1. TOR Signaling Regulates Ribosome and tRNA Synthesis via LAMMER/Clk and GSK-3 Family Kinases 
Jaehoon Lee, Robyn D. Moir, Kerri B. McIntosh, Ian M. Willis 
Molecular Cell 
Volume 45, Issue 6, Pages (March 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2012 45, 836-843DOI: (10.1016/j.molcel.2012.01.018)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Hyperphosphorylation of Rpc53 under Conditions that Repress Ribosome and tRNA Synthesis Requires the Kinases Kns1 and Mck1
(A) Rpc53-HA immunoblotting of extracts prepared from a wild-type yeast strain (W303, OD600 ∼0.5) before or after treatment with rapamycin (Rap), methyl methane sulfonate (MMS), chlorpromazine (CPZ), or tunicamycin (Tu) or after growth to stationary phase (OD600 ∼10). The stationary phase extract was further treated with alkaline phosphatase in the presence or absence of a phosphatase inhibitor cocktail.
(B) Scheme to identify regulators of Rpc53 phosphorylation. An RPC53-HA allele was introduced into 272 mutant strains containing gene-deletions of kinases, phosphatases, and their regulators (Table S2) using SGA methods. Haploid double drug resistant strains were grown as indicated for extract preparation and immunoblotting. Deletions of only two genes (KNS1 and MCK1) were found to compromise the phosphorylation of Rpc53. A representative western blot is shown for wild-type, kns1Δ, and mck1Δ strains.
(C) Extracts of strains deleted for different GSK3 family members were prepared before or after treatment with rapamycin.
(D) Extracts of wild-type and kns1Δ strains were prepared before or after treatment with different repressing conditions as in (A).
(E) Extracts of an mck1Δ strain were prepared before or after treatment with different repressing conditions as in (A). See Supplemental Experimental Procedures for details.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Deletion of KNS1 and MCK1 Compromises Rapamycin-Mediated Repression of Ribosome and tRNA Synthesis
(A) Northern analysis of a short-lived tRNALeu precursor and the stable U3 snRNA was performed with RNA from early log phase cultures of wild-type, single deletion, and double deletion strains before and after rapamycin treatment (left panel). Normalized pre-tRNALeu band intensities were used to determine the level of transcriptional repression relative to the untreated wild-type strain. Results from three biological replicates are plotted (right panel) ± SD.
(B) Northern analysis of the short-lived 20S precursor rRNA and mature 18S rRNA was performed with RNA from the log phase cultures in (A) (left panel). Normalized ratios of 20S:18S rRNA were used to determine the level of transcriptional repression relative to the untreated wild-type strain. Results from biological replicates are plotted (right panel) ± SD.
(C) Quantitation of ribosomal protein (RP) mRNA abundance. RNA preparations were analyzed by RT-PCR for various RP mRNAs using the 2-ΔΔCT method and U1 snRNA as a control across all experiments. Results are expressed relative to the untreated wild-type strain (set to 1.0). For each strain and condition, triplicate technical replicates were analyzed from three biological replicates. The data are plotted ± SD. Wild-type and mutant strains are color-coded following the scheme used in (A) and (B). Control mRNAs for ACT1 and TUB1 showed no significant change in expression with deletion of Kns1 and/or Mck1 in the presence or absence of rapamycin.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
TORC1 Activity Determines the Expression, Phosphorylation State, and Localization of Kns1
(A) Phos-tag™ acrylamide gel analysis of a W303 strain containing wild-type Kns1-myc or a kinase dead (KD) mutant on pRS425. Cells grown to mid-log phase were treated with rapamycin or drug vehicle before extract preparation and myc immunoblotting. Sample loading was normalized for total Kns1 protein (see Figure S3C).
(B) Kns1-myc localization was determined by indirect immunofluoresence (Alexa Fluor 488) in a W303-derived strain where KNS1 expression was driven by the TDH3 promoter. Kns1 nuclear localization is confirmed by the overlap with DAPI nuclear staining (merge). Cells were mock-treated (left panels) or rapamycin-treated (right panels) for 60 min. Images are representative of multiple independent experiments.
