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 posttreatment 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.

Recovery from Rapamycin

“…We adapted a quantitative assay for rapamycin using mass spectrometry to detect the cell-associated pool of the drug in yeast cells (see under "Experimental Procedures") (31). This assay detects the parent ion of rapamycin and thus only chemically unmodified drug.…”

Conditional Nuclear Import and Export of Yeast Proteins Using a Chemical Inducer of Dimerization

“…Using these reagents, we showed that vYFP-FRB could be conditionally imported or exported from the nucleus and that the kinetics is rapid, achieving completion within 10-15 min [4]. Importantly, we also generated a strain that is resistant to rapamycin, which permits use of this inexpensive, commercially available CID at high concentrations (at least 10 lM).…”

“…Inspired by the reversibility of natural trafficking pathways, we recently developed a synthetic way to control protein position in the budding yeast, Saccharomyces cerevisae [4]. This method was adapted from work in mammalian models [5][6][7] and pioneering efforts by the Crabtree and Schreiber groups on chemical inducers of dimerization (CIDs; reviewed in [8][9][10]).…”

“…To adapt the CID-based import/export components for use in yeast, we generated Gateway cassettes [15] for the rapid creation of FRB and FKBP fusions; the FRB vector allows installation of the Venus variant of yellow fluorescent protein (vYFP) and FRB to the C-terminus of the gene-of-interest, while the FKBP vector can be used to produce FKBP-NES and FKBP-NLS chimeras [4]. Using these reagents, we showed that vYFP-FRB could be conditionally imported or exported from the nucleus and that the kinetics is rapid, achieving completion within 10-15 min [4].…”

Working with Proteins in silico: A Review of Online Available Tools for Basic Identification of Proteins

“…Determination of the subcellular localization of a protein is important to correctly define its function, especially for receptor proteins that reside in membrane systems (Geda et al, 2008). Experimental prediction of a subcellular localization of the protein of interest requires labeling with a dye, mostly EGFPs (Green Fluorescent Protein), and approaches like transient expression in a model plant system (Llopis et al, 1998).…”