical density was measured at the indicated time points. (C) Reversibility of the depletion in the TetO7-Ubi-Leu-Has1 strain. Doxycycline or ethanol (solvent) was added to exponentially growing yeast to a final concentration of 2 μg/ml at time 0. After 8 h, the doxycycline/ethanol was washed away, and the cultures were monitored until the exponential growth was restored. (D) Viability of cells after 8 h of depletion. Two hundred cells from the TetO7-Ubi-Leu-Has1 strain were grown for 8 h with or without doxycycline and then plated on YPD agar; growing colonies were counted after 2 d. For all experiments, a mean of three independent biological replicas is shown; error bars represent SDs. The y-axes in B and C are in logarithmic scale.
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Figure 1: Comparison of different depletion systems. (A) Schematic representation of the depletion cassettes used. The promoter is indicated by a broken arrow; DAA, destabilizing amino acid; tc, tetracycline aptamer. (B) Growth of strains with the standard TetO7 promoter and the new TetO7-Ubi-Leu cassette during depletion. Doxycycline or ethanol (solvent) was added to exponentially growing cultures to a final concentration of 2 μg/ml, and optical density was measured at the indicated time points. (C) Reversibility of the depletion in the TetO7-Ubi-Leu-Has1 strain. Doxycycline or ethanol (solvent) was added to exponentially growing yeast to a final concentration of 2 μg/ml at time 0. After 8 h, the doxycycline/ethanol was washed away, and the cultures were monitored until the exponential growth was restored. (D) Viability of cells after 8 h of depletion. Two hundred cells from the TetO7-Ubi-Leu-Has1 strain were grown for 8 h with or without doxycycline and then plated on YPD agar; growing colonies were counted after 2 d. For all experiments, a mean of three independent biological replicas is shown; error bars represent SDs. The y-axes in B and C are in logarithmic scale.

Mentions: To constitutively destabilize the target protein, we modified the existing TetO7-3HA cassette on a plasmid pMK140 (Alexander et al., 2010) by insertion of the ubiquitin-coding sequence followed by a codon for a destabilizing amino acid (DAA) upstream of the 3× hemagglutinin (HA; 3HA) tag (Figure 1A). The resulting depletion cassette can be integrated in the yeast genome, in frame with the open reading frame of interest. The expression of the resulting fusion protein ubiquitin-DAA-3×HA-proteinX is under the control of the TetO7 promoter. We included the 3×HA tag in the cassettes to allow simple detection of the targeted protein. Although we have not yet observed any adverse effects of the widely used 3×HA tag on the functionality of the several dozen proteins we have studied, an N-terminal tag can negatively affect the function of some fusion proteins. In this case, the integration cassettes can also be amplified using a reverse primer that base pairs upstream of the 3×HA tag and thus fuses only the DAA to the protein of interest. All the experiments were performed in the strain YMK118, in which TetO7 promoter is repressed by doxycycline added to the media (Alexander et al., 2010). Three constructs were created, with leucine, isoleucine, or alanine as DAA to allow titration of the fusion proteins' expression levels and of the rate of depletion. According to the N-end rule, proteins with leucine and isoleucine residues at their N-ends have expected half-lives of 10 and 30 min, respectively. Proteins with alanine as their N-terminal amino acid are considered stable (Bachmair et al., 1986).


Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Comparison of different depletion systems. (A) Schematic representation of the depletion cassettes used. The promoter is indicated by a broken arrow; DAA, destabilizing amino acid; tc, tetracycline aptamer. (B) Growth of strains with the standard TetO7 promoter and the new TetO7-Ubi-Leu cassette during depletion. Doxycycline or ethanol (solvent) was added to exponentially growing cultures to a final concentration of 2 μg/ml, and optical density was measured at the indicated time points. (C) Reversibility of the depletion in the TetO7-Ubi-Leu-Has1 strain. Doxycycline or ethanol (solvent) was added to exponentially growing yeast to a final concentration of 2 μg/ml at time 0. After 8 h, the doxycycline/ethanol was washed away, and the cultures were monitored until the exponential growth was restored. (D) Viability of cells after 8 h of depletion. Two hundred cells from the TetO7-Ubi-Leu-Has1 strain were grown for 8 h with or without doxycycline and then plated on YPD agar; growing colonies were counted after 2 d. For all experiments, a mean of three independent biological replicas is shown; error bars represent SDs. The y-axes in B and C are in logarithmic scale.
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Related In: Results  -  Collection

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Figure 1: Comparison of different depletion systems. (A) Schematic representation of the depletion cassettes used. The promoter is indicated by a broken arrow; DAA, destabilizing amino acid; tc, tetracycline aptamer. (B) Growth of strains with the standard TetO7 promoter and the new TetO7-Ubi-Leu cassette during depletion. Doxycycline or ethanol (solvent) was added to exponentially growing cultures to a final concentration of 2 μg/ml, and optical density was measured at the indicated time points. (C) Reversibility of the depletion in the TetO7-Ubi-Leu-Has1 strain. Doxycycline or ethanol (solvent) was added to exponentially growing yeast to a final concentration of 2 μg/ml at time 0. After 8 h, the doxycycline/ethanol was washed away, and the cultures were monitored until the exponential growth was restored. (D) Viability of cells after 8 h of depletion. Two hundred cells from the TetO7-Ubi-Leu-Has1 strain were grown for 8 h with or without doxycycline and then plated on YPD agar; growing colonies were counted after 2 d. For all experiments, a mean of three independent biological replicas is shown; error bars represent SDs. The y-axes in B and C are in logarithmic scale.
Mentions: To constitutively destabilize the target protein, we modified the existing TetO7-3HA cassette on a plasmid pMK140 (Alexander et al., 2010) by insertion of the ubiquitin-coding sequence followed by a codon for a destabilizing amino acid (DAA) upstream of the 3× hemagglutinin (HA; 3HA) tag (Figure 1A). The resulting depletion cassette can be integrated in the yeast genome, in frame with the open reading frame of interest. The expression of the resulting fusion protein ubiquitin-DAA-3×HA-proteinX is under the control of the TetO7 promoter. We included the 3×HA tag in the cassettes to allow simple detection of the targeted protein. Although we have not yet observed any adverse effects of the widely used 3×HA tag on the functionality of the several dozen proteins we have studied, an N-terminal tag can negatively affect the function of some fusion proteins. In this case, the integration cassettes can also be amplified using a reverse primer that base pairs upstream of the 3×HA tag and thus fuses only the DAA to the protein of interest. All the experiments were performed in the strain YMK118, in which TetO7 promoter is repressed by doxycycline added to the media (Alexander et al., 2010). Three constructs were created, with leucine, isoleucine, or alanine as DAA to allow titration of the fusion proteins' expression levels and of the rate of depletion. According to the N-end rule, proteins with leucine and isoleucine residues at their N-ends have expected half-lives of 10 and 30 min, respectively. Proteins with alanine as their N-terminal amino acid are considered stable (Bachmair et al., 1986).

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH
Related in: MedlinePlus