Genes & Development



Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central regulator of protein synthesis - Supplementary material

Table S1 Strains

|Strain |Genotype |Source |Figure |

|YAL6B |MATa; his3Δ leu2Δ met15Δ ura3Δ lys1::KanMX6 arg4::KanMX4 |(Gruhler et al. 2005) |1C-D |

|AH035 |MATa; SCH93E (T737E, S758E, S765E) [YAL6B] |This study |1C-D |

|AH105 |MATa; tap42::HphMX4 [YAL6B] |This study |1C-D |

|TB50a |MATa; trp1 leu2 ura3 his3 rme1 |wt |2A, 2C, S3A |

|AH132 |MATa; KSP1-3HA ::HIS3MX6 [TB50] |This study |2A, 2C |

|AH134 |MATa; RPH1-3HA ::HIS3MX6 [TB50] |This study |2A, 2C |

|AH141 |MATa; HIS3MX6::PADH1-3HA-STB3 [TB50] |This study |2A, 2C, S1B |

|MS036 |MATa; PAR32-5HA ::HIS3MX6 [TB50] |This study |2A, 2C |

|MS037 |MATa; SKY1-5HA ::HIS3MX6 [TB50] |This study |2A, 2C |

|MS035 |MATa; AVT1-5HA ::HIS3MX6 [TB50] |This study |2A |

|MS041 |MATa; SYG1-5HA ::HIS3MX6 [TB50] |This study |2A |

|AU046 |MATa; PIN4-3HA::HIS3MX6 [TB50] |This study |2A, 2C |

|AH090-7a |MATa; sch9::KanMX6 tap42::HphMX4 [TB50] |This study |1A, 2B, 3D, 4A, 5A-B, |

| | | |S9A |

|RL215-4c |MATa; sch9::KanMX6 [TB50] |This study |3B, 4F, 5D, S9B |

|AH191-9b |MAT?; tpk1::KanMX6 tpk2::HIS3MX6 tpk3::HphMX4 yak1::NatMX4 |This study |3C, S4C |

|AH149-4c |MATα; sch9::KanMX6 maf1::HphMX4 [TB50] |This study |4B-F, 5C, S6A |

|AH220 |MAT?; sch9:: KanMX6 HHF2-mCherry::HphMX4 [TB50] |This study |S7A-D |

|AH158 |MATa; sch9::KanMX6 RPC82-TAP::HIS3MX6 [TB50] |This study |4F, S8A |

|AH206 |MATa; sch9::KanMX6 tap42::HphMX4 RPA190-13myc::HIS3MX6 [TB50] |This study |5E |

|AH208 |MATa; sch9::KanMX6 tap42::HphMX4 RPA190-TAP::HIS3MX6 [TB50] |This study |5F |

|AH131 |MATa; DED1-3HA::HIS3MX6 [TB50] |This study |S1B |

|AU047 |MATa; REG1-5HA::HIS3MX6 [TB50] |This study |S1B |

|MS066 |MATa; par32::NatMX4 [TB50] |This study |S3A |

|AU071 |MATa; pin4::NatMX4 [TB50] |This study |S3A |

|AU076 |MATa; rph1::NatMX4 [TB50] |This study |S3A |

|AU045 |MATa; yak1::NatMX4 [TB50] |This study |S3A |

|RL276-2b |MATa; HIS3 [TB50] |This study |S3B, S4C |

|MS040 |MATa; sky1::HIS3MX6 [TB50] |This study |S3B |

|AH148 |MATa; stb3::HIS3MX6 [TB50] |This study |S3B |

|AH174-2d |MATa; dot6::HphMX4 [TB50] tod6::HIS3MX6 [TB50] |This study |S3B |

|RL276-5a |MATα; TRP1 [TB50] |This study |S3C |

|JU513 |MATα; ksp1::TRP1 [TB50] |This study |S3C |

|BY4741 |MATa; his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 |wt |S3D |

|- |Deletion strains in the BY4741 background |(Winzeler et al. 1999)|S3D |

|W303α |MATα; ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 |wt |S4A-B, S8B |

|AH091 |MATα; sch9as (T492G) [W303] |This study |S4A-B, S8B |

|Y3527 |MATα; tpk1as (M164G) tpk2as (M147G) tpk3as (M165G) [W303] |(Yorimitsu et al. |S4A-B, S8B |

| | |2007) | |

|Y3528 |MATα; tpkas (M164G) tpk2as (M147G) tpk3as (M165G) sch9as (T492G) [W303] |(Yorimitsu et al. |S4A-B, S8B-C |

