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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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Western blot analysis of Has1p levels in strains with different depletion cassettes. Protein samples from strains were collected at the indicated time points following the addition of doxycycline. The proteins were detected using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is plotted on the right; a mean of two independent experiments is shown. The following strains were used: (A) TetO7-Has1 and TetO7-Ubi-Leu-Has1, (B) TetO7-Ubi-Ile-Has1 and TetO7-Ubi-Ala-Has1, and (C) TetO7-tc-Ubi-Leu-Has1 with added tetracycline aptamer.
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Figure 2: Western blot analysis of Has1p levels in strains with different depletion cassettes. Protein samples from strains were collected at the indicated time points following the addition of doxycycline. The proteins were detected using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is plotted on the right; a mean of two independent experiments is shown. The following strains were used: (A) TetO7-Has1 and TetO7-Ubi-Leu-Has1, (B) TetO7-Ubi-Ile-Has1 and TetO7-Ubi-Ala-Has1, and (C) TetO7-tc-Ubi-Leu-Has1 with added tetracycline aptamer.

Mentions: The rates of Has1p protein depletion in TetO7 and TetO7-Ubi-Leu strains were analyzed by Western blotting (Figure 2A). We observed a rapid drop of Has1p levels to below the 20% level within 2 h of depletion in the TetO7-Ubi-Leu, while ∼75% of the protein remained in the TetO7 strain at the same time point, in agreement with the observed differences in the growth rates of the strains. Next we compared the Has1p depletion using the TetO7-Ubi-DAA with leucine, isoleucine, and alanine as destabilizing residues. The Has1p protein was depleted fastest in the TetO7-Ubi-Leu strain and with an intermediate rate in the TetO7-Ubi-Ile strain. In the TetO7-Ubi-Ala strain, which is expected to produce a stable protein, the depletion rate was very similar to the standard TetO7 strain (Figure 2B). Interestingly, the Has1p protein depletion did not show a simple exponential behavior as would be expected in an ideal case. An initial rapid drop in the Has1p levels was followed by a much slower reduction as the strain growth rate reduces. Furthermore, the Leu-Has1p half-life was ∼60 min, considerably longer than the 10 min predicted by the N-end rule. As a component of large multiprotein complexes, Has1p might not be readily accessible to proteasomes. Therefore only the accessible pool of Has1p is degraded rapidly and the depletion of the remaining Has1p, present in complexes, is much slower and follows duplication of the cells.


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

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

Western blot analysis of Has1p levels in strains with different depletion cassettes. Protein samples from strains were collected at the indicated time points following the addition of doxycycline. The proteins were detected using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is plotted on the right; a mean of two independent experiments is shown. The following strains were used: (A) TetO7-Has1 and TetO7-Ubi-Leu-Has1, (B) TetO7-Ubi-Ile-Has1 and TetO7-Ubi-Ala-Has1, and (C) TetO7-tc-Ubi-Leu-Has1 with added tetracycline aptamer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325845&req=5

Figure 2: Western blot analysis of Has1p levels in strains with different depletion cassettes. Protein samples from strains were collected at the indicated time points following the addition of doxycycline. The proteins were detected using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is plotted on the right; a mean of two independent experiments is shown. The following strains were used: (A) TetO7-Has1 and TetO7-Ubi-Leu-Has1, (B) TetO7-Ubi-Ile-Has1 and TetO7-Ubi-Ala-Has1, and (C) TetO7-tc-Ubi-Leu-Has1 with added tetracycline aptamer.
Mentions: The rates of Has1p protein depletion in TetO7 and TetO7-Ubi-Leu strains were analyzed by Western blotting (Figure 2A). We observed a rapid drop of Has1p levels to below the 20% level within 2 h of depletion in the TetO7-Ubi-Leu, while ∼75% of the protein remained in the TetO7 strain at the same time point, in agreement with the observed differences in the growth rates of the strains. Next we compared the Has1p depletion using the TetO7-Ubi-DAA with leucine, isoleucine, and alanine as destabilizing residues. The Has1p protein was depleted fastest in the TetO7-Ubi-Leu strain and with an intermediate rate in the TetO7-Ubi-Ile strain. In the TetO7-Ubi-Ala strain, which is expected to produce a stable protein, the depletion rate was very similar to the standard TetO7 strain (Figure 2B). Interestingly, the Has1p protein depletion did not show a simple exponential behavior as would be expected in an ideal case. An initial rapid drop in the Has1p levels was followed by a much slower reduction as the strain growth rate reduces. Furthermore, the Leu-Has1p half-life was ∼60 min, considerably longer than the 10 min predicted by the N-end rule. As a component of large multiprotein complexes, Has1p might not be readily accessible to proteasomes. Therefore only the accessible pool of Has1p is degraded rapidly and the depletion of the remaining Has1p, present in complexes, is much slower and follows duplication of the cells.

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