title: A small molecule-directed approach to control protein localization and function.
abstract: 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.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2216, USA.
authors.forename: Prasanthi
authors.initials: P
authors.lastname: Geda
authors.forename: Srikanth
authors.initials: S
authors.lastname: Patury
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.forename: Nike
authors.initials: N
authors.lastname: Bharucha
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Dobry
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Lawson
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: Anuj
authors.initials: A
authors.lastname: Kumar
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006360:Heat-Shock Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D020123:Sirolimus / Q000493:pharmacokinetics / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C109868:CRZ1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D019913:Nuclear Localization Signals; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D020123:Sirolimus