| | |2007) | |

|AH311-1b |MATα; sch9as::TRP1 [TB50] |This study |S5 |

|AH311-3a |MATα; sch9as::TRP1 maf1::HphMX4 [TB50] |This study |S5 |

|AH310-5c |MATα; SCH9::TRP1 maf1::HphMX4 [TB50] |This study |S6B |

|AH477 |MATα; RPC82-TAP::HIS3MX6 [W303] |This study |S8C |

|AH478 |MATα; sch9as (T492G) RPC82-TAP::HIS3MX6 [W303] |This study |S8C |

|AH479 |MATα; tpk1as (M164G) tpk2as (M147G) tpk3as (M165G) RPC82-TAP::HIS3MX6 [W303] |This study |S8C |

|AH480 |MATα; tpkas (M164G) tpk2as (M147G) tpk3as (M165G) sch9as (T492G) |This study |S8C |

| |RPC82-TAP::HIS3MX6 [W303] | | |

Table S2 Plasmids

|Plasmid |Vector::insert |Source |Figure |

|pRS415 |pRS415 | |1B-C, 4B-E, 5C, S6A-B |

|pRS416 |pRS416 | |5D, S9B |

|pRS413 |pRS413 | |S4F |

|pAH123 |pRS415::TAP42 |This study |1A-C, 2B, 3D, 4A, 5A-B, 5E, 7A, S9A |

|pAH124 |pRS415::tap42-11 |This study |1A-C, 2B, 3D, 4A, 5A-B, 5E, 7A, S9A |

|pAH144 |pRS414::SCH9 |This study |1A, 2B, 3B, 3D, 4A-E, 5A-E, 7A, S6A, S7A-D, S8A, |

| | | |S9A-B |

|pAH145 |pRS414::sch9as (T492G) |This study |3B, 4B-E, 5C-D, S6A, S7C-D, S8A, S9B |

|pAH146 |pRS414::SCH9DE (T723D, S726D, T737E, S758E, S765E)|This study |1A, 1D-E, 2B, 3D, 4A, 4F, 5A-B, 5E, 7A, S7A-B, S9A |

|pAH149 |pRS416::HIS3 |This study |3D, 4A-E, 5A-C, S6A-B, S9A |

|pAH152 |pRS415::HIS3 |This study |5D, S6B, S9B |

|pAH212 |pRS426::TOD6-5HA |This study |2B |

|pAH175 |pRS416::DOT6-5HA |This study |2B, S2B |

|pMS034 |pRS416::PAR32-5HA |This study |2B |

|pAH099 |pRS416::MAF1-3HA |This study |2B, 3B-D, 4F, S4A-B, S8A-C |

|pAH107 |pRS426::PGAL1-GST-SCH9kd |(Urban et al. 2007) |3E |

|pAH108 |pRS426::PGAL1-GST-SCH93E |This study |3E |

|pAH216 |pGEX6P1::MAF1 |This study |3E |

|pAH229 |pGEX6P1::MAF16A1 (S90A, S101A, S177A, S178A, |This study |3E |

| |S209A, S210A) | | |

|pAH237 |pGEX6P1::MAF16A2 (S90A, S101A, S178A, S179A, |This study |3E |

| |S209A, S210A) | | |

|pAH236 |pGEX6P1::MAF17A (S90A, S101A, S177A, S178A, S179A,|This study |3E |

| |S209A, S210A) | | |

|pAH095 |pRS415::MAF1 |This study |4B-E, 6C, S6A-B |

|pAH238 |pRS415::MAF17E (S90E, S101E, S177E, S178E, S179E, |This study |4C-D, S6A-B |

| |S209E, S210E) | | |

|pAH247 |pRS415::MAF17A (S90A, S101A, S177A, S178A, S179A, |This study |4C-D, S6A-B |

| |S209A, S210A) | | |

|pAH240 |pRS416::MAF17E-3HA (S90E, S101E, S177E, S178E, |This study |4F |

| |S179E, S209E, S210E) | | |

|pAH205 |pRS416::RRN3-5HA |This study |5F |

|pAH262 |pRS416::TOD6-5HA |This study |S1A |

|pAH268 |pRS416::TOD66A-5HA (S280A S298A S308A S318A S333A |This study |S1A |

| |S346A) | | |

|pAH272 |pRS416::DOT65A-5HA (S247A S282A S313A S335A S368A)|This study |S1A |

|pAH217 |pRS416::MAF1-GFP |This study |S7A-D |

|pAH248 |pRS416::MAF17A-3HA (S90A, S101A, S177A, S178A, |This study |S6A |

| |S179A, S209A, S210A) | | |

SCH9 and MAF1 plasmids were checked for complementation of the relevant deletion mutant. Point mutagenesis was performed by gene synthesis (Genscript corp.) or fusion PCR according to standard protocols and checked by sequencing.