(C) Quantitation of Kns1-myc localization. Localization was assigned by manual inspection of >120 untreated cells (black bars) and >170 rapamycin-treated cells (green bars) over several independent fields to the nucleus (colocalization with DAPI), the cytoplasm (outside the DAPI signal), or both compartments (localization throughout the cell). Lower and upper limits of fluorescence intensity were set to the background level (no primary antibody) and to the maximum signal per field, respectively.
(D) Time course of Kns1 induction by rapamycin. Samples were resolved by SDS-PAGE (no Phos-tag), and Kns1-myc was quantified and normalized to Tfc1 (as in Figure S3A). The fold change in Kns1 is indicated.
(E) Induction of Kns1 by MMS was determined as in (D).
(F) Overexpression of Kns1 causes hyperphosphorylation of Rpc53. Wild-type and TDH3pr-Kns1 strains (derived from RPC53 del) were treated with rapamycin for 1 hr and extracts were blotted for RPC53-HA. Data in (D), (E), and (F) are representative of multiple experiments.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Kns1 Is a Priming Kinase for Mck1 Phosphorylation of Rpc53
(A) Equal amounts of wild-type or kinase-dead (KD) GST-Kns1, detected by immunoblotting (α-GST), were used to phosphorylate recombinant Rpc53 (C53-His) with [γ-32P]ATP.
(B) Equal amounts of wild-type or kinase-dead GST-Mck1, detected by immunoblotting (α-His5), were used to phosphorylate Rpc53 as in (A). Note that the low level of GST-Mck1 and Rpc53 phosphorylation seen with kinase-dead GST-Mck1 is due to a copurifying kinase.
(C) Schematic showing the known phosphosites in Rpc53 and the location of mutants M1A and M2A, which contained triple phosphosite substitutions to alanine. The sequence around the M1 site is shown with a proposed Kns1 motif (RXXS/TP) underlined and the phosphosite boxed in gray. Two overlapping GSK3 motifs (S/TXXXS/T) direct Mck1 phosphorylation of primed substrates (blue arrows) at phosphosites boxed in gray.
(D) In vitro phosphorylation of wild-type and mutant Rpc53 proteins by recombinant wild-type and kinase-dead Kns1 with [γ-32P]ATP. Equal loading of Rpc53 is shown by western blot (WB).
(E) Rpc53 phosphorylation was performed as above by titrating equal amounts of wild-type or kinase-dead GST-Mck1 in the presence of a constant amount of recombinant Kns1.
(F) Plasmid shuffling was used to introduce the indicated RPC53-HA alleles into a BY4741-related strain containing a chromosomal deletion of RPC53. Extracts were analyzed by immunblotting before and after rapamycin treatment.
(G) The wild-type and M1A mutant Rpc53 strains shown in (F) were transformed with a plasmid containing S. pombe Rpc11 under GAL promoter control followed by deletion of the chromosomal S. cerevisiae RPC11 gene (rpc11Δ::natR). Northern analysis of pre-tRNALeu and U4 snRNA was performed before and after rapamycin treatment.
(H) Normalized pre-tRNALeu band intensities were used to determine the level of transcription relative to the untreated wild-type Rpc53 strain. Results from three biological replicates are plotted ± SD.
(I) Model of TOR regulation of pol III transcription. Nutrients and stress regulate Maf1 via Sch9 and PKA and regulate pol III via Kns1 and Mck1. Under nutrient-replete conditions, Sch9 and PKA phosphorylate Maf1 and inhibit its ability to repress transcription. This inhibition of Maf1 is relieved under stress conditions which also result in the phosphorylation of Rpc53 by Kns1 and Mck1 and changes in polymerase function. Rpc53 and its partner Rpc37 function in transcription elongation and interact with Rpc11, which functions in proofreading, termination, and facilitated recycling and cooperates with Rpc53 in enabling repression by Maf1. The inability of Maf1 to inhibit elongating or recycling polymerases suggests that Kns1 and Mck1 phosphorylation of Rpc53 leads to inhibition of recycling or promotes polymerase dissociation after termination to allow interactions with Maf1 that prevent subsequent reinitiation (see text for details). Dotted lines represent interactions that are indirect (e.g., Sch9 to PKA [Soulard et al., 2010] and TORC1-regulated expression of Kns1, Figure 3D). Arrows and bars indicate positive and negative consequences, respectively, on a protein function or process.
Molecular Cell  , DOI: ( /j.molcel )
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