1. Volume 33, Issue 6, Pages 704-716 (March 2009)
Sfp1 Interaction with TORC1 and Mrs6 Reveals Feedback Regulation on TOR Signaling 
Harri Lempiäinen, Aino Uotila, Jörg Urban, Ilse Dohnal, Gustav Ammerer, Robbie Loewith, David Shore 
Molecular Cell 
Volume 33, Issue 6, Pages (March 2009)
DOI: /j.molcel
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2. Figure 1
Purification and Identification of Sfp1 In Vivo Interacting Proteins
(A) SDS-PAGE of Sfp1-TAP-associated proteins, stained with silver; mock purification from untagged cells was performed as control. Protein bands identified by mass spectrometry are indicated.
(B) Table of Sfp1-interacting proteins identified. Score values, a measure of the significance of protein identifications, were derived by using Mascot software (see the Supplemental Data).
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3. Figure 2 Sfp1 Interacts with TORC1 Components In Vivo
(A) Tor1 and Kog1 interact with Sfp1 in a rapamycin-sensitive manner. Lysates from cells expressing 3HA-Tor1 or Kog1-3HA alone, or together with Sfp1-TAP, were subject to a single-step TAP purification. For indicated samples, rapamycin (200 ng/ml) was added 30 min prior to cell harvesting. Proteins were separated by SDS-PAGE and detected by immunoblotting with the indicated antibodies. Act1 served as control.
(B) Lst8 interacts with Sfp1 in a rapamycin-sensitive manner. Lysates from cells expressing Lst8-3HA alone or together with Sfp1-TAP were analyzed as in (A).
(C) Sfp1 associates with Kog1 and Tco89. Lysates from cells expressing the indicated TAP-tagged proteins were subjected to single-step TAP purification and analyzed as in (A).
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4. Figure 3
Sfp1 Phosphorylation, Nuclear Localization, and Interaction with Tor1 Are Controlled in a TORC1-Dependent Manner
(A) Sfp1 is dephosphorylated upon rapamycin treatment, whereas cycloheximide (CHX) causes Sfp1 hyperphosphorylation and increased interaction with Tor1. Sfp1 was purified from cells expressing Sfp1-TAP and 3HA-Tor1; cells were treated prior to harvesting with CHX (30 min, 25 μg/ml), rapamycin (rapa; 200 ng/ml for indicated times), or drug vehicle. Treatment with λ phosphatase (PP) in the absence or presence of phosphatase inhibitor (PPi) (lanes 1 and 2) was done after purification. Proteins were separated by SDS-PAGE and stained with Pro-Q Diamond Phospho-protein Stain (Phospho-protein), followed by Sybro Ruby protein gel stain (Total protein). Relative phosphorylation was determined as described in the Experimental Procedures; data are reported as averages (bars) from three experiments, with standard deviations indicated by the lines above (except lane 1). Coimmunoprecipitated 3HA-Tor1 was detected by anti-HA western. Protein inputs for the experiment are shown in Figure S2A.
(B) Wortmannin, caffeine, and rapamycin differentially regulate Sfp1 phosphorylation and interaction with TORC1, whereas nutrient starvation has no effect. Cells used for samples 1–4 were collected by centrifugation and cultured in either YPAD or H2O for the indicated times. For samples 6–8, wortmannin (10 μg/ml), caffeine (20 mM), or rapamycin (200 ng/ml) were added for 20 min. Protein inputs are shown in Figure S2B.
(C) TORC1 inhibitors cause Sfp1 relocalization to the cytoplasm, whereas CHX increases Sfp1 nuclear concentration. Cells carrying a genomic GFP-tagged SFP1 and mCherry-tagged HHF2 (Histone H4) were grown to exponential phase, and the indicated drugs (or vehicle) were added for 45 min (rapamycin and caffeine) or 30 min (wortmannin and CHX). For the -nutrient sample, cells were collected by centrifugation, and H2O was added for 45 min. Sfp1 colocalization with Histone H4 (nuclear marker) was quantified and is presented as a ratio of nuclear (colocalized) versus cytoplasmic (noncolocalized) Sfp1-GFP pools. For each condition, 50–100 cells were used for the quantification. Data are reported as averages (bars) from two experiments, with standard deviations indicated by the lines above.
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5. Figure 4 TORC1 Directly Phosphorylates Sfp1 at Multiple Residues
(A) TORC1-dependent in vitro phosphorylation of Sfp1. Rapamycin (5 μM + FKBP12 20 ng/μl), caffeine (10 mM), wortmannin (0.23 mM), or CKII inhibitor (0.46 mM) were added after purification and just before initiating the in vitro kinase reaction (for the control sample, DMSO was added to a 1.5% final concentration).
(B) Location of phosphorylated residues (P) in Sfp1 together with predicted Zn-finger domains (Fingerman et al., 2003) (black boxes). In the case of the two paired residues S181/S183 and T227/S228, it is not known which one of the two residues is actually phosphorylated.
(C) Mutation of Sfp1-phosphorylated residues to alanine (sfp1-1) leads to decreased in vivo phosphorylation of Sfp1 and reduced response to rapamycin. Wild-type Sfp1-TAP and sfp1-1-TAP were purified from cells treated with rapamycin (200 ng/ml for indicated times) or drug vehicle. Samples were analyzed as in Figure 3A. Data are reported as averages (bars) from three experiments, with standard deviations indicated by the lines above.
(D) sfp1-1 mutation reduces Sfp1 phosphorylation in vitro. Wild-type Sfp1-TAP (SFP1) and sfp1-1-TAP (sfp1-1) were purified from rapamycin- (200 ng/ml, 30 min) or drug vehicle-treated cells and were used for in vitro kinase assay.