Table S3 Screens overview

| |Total detected |Number phosphorylation patterns / Common |Total distinct phosphopeptidesd|Total distinct phosphoproteins |

| |features |features over all patterns | | |

|Screen 1a |47,515 |6 / 7,832 |924 |521 |

|Screen 2b |65,570 |12 / 5,263 |1,156 |604 |

|Screen 3c |67,150 |12 / 5,216 |1,470 |684 |

a wt cells vs. wt cells treated with cycloheximide

b wt cells +/- rapamycin vs. SCH93E cells +/- rapamycin

c wt cells +/- rapamycin vs. tap42-11 cells +/- rapamycin

d Identified with a PeptideProphet (Keller et al. 2002) score > 0.4 and mapped to features with S/N > 10

Table S4 Summary of the phosphoproteomic screens

| |Total distinct |Downregulated |Upregulated |SCH9-dependent |TAP42-dependent |

| |phosphopeptides (proteins)|phosphopeptidesa |phosphopeptidesb |phosphopeptides (proteins)|phosphopeptides (proteins)|

| | |(proteins) |(proteins) | | |

|Screen 1 |924 (521) |57 (49) |49 (38) |n.a. |n.a. |

|Screen 2 |1,156 (604) |68 (39) |31 (28) |21 (16) |n.a. |

|Screen 3 |1,470 (684) |75 (50) |60 (46) |n.a. |40 (29) |

a Screen 1: ≥2-fold downregulated by CHX; Screen 2 and 3: ≥2-fold downregulated by rapamycin

b Screen 1: ≥2-fold upregulated by CHX; Screen 2 and 3: ≥2-fold upregulated by rapamycin

Table S5 Rapamycin sensitivities of yeast strains (BY4741 background) harboring deletions in TORC1 effectors identified in the phosphoproteomic screens.

ORF |Name |Scorea | |ORF |Name |Score | |ORF |Name |Score | |YAL021C |CCR4 |--- | |YGL019W |CKB1 |+/- | |YMR196W |  |+ | |YAR002W |NUP60 |+/- | |YGL023C |PIB2 |+/- | |YMR205C |PFK2 |+ | |YBL007C |SLA1 |-- | |YGL255W |ZRT1 |+ | |YMR216C |SKY1 |++ | |YBL008W |HIR1 |+ | |YGR038W |ORM1 |+ | |YMR230W |RPS10B |+ | |YBL054W |TOD6 |+ | |YGR125W |  |+ | |YMR243C |ZRC1 |+ | |YBL058W |SHP1 |-- | |YGR162W |TIF4631 |+/- | |YMR275C |BUL1 |++ | |YBL103C |RTG3 |++ | |YGR237C |  |++ | |YNL074C |MLF3 |++ | |YBR181C |RPS6B |+/- | |YGR240C |PFK1 |+ | |YNL076W |MKS1 |+++ | |YBR197C | |+ | |YHL016C |DUR3 |+ | |YNL096C |RPS7B |++ | |YCL011C |GBP2 |++ | |YHR082C |KSP1 |++ | |YNL098C |RAS2 |+ | |YCR077C |PAT1 |--- | |YHR097C |  |++ | |YNL101W |AVT4 |+ | |YDL019C |OSH2 |+ | |YIL038C |NOT3 |- | |YNL127W |FAR11 |++ | |YDL051W |LHP1 |+ | |YIL047C |SYG1 |++ | |YNL265C |IST1 |+ | |YDL173W |PAR32 |+ | |YIL094C |LYS12 |+ | |YNL321W | |+/- | |YDL204W |RTN2 |+ | |YIL095W |PRK1 |++ | |YNR024W |  |++ | |YDR005C |MAF1 |+ | |YIL135C |VHS2 |++ | |YOL019W | |-- | |YDR028C |REG1 |+/- | |YIR023W |DAL81 |+/- | |YOL036W |  |+ | |YDR093W |DNF2 |+/- | |YJL036W |SNX4 |+/- | |YOL051W |GAL11 |+++ | |YDR137W |RGP1 |+/- | |YJL165C |HAL5 |+/- | |YOL060C |MAM3 |++ | |YDR169C |STB3 |++ | |YJR001W |AVT1 |+ | |YOL061W |PRS5 |+/- | |YDR332W | |+ | |YJR062C |NTA1 |+ | |YOR066W |  |+ | |YDR345C |HXT3 |+/- | |YKL062W |MSN4 |+ | |YOR081C |TGL5 |+ | |YDR348C | |+ | |YKL064W |MNR2 |+ | |YOR083W |WHI5 |+/- | |YDR352W | |++ | |YKR092C |SRP40 |++ | |YOR084W | |+ | |YDR363W |ESC2 |+ | |YLL021W |SPA2 |+/- | |YOR153W |PDR5 |+++ | |YDR405W |MRP20 |++ | |YLL028W |TPO1 |+/- | |YOR308C |SNU66 |+ | |YDR507C |GIN4 |++ | |YLR237W |THI7 |- | |YOR311C |HSD1 |+ | |YDR508C |GNP1 |++ | |YLR240W |VPS34 |--- | |YOR322C |LDB19 |+/- | |YER040W |GLN3 |+++ | |YLR257W |  |+ | |YPL023C |MET12 |+ | |YER052C |HOM3 |+/- | |YML035C |AMD1 |+/- | |YPL049C |DIG1 |++ | |YER088C |DOT6 |++ | |YML072C |TCB3 |+ | |YPL180W |TCO89 |-- | |YER169W |RPH1 |++ | |YML119W | |+/- | |YPL181W |CTI6 |++ | |YFL021W |GAT1 |++ | |YML123C |PHO84 |+ | |YPR156C |TPO3 |++ | |YFR053C |HXK1 |+ | |YMR086W | |+ | |YPR185W |ATG13 |++ | |a Rapamycin resistance/sensitivity: --- strong sensitivity; -- moderate sensitivity; - slight sensitivity; +/- no phenotype; + slight resistance; ++ moderate resistance; +++ strong resistance. Representative spot assays are shown in Fig. S3D.

