The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes  Hirohito Haruki, Junichi Nishikawa, Ulrich K. Laemmli  Molecular Cell  Volume 31, Issue 6, Pages 925-932 (September 2008) DOI: 10.1016/j.molcel.2008.07.020 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Scheme of the AA Technique: Anchor Selection (A) The anchor and target C-terminally fused to the FKBP12 and FRB domains, respectively, and the ternary complex formed upon addition of rapamycin. (B) Two main schemes were successfully applied to deplete the nucleus of target proteins. The static scheme 1 (left half of the depicted cell) uses the abundant, essential plasma membrane H+-ATPase Pma1p as an anchor. The PMA1 anchor fused to FKBP12 was found to rapidly inactivate, in a ligand-dependent manner, proteins that shuttle between the nucleus and cytoplasm, such as proteins involved in nuclear transport. This anchor, though, does not work with typical nonshuttling nuclear proteins. The mobile scheme 2 (right half of the depicted cell) uses a ribosomal protein anchor, RPL13A-FKBP12, taking advantage of the large flux of ribosomal proteins transiting the nucleus during their assembly process to the 40S and 60S particles, to deplete the nucleus of the target proteins. Scheme 2 worked for all genes tested that code for nuclear proteins. (C) Exploratory spot-test series in which the NLS-cargo import function of KAP95-FRB (importin-β), which is inhibited with the nucleopore (NIC96-FKBP12), core histone (HTB2-FKBP12), and H+-ATPases (PMA1-FKBP12) anchors. Control (−RAP) and rapamycin (+RAP) containing plates are shown. The strains tested were TD422, expressing KAP95-FRB lacking an anchor, and derivative thereof, expressing the NIC96 (HHY50), the HTB2 (HHY51), or the PMA1 anchor (HHY118). Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Rescue by Complementation and Imaging of the Anchor-Target Complex (A) Shows spot-test on control (−URA or −LEU) and rapamycin (−URA or −LEU +RAP) containing plates. Note that anchoring in the PMA1 anchor strain by addition of rapamycin results in lethality for the three transport genes, KAP95 (importin-β), SRP1 (importin-α) and MEX67. But lethality is complemented/rescued by coexpression of the endogenously FRB-tagged and of the untagged gene product from a CEN plasmid. Strains used are the parental PMA1 anchor strains HHY110, HHY118 (KAP95-FRB), HHY122 (SRP1-FRB), and HHY133 (MEX67-FRB). (B) Exponential culture of PMA1 anchor strain HHY225 expressing KAP95-FRB-GFP was treated with rapamycin and fixed at indicated time points. Fluorescence micrograph shows the rapid displacement by exposure to rapamycin for 15 min of KAP95-FRB-GFP from its normal location at the nuclear periphery (top left, −RAP) to the plasma membrane anchor PMA1-FKBP12 (top right, +RAP). The bottom row shows the nuclear DAPI signals. Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Anchoring Results in Specific Import/Export Phenotypes (A) PMA1 anchor strains HHY118 and HHY133 expressing KAP95-FRB (top row) and MEX67-FRB (bottom row), respectively, were transformed with a multicopy (2 μm) plasmid that express NLS-GFP. Left micrographs show live images of the NSL-GFP cargo dominantly localized in the nucleus due to normal import (−RAP, 0 min.). However, exposure of the cells for 3 min to rapamycin blocks import. This is manifested by a general redistribution of NLS-GFP throughout the cell (top, +RAP, 3 min.). The bottom row shows a specificity control, demonstrating that anchoring MEX67-FRB does not inhibit the normal nuclear signal of NLS-GFP as expected. (B) Poly(A)+ was visualized by in situ hybridization with an oligo(dT)50 probe in HHY133 expressing MEX67-FRB. The top row shows, as a control, the predominantly cytoplasmic poly(A)+ signal resulting from normal RNA export (−RAP, 0 min.). However, exposure of the cells for 30 min to rapamycin blocks RNA export (+RAP, 30 min.). This is manifested by the retention of the poly(A)+ signal in the nucleus. The bottom row shows the DAPI signal of the nucleus. (C) Identical to (A), but carried out with the ribosome anchor strain HHY208. (D) Immunoblot of KAP95-FRB and TBP-FRB in the PMA1 (lanes 1 and 2, HHY118) and ribosome anchor (lanes 3 and 4, HHY154) strains. The blot shows that exposure to rapamycin (+RAP) for 1 hr (1H) does not result in degradation of the targets and anchors. Top filter was blotted with the anti-FRB (α-FRB) and the bottom one with anti-FKBP12 (α-FKBP12). Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Specific Transcription Phenotype by Anchoring TBP (A) Bar graph shows the rapid inhibition of GAL1 transcription by anchoring TBP-FRB. Ribosome anchor strain HHY154 expressing TBP-FRB was exponentially cultured in YPAR. Rapamycin was added concomitantly (0) or prior to galactose at −15, −30, −60 min, as indicated. The mRNA level of GAL1 and ACT1 was measured after 60 min of galactose induction using reverse transcriptase-mediated real-time PCR. The fold inhibition by exposure to rapamycin is indicated. (B) Spot-test series of various FRB constructs in the ribosome anchor strain on control and rapamycin plates. Note that coexpression of untagged gene products results in full complementation of the lethal phenotype (normal growth on RAP plates). The plate contains glucose for strains HHY168 (parental ribosome anchor strain), HHY208 (KAP95-FRB), HHY195 (SRP1-FRB), HHY182 (MEX67-FRB), and HHY154 (TBP-FRB), while galactose is used for HHY183 (GAL4-FRB). (C) Exponential culture of ribosome anchor strain HHY209 expressing TBP-FRB-GFP was treated with rapamycin and fixed at indicated time points thereafter. GFP micrographs in the top row show the time course of the depletion of TBP-FRB-GFP from the nucleus to the cytoplasm by anchoring. Bottom row shows the DAPI signal of the nuclei. Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 The AA Technique Works for All Genes Tested (A) Spot-test and complementation results for many different genes involved in diverse functions, as indicated. Complementation was observed for all constructs by mating (Mated +RAP). The fold inhibition of GAL1 transcription following preexposure to rapamycin for 60 min is listed. Note that anchoring GAL4-FRB did not lead to lethality on rapamycin plates, since media used for the spot test contained glucose, but see Figure 4B. However, anchoring this protein resulted in the galactose media in transcription inhibition of GAl1 down to background level (see [B]). (B) Bar graph shows the inhibition of GAL1 transcription by anchoring several gene products involved in transcription. These are GAL4-FRB (HHY183), SPT20-FRB (HHY189), SRB5-FRB (HHY178), SNF2-FRB (HHY190), TBP-FRB (HHY154), and RPO21-FRB (HHY170). Rapamycin was added together with galactose, or 15, 30, and 60 min prior to galactose (0 min, indicated by a wedge). The mRNA level of GAL1 was measured after 60 min galactose induction, and the maximum value was normalized to 100%. Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Examples of Specific Phenotypes (A) Spot test of cells containing a URA3 reporter inserted at silent mating-type locus HML on plates containing rapamycin alone (+RAP) or this ligand and 5′-fluoroorotic acid (+FOA, +RAP). FOA kills cells expressing this reporter. Note that the untagged strain (Sir3p, JNY356) grows normally on either plate. In contrast, the tagged strain (SIR3-FRB, JNY365) is killed on the double-selection plate (+RAP +FOA) due to the desilencing of URA3. The expression state of URA3 is indicated (silenced/desilenced). (B) Anchoring of the cohesin subunit SCC1-FRB results in sister chromatid separation (doublets) in about 28% of the cells. Exposure to rapamycin was 120 min. All strains contained a LacO array inserted near GAL1 highlighted with the help of GFP-LacI. Only about 2%–3% doublets were observed in the controls, including anchoring of SMC2-FRB or TOP2-FRB. Inset shows a field of cells enriched for doublets. Molecular Cell 2008 31, 925-932DOI: (10.1016/j.molcel.2008.07.020) Copyright © 2008 Elsevier Inc. Terms and Conditions