Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

Abstract

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.

Figure 1

Overview of this approach using chemical-induced dimerization to modulate the nuclear import and/or export of target proteins in yeast. (a) Schematic representation of the components in the conditional mislocalization system. For simplicity, only drug-directed nuclear import is shown here. Genetic modifications necessary to adapt this procedure for yeast are indicated in the inset box. The underlying mutation in TOR1-1 is indicated with a star in the diagram; chromosomal coordinates of the TOR1 gene are shown. Replacement of the FPR1 gene with the kanMX6 cassette is also indicated; the chromosomal sequence (ten nucleotides) immediately upstream of the kanMX replacement is shown. W, Watson strand; C, Crick strand. (b) Scheme for the synthesis of the rapamycin analog, C20-methallylrapamycin (C20-MaRap).

Figure 2

Workflow for application of the drug-directed nuclear trafficking system in yeast. Numbers correspond to the steps in the protocol as presented here. Steps 1–9 are used to generate a strain of yeast suitable for this analysis. Steps 10–14 enable identification of the optimal dosage of rapamycin or C20-MaRap to be used; rapamycin is shown in the upper right box, with the face of the molecule that interacts with FRB domain shown in blue. Steps 15–21 detail the small molecule treatment, cell imaging and basic data analysis. yfg, your favorite gene.

Figure 3

Coding sequence for the FRB^PLF, FKBP12-NES and FKBP12-NLS fusions. (a) DNA sequence of the FRB^PLF domain used in our studies. Mutated sequences encoding the P, L and F residues are indicated in lower case and shaded in orange. (b) DNA and amino acid sequences of the FKBP12-NES and FKBP12-NLS fusions are shown. As indicated, the NES is derived from the HIV-1 REV protein, and the NLS is derived from the SV40 large T-antigen. The HIV-1 REV NES is highlighted in blue. The SV40 NLS sequence is highlighted in red.

Figure 4

Sample quantified results from applications of chemical-induced mislocalization. (a) shows successful and (b) shows unsuccessful examples. Bar graphs indicate percent nuclear fluorescence. FKBP12 chimeras used in each experiment are indicated above the respective bar graphs. Sky1p is a SRPK1-like kinase, and Elf1p is a transcription elongation factor. Pho4p is a phosphate-regulated transcription factor and Fun20p is a pre-mRNA splicing protein. Rap, rapamycin.

Figure 5

Sample microscope images indicating successful and unsuccessful applications of this system for drug-dependent control of nuclear protein localization in yeast. (a) Sample images of successful rapamycin-mediated nuclear import of vYFP. DAPI staining indicates the nucleus, and imaging by differential interference contrast (DIC) microscopy indicates the cell outline. Note the shift in vYFP fluorescence into the nucleus on rapamycin treatment. (b) Unsuccessful application of the rapamycin system to direct Pho4p into the nucleus under normal growth conditions. As shown, strong nuclear localization is not evident on rapamycin treatment.