Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.
Bottom Line: A protein depletion by promoter shutoff or protein destabilization is an important tool in investigation of functions of essential genes.Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.A depletion time of <40 min was sufficient to achieve a robust phenotype.
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.Show MeSH
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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.
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.