Figure S1 Assay of Stb3, Ded1 and Reg1 phosphorylation with fluorescent dyes

A. Cells expressing the indicated alleles of TOD6-5HA and DOT6-5HA were grown at 30 °C in YPD and subjected to a 15 min drug vehicle or rapamycin treatment. Proteins were extracted under denaturing conditions and Tod6 and Dot6 migration in SDS-PAGE was assayed by western blotting. Mutation of the R[R/K]xS motifs in the two proteins (TOD66A and DOT65A) yielded versions of the proteins that co-migrated with their respective wt versions after rapamycin treatment. B. HA-tagged Stb3, Ded1 and Reg1 were immunoprecipitated from extracts prepared from cells treated with rapamycin or mock-treated with drug vehicle for 20 min, resolved by SDS-PAGE and stained for phosphorylated residues (ProQ Diamond) and total protein levels (SYPRO Ruby). Strains expressing untagged proteins were used as controls. Stb3 becomes dephosphorylated after rapamycin treatment. Ded1 becomes hyperphosphorylated after rapamycin treatment (as predicted in the phosphoproteomic screens). Reg1 shows no change in phosphorylation after rapamycin treatment. This could be due to the fact that Reg1 is phosphorylated at many positions which appear to be rapamycin-insensitive.

[pic]

Figure S2 Venn diagram of Sch9- and Tap42-dependent phosphopeptides

Sch9 and Tap42 control largely independent subsets of rapamycin-dependent phosphopeptides. Venn diagram of phosphopeptides found in both rapamycin screens. The four phosphopeptides predicted to be dependant on both Sch9 and Tap42 belonged to Rtg3, Tod6, Vhs2 and Stb3.

[pic]

Figure S3 Genes identified in the phosphoproteomic screens modulate rapamycin sensitivity

A-C. 10-fold serial dilutions of yeast cells (TB50a background) of the indicated genotype were spotted on YPD supplemented with indicated concentrations of rapamycin and grown for 2-3 days at 30°C. The PIN4 null mutant is hypersensitive to rapamycin. Deletion of RPH1, SKY1, STB3, KSP1 or TOD6 and DOT6 confer resistance to rapamycin. D. Representative plates indicating rapamycin sensitivities of yeast strains (BY4741 background) harboring deletions in TORC1 effectors identified in the phosphoproteomic screens. A comprehensive summary of this data can be found in Table S5.