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6. Figure 5
TORC1 Phosphorylation of Sfp1 Regulates Nuclear Localization and the RP Promoter Interaction
(A) Decreased nuclear localization of sfp1-1. Cells carrying a genomic GFP-tagged SFP1 or sfp1-1 allele and mCherry-tagged HHF2 were used for the experiment as described in Figure 3C. Rapamycin (rapa; 200 ng/ml, 45 min) was added to the indicated samples. Quantification data are reported as averages (bars) from two experiments, with standard deviations indicated by the lines above.
(B) Reduced binding of sfp1-1 to the RP gene promoter. Sfp1 recruitment to the RPL2B promoter was analyzed by ChIP in a strain carrying a TAP-tagged endogenous allele of SFP1 or sfp1-1. Samples were drawn from unstressed (−) and rapamycin (200 ng/ml 45 min; rapa)-treated cells. Data are reported as averages (bars) from three experiments, with standard deviations indicated by the lines above.
(C) Tetrad analysis shows synthetic growth defect of sfp1-1 sch9Δ cells. Spore colonies were imaged after 4 days of incubation at 30°C.
(D) CHX hypersensitivities of sfp1 and sch9 mutants. CHX sensitivity of indicated strains was determined by halo assays (examples in panel above). The area of inhibition (cm2) was measured for three independent cultures and is shown as an average value (bar), with standard deviation indicated by the lines above. A two-tailed, paired Student's t test was performed, and p values were calculated comparing the indicated strains (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).
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7. Figure 6 Sfp1 Regulates TORC1 Activity toward Sch9
(A) Deletion of SFP1 hyperactivates TORC1. Sch9 phosphorylation in wild-type and sfp1Δ cells containing Sch9-5HA plasmid was measured by using NTCB chemical fragmentation analysis (Urban et al., 2007). Sch9 is dephosphorylated in response to rapamycin (rapa; 200 ng/ml, 10 min) and hyperactivated by CHX (25 μg/ml, 10 min). Sch9-5HA was detected by anti-HA western. Relative ratios (normalized to untreated wild-type sample) between phosphorylated (+Pi) and dephosphorylated (−Pi) fractions of Sch9 are shown below the lanes.
(B) Sfp1 overexpression represses TORC1 activity. Sfp1 was overexpressed by adding galactose (2%) for indicated times to cells containing an integrated GAL1-promoter-driven SFP1 (GAL1-SFP1) and a plasmid expressing Sch9-5HA. An otherwise isogenic strain (SFP1) lacking the GAL1-SFP1 construct was used as control. The kinetics of Sfp1 expression from the GAL1 promoter is shown in Figure S6A.
(C) Mutations in the C-terminal Zn-finger region of Sfp1 hyperactivate TORC1-dependent phosphorylation of Sch9. Sch9 phosphorylation in wild-type and indicated SFP1 mutant cells containing a Sch9-5HA-expressing plasmid was measured by using NTCB chemical fragmentation analysis (as before).
(D) Reduced binding of Sfp1 Zn-finger mutants to an RP gene promoter. ChIP analysis of SFP1 wild-type and indicated Zn mutant strains was done as described in Figure 5B and the Experimental Procedures. Data are reported as averages (bars) from three experiments, with standard deviations indicated by the lines above.
(E) Sfp1 Zn-finger mutations do not affect the TORC1 interaction. Lysates from cells expressing the indicated TAP-tagged SFP1 alleles and Kog1-3HA were subjected to single-step TAP purification and analyzed as in Figure 2A.
(F) Effect of SCH9 mutations on TORC1 activity toward the Sch9 C terminus. TORC1 activity toward an sch9-T570A-6HA reporter construct was assayed by using NTCB chemical fragmentation analysis (as before). In addition to the catalytically inactive Sch9 reporter, cells expressed an additional allele of SCH9 as indicated.
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8. Figure 7 Mrs6 Regulates TORC1 Signaling to Sfp1 and Sch9
(A) Reduced phosphorylation and TORC1 binding of Sfp1 in mrs6-2 cells. Sfp1-TAP was purified from MRS6 or mrs6-2 cells at indicated temperatures. Cells were treated with rapamycin (rapa; 200 ng/ml, 45 min) or drug vehicle prior to harvesting. Samples were analyzed as in Figure 3A. Protein inputs for the experiment are shown in Figure S7B.
(B) Reduced nuclear localization of Sfp1 in mrs6-2 cells. Cells carrying a genomic GFP-tagged SFP1 and mCherry-tagged HHF2 in MRS6 or mrs6-2 backgrounds were used for the experiment at indicated temperatures. Details of microscopy and quantification are described in Figure 3C. Quantification data are reported as averages (bars) from two experiments, with standard deviations indicated by the lines above.
(C) Interaction of sfp1 Zn-finger mutants with Mrs6. Lysates from cells expressing the indicated TAP-tagged SFP1 alleles and Mrs6-3HA were subjected to single-step TAP purification and analyzed as described in Figure 2A.
(D) Localization of SFP1 Zn-finger mutants. Cells carrying indicated genomic GFP-tagged SFP1 alleles and mCherry-tagged HHF2 were analyzed as in (B). Quantification data are reported as averages (bars) from two experiments, with standard deviations indicated by the lines above.
(E) Mrs6 regulates Sch9 phosphorylation. Sch9 phosphorylation in MRS6 or mrs6-2 cells (grown at indicated temperatures) containing Sch9-5HA plasmid was measured by using NTCB chemical fragmentation analysis.
(F) Schematic model of connections between TORC1, Sfp1, Sch9, and Mrs6 signaling.
Molecular Cell  , DOI: ( /j.molcel )
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