4.2 Article

A small molecule-directed approach to control protein localization and function

期刊

YEAST
卷 25, 期 8, 页码 577-594

出版社

JOHN WILEY & SONS LTD
DOI: 10.1002/yea.1610

关键词

yeast; chemical inducers of dimerization; chemical biology; rapamycin; subcellular localization; bifunctional molecules

资金

  1. American Cancer Society [RSG-06-179-01-MBC]
  2. National Science Foundation [DBI 0543017]
  3. Basil O'Connor Award [5-FY05-1224]

向作者/读者索取更多资源

Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min post-treatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale. Copyright (c) 2008 John Wiley & Sons, Ltd.

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