[pic]

Figure S4 Sch9 is the major physiological Maf1 kinase at 30 °C

A. 10-fold serial dilutions of W303α cells of the indicated genotype were spotted on the indicated media grown for 2 days at 30°C B. Sch9, rather than PKA, is the major in vivo Maf1 kinase at 30 °C. Strains in A were transformed with a Maf1-3HA reporter plasmid, grown in YPD and assayed for Maf1 phosphorylation upon treatment with 200 nM 1NM-PP1. C. Maf1 phosphorylation upon diauxic shift and entry into stationary phase. 1.414-fold serial dilutions of cells of the indicated genotype and expressing Maf1-3HA were grown in SC –Ura. Plotted are the optical densities vs. dilution at the time when the cultures were harvested. Protein extracts were analyzed by western blotting. Sch9 phosphorylation was probed using a phosphospecific antibody raised against phosphorylated T737 (TORC1 site). Total Sch9 levels were detected using an antibody raised against Sch9 activation loop (phosphorylated T570), constitutively phosphorylated by Pkh1/2 (Urban et al. 2007). Note that Maf1 dephosphorylation roughly parallels Sch9 T737 dephosphorylation in wt cells and that Maf1 dephosphorylation is not accelerated in the pka background.

[pic]

Figure S5 Sch9 regulates pre-tRNA levels

Cells of the indicated genotype were grown at 30 °C in YPD and treated with 300 nM 1NMPP1 for the indicated times. Aliquots were withdrawn, total RNA was extracted and the levels of ACT1 mRNA and pre-tRNAPro were measured by quantitative RT-PCRs. Data are means of three independent experiments +/- s.d. Pre-tRNAPro levels dropped upon Sch9as inhibition by 1NMPP1 in a Maf1-dependent manner. *** P < 0.001 vs. the sch9as MAF1 control. ### P < 0.001 vs. the untreated isogenic control.

[pic]

Figure S6 Maf1 phosphorylation sites mutants largely, but not completely, complement the loss of MAF1.

A. Inhibition of Sch9 results in a Maf1-dependent drop in the synthesis of tRNA and 5S rRNA. Cells expressing Maf17E or Maf17A display phenotypes intermediate between maf1 and MAF1 cells. B. Rapamycin treatment results in a Maf1-dependent drop in the synthesis of tRNA and 5S rRNA. Cells expressing Maf17E or Maf17A display phenotypes intermediate between maf1 and MAF1 cells. The * indicates the migration of a probable 5S degradation product.

[pic]

Figure S7 Sch9 regulates Maf1 localization

A. Rapamycin-induced Maf1 nuclear accumulation is not blocked by Sch9DE. AH220 (sch9 HHF2-mCherry) cells, complemented with pAH144 (SCH9wt) or pAH146 (SCH9DE) and transformed with pAH217 (MAF1-GFP), were grown in SC –URA –TRP 0.2% Gln and processed for fluorescence microscopy before and after 15 min rapamycin treatment. B. Quantification of Maf1 nuclear vs. cytoplasmic localization in A. Data are means from at least 20 cells +/- s.d. C. Sch9 function is required for cytoplasmic Maf1 localization. AH220 (sch9 HHF2-mCherry) cells, complemented as in figure 3B and transformed with pAH217 (MAF1-GFP), were grown in SC –URA –TRP 0.2% Gln and processed for fluorescence microscopy before and after 15 min 1NM-PP1 treatment D. Quantification of Maf1 nuclear vs. cytoplasmic localization in C. Data are means from at least 20 cells +/- s.d. Statistical significances for B and D: *** P < 0.001 vs. wt control; # P 0.125 is considered to have a correctly located phosphorylation site. For the phosphopeptides shown in Supplemental File F1 a predicted false positive rate of 5% was accepted.

4 Protein extraction

PPi: 10 mM NaF, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.

Denaturing protein extracts were performed by the TCA-Urea method as described previously (Urban et al. 2007).

Native protein extracts were prepared from 100 ml cultures grown to OD600 0.6-0.9 and treated as described in the text. Cells were cooled on ice/water in falcon tubes for 10 min and collected by centrifugation at 2000 x g. They were washed with ice-cold water, centrifuged again, resuspended in 1.6 ml protein lysis buffer (PBS, 10% glycerol, 0.5% Tween-20) supplemented with 1mM PMSF, 1x Pi and 1x PPi and lysed with glass beads 5x 30 s at max speed with pauses on ice (Loewith et al. 2002). Lysates were cleared by centrifugation at 7000 x g.

For the phosphoproteomic screens, biochemical activity was quenched by direct addition of 6% trichloroacetic acid to the cell culture. Cells were then cooled and pelleted (2000 x g), washed twice with acetone, dried under vacuum and resuspended in 50 mM Tris-Cl pH 7.5, 7M urea, 1x PPi, 1x Pi. Cells were lysed with glass beads (0.5 mm) in a Fast prep machine (Bio101; 6 x 45 s at max speed). Lysates were cleared by centrifugation at 21,000 x g for 10 min at 4°C.

5 Immunoprecipitation, phosphostaining and lambda phosphatase treatment

HA-tagged proteins were immunoprecipitated from cleared native protein extracts with Protein-A CL4B sepharose (GE Healthcare) bound and crosslinked to anti-HA antibodies (12CA5) for 90 min at 4 °C. Unbound proteins were washed from the beads with lysis buffer.

For phosphostaining, immunoprecipitated proteins were resolved by SDS-PAGE and stained with ProQ Diamond (GE Healthcare) to probe for total phosphorylation. SYPRO Ruby was used as a post stain to control for total protein levels.

For lambda phosphatase treatment, beads were washed two times with with lambda buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.1 mM EGTA, 2 mM DTT, 0.01% Brij-35). Beads were split in three aliquots which were either directly resuspended in sample buffer and heated at 95 °C, or treated 15 min at 30 °C with 400 U lambda phosphatase (NEB) in lambda buffer + 2.5 mM MnCl2 in presence or absence of 15 mM Na3VO4 and 50 mM NaF. Reactions were stopped by adjustment to 1x sample buffer and denaturation at 95 °C.

6 RNA extraction and analysis, cDNA synthesis

Total RNA extraction was performed as described previously (Laferte et al. 2006) with minor modifications. Briefly, cultures were mixed with two volumes of ice-cold water and were collected by centrifugation at 2,000 x g. Cells were resuspended in 500 µl AE buffer (50 mM sodium acetate pH 5.3, 10 mM EDTA) + 0.1% [w/v] SDS and lysed by shaking with glass beads in presence of 500 µl phenol solution (Sigma). Lysates were centrifuged at 21,000 x g. 450 µl of the aqueous phase was collected and extracted with 450 µl phenol-chloroform-isoamylalcohol (25:24:1; Equilibrated in AE buffer). RNA was precipitated overnight at -20°C from 400 µl of the aqueous phase mixed with 40 µl 3M sodium acetate pH 5.2 and 1 ml ethanol. RNA was pelleted by centrifugation at 21,000 x g, washed with 1 ml 80% EtOH and resuspended in H2O.

For 3H-uracil incorporation analysis, RNA species were resolved on agarose or polyacrylamide gels, stained with ethidium bromide (EtBr) as a loading control and blotted before being exposed to a Cyclone imager screen.

cDNA was synthesized with a mixture of random and oligo-dT primers using Bio-Rad’s iScript system. Reactions in which the enzyme was omitted were performed to control for genomic DNA contaminations in SYBR Green quantitative PCR reactions (Applied biosystems) using primers for ACT1 and pre-tRNAPro (ACT1 Fw 5’-GAATT GAGAG TTGCC CCAGA-3’; Rev 5’-AGAAG GCTGG AACGT TGAAA-3; pre-tRNAPro Fw 5’-GCTTTGGGCGACTTCCTG-3’; Rev 5’-GGGGCGAGCTGGGAATTGAA-3’).

7 Fluorescent microscopy

Cells expressing Maf1-GFP, and Hhf2-mCherry as a nuclear marker, were grown overnight to exponential phase in selective synthetic medium and treated as described in the text. Cells were then immediately mounted on glass slides with cover slips and observed using a Leica AF 6000 LX fluorescent microscope fitted with a camera. Signal intensity quantifications in the cytoplasm and the nucleus were performed using ImageJ and were corrected for background fluorescence (Abramoff et al. 2004).

8 Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed with Dynabeads M280 coupled with sheep anti-mouse IgG (Dynal) and quantified by real-time PCR using the SYBR Green system as described previously (Bianchi et al. 2004). IPs were quantified using primers for the rDNA locus (rDNA4) described earlier (Beckouet et al. 2008) and normalized by RT-PCRs using DNA purified from the IP input. IP efficiency was normalized with similar quantification for the ACT1 locus (see primers above) as a control.

9 Primers extension assays

0.5 μg total RNA was mixed with 3 pmole of each primer, denatured at 70°C and directly placed on ice. Reactions were initiated by addition of 100 U M-MLV reverse transcriptase (Invitrogen), its supplied buffer, 500 μM dCTP, 500 μM dGTP, 500 μM dTTP, 5 μM dATP and 5 μCi α-32P-dATP. Reactions were shaken for 30 min at 45°C and terminated by addition of 20 μl 0.01% bromophenol blue in formamide. Extension products were resolved by PAGE and analyzed using a BioRad Molecular Imager. Assays with 0.25 μg RNA or with single primer alone were performed to control for linearity and absence of primer interferences. Sequences of primers used for 35S and 18S extension are respectively 5'-ACACG CTGTA TAGAG ACTAG GC-3' and 5'-GCTTA TACTT AGACA TGCAT GGC-3'. Values for the 35S rRNA was normalized by the 18S levels and by the average of all values of the experiments.

10 SuperHirn parameters

//-------------------------------------------------------------------------------------------------------

//

// GENERAL:

//

// retention time tolerance: tolerance with which lc-peaks will be merged

// AFTER the alignment of the spectra [min]

MS1 retention time tolerance=3

//

// mass time tolerance: mass tolerance with which lc-peaks will be merged

// AFTER the alignment of the spectra [Da]

MS1 m/z tolerance=0.008

//

// MS2 m/z tolerance: mass tolerance with which MS2 identifications will be associated

// to a defined MS1 LC elution peak [Da]

MS2 m/z tolerance=0.004

//

// MS2 mass matching modus: define which modus used to match ms2 assignments to ms1 peaks

// - theoretical mass [1] : use theoretical mass calculated from sequence

// - MS1 precursor mass [0]: use measured ms1 mass of precursor ion

MS2 mass matching modus=1

//

//

// Peptide Prophet Threshold: threshold used in clustering peptides into proteins

//

Peptide Prophet Threshold=0.5

//

// MS2 SCAN tolerance: SCAN tolerance with which MS2 identifications will be associated

// to a defined MS1 LC elution peak []

MS2 SCAN tolerance=200

//

// MS2 retention time tolerance: retention time tolerance with which MS2 identifications will be associated

// to a defined MS1 LC elution peak [min]

// (if set to -1, then the MS1 retention time tolerance will be used

MS2 retention time tolerance=5

//

// IL MS2 SCAN tolerance: SCAN tolerance with which MS2 info FROM INCLUSION LIST will be associated

// to a defined MS1 LC elution peak []

INCLUSIONS LIST MS2 SCAN tolerance=140

//

//-------------------------------------------------------------------------------------------------------

//

// MS1 feature selection options

// these options apply to the selection of MS1 feature from the XML/APML format

// they do not apply to the basic extraction of features from the raw mzXML data

//

// elution window: enables to only process a period of the

// elution gradient, defines by start / end

// only peaks within this region are accepted!!!, [min]

start elution window=20.0

end elution window=90.0

//

// LC peak score cutoff: above which are LC peaks accepted,otherwise discarted

LC peak score cutoff=10000

//

// LC peak intensity cutoff: only MS1 feature at or over this intensity level are accepted, otherwise discarted

MS1 feature intensity cutoff=10000

//

// Charge state min: For the selection of MS1 features by charge state, here its, the minimal charge state:

MS1 feature CHRG range min=2

//

// Charge state max: For the selection of MS1 features by charge state, here its, the maximal charge state:

MS1 feature CHRG range max=5

//

// M/z min: For the selection of MS1 features by m/z, here its, the minimal m/z value:

MS1 feature mz range min=300

//

// M/z max: For the selection of MS1 features by m/z, here its, the maximal m/z value:

MS1 feature mz range max=1600

//-------------------------------------------------------------------------------------------------------

//

// PRINT AND VISUALIZE OPTIONS:

// ( 0 = no, 1 = yes )

// pairwise alignment output : print the TR vs. delta Tr plots for the pairwise copmarison

pairwise alignment plotting=0

//

// pairwise alignment output : print the TR vs. delta Tr plots for the MASTER alignment

MASTER alignment plotting=0

//

// pairwise LC/MS correlation : print analysis results from the pairwise LC/MS correlation

pairwise correlation analysis=0

//

// similarity matrix : print the similarity matrix from the pairwise LC/MS correlation

similarity matrix=1

//

// alignment tree : print the constructed alignment tree into a file

print alignment tree=1

//

// Background correction profiles : prints the coefficient profiles from the background

// for the different runs to the screen:

print background correction profiles=1

//

// gnuplot plot gerenator: if plots should be generate through out the whole program rountine

gnuplot plot generator=1

//-------------------------------------------------------------------------------------------------------

//

// STORAGE OF DATA IN THE XML MASTER AND LC-MS FILE:

// ( 0 = no, 1 = yes )

//

// store only best MS2 per feature : only the best MS2 scan / feature will be store in the XML file

// (LC-MS runs and MasterMap) use to reduce XML file size

store only best MS2 per feature=0

//

// store only best MS2 per ALIGNED feature : only the best MS2 scan / ALIGNED feature will be store in the XML file

// (LC-MS runs and MasterMap) use to reduce XML file size

store only best MS2 per ALIGNED feature=0

//

// nb. max. alternative protein names : max. number of alternative proteins that will be store in the XML file

// for a non proteotypic peptide

nb. max. alternative protein names=5

//-------------------------------------------------------------------------------------------------------

//

// ALIGNMENT OF LC_MS SPECTRA:

//

// Window retention time: retention time window (min) to search

// for common peaks BEFORE the alignment.[min]

retention time window=5.5

//

// mass window : mass window (DA) to search for common

// peaks BEFORE the alignment. [Da]

mass / charge window=0.008

//

// smoothing error TR window: used to copmute the alignment error, use a tr window to

// calculate the standard deviations of raw data to predicted

// delta shift [min]

smoothing error TR window=1.0

//

// max. nb. stripes: in the plot of TR A vs TR B, there are off diagnal

// horizontal and vertical stripes, which come from

// high abundance long eluting peptides.

// allow only such stripes of max. length around the diagonal [#]

max. nb. stripes=1

//

// sequence alignment comparsion: defines the weight with which peptide identification information

// is used in the matching of common lc/ms peaks between runs ( 0(not used) - 5000)

MS2 info alignment weight=0

//

// maximal smoothing error: when calculating the upper / lower error of the fitted delta

// do not allow an error that is bigger then this paramater [min]

maximal smoothing error=3.0

//

// % outside error delta points: how many percentage of points can still lay outside the alignment error

// borders in order to stop the alignment iterations

//

perc. outside error delta points=0.75

//-------------------------------------------------------------------------------------------------------

//

// LC-MS correlations

//

// intensity bin size: used to correlate 2 LC-MS peaks also by their intensity

// compares in which bin the 2 peaks are, for this use a bin size

intensity bin size=2000

//

// intensity bin tolerance: in the comparison of intensity bins, how far to bins can be appart

// and still be accepted for same

intensity bin tolerance=2

//

// min. LC/MS correlation score: represents the worst score possible, this one will be used to

// normaize the observed scores between 0(bad) and 1(good) [ 0 ... 1]

minimal LC/MS score=0.1

//

// LC/MS sim. score modus: which scoring system to use for LC/MS similarity:

// - [ALIGN]: asssessment of uncertainty in the alignment

// - [INTNES]: asssessment of ranking correlation of peak areas

// _ [PEAK_MATCHING]: according to how many features overlap

// _ [TOTAL]: combination of all scores:

// _ [NORM_TOTAL]: normallized score of total score:

LC/MS sim. score modus=TOTAL

//-------------------------------------------------------------------------------------------------------

//

// MS1 PEAK DETECTION PARAMETERS FOR THE DIFFERENT FILTER METHODS:

//

// Create monoisotopic LC profile: to create and store the original profile of the detected

// monosiotopic pecursors in the XML (!!! increases the

// XML file size!!! (on[1]/off[0])

Create monoisotopic LC profile=1

//

//

// FT MS1 data centroid data : define if ipnut FT-LTQ data is in centriod mode (1)

// or ectract data from profile mzXMLs (0)

FT MS1 data centroid data=0

//

// mz cluster tolerance : defines which tolerance is used to cluster different

// m/z values into a m/z cluster

FT peak detect MS1 m/z tolerance=0.01

//

//

// MS1 minimal # peak members: minimal number of members in an LC elution peak, if

// an elution peaks is discarded if it has less member

FT peak detect MS1 min nb peak members=4

//

// MS1 minimal intensity : all peaks with small intensity are not considered

FT peak detect MS1 intensity min threshold=20000

//

// MS1 intensity cut off : used to discard peak with too low intensity in a

// LC elution cluster. peak which are less x% of the

// cluster apex peak intensity are removed [ 1 .. 0]

MS1 intensity apex percentil cutoff=0.1

//

// MS1 max scan member distance: defines how many scans can be between members of

// a LC elution peak (MS2 scans are not inlcuded!!!)

MS1 max inter scan distance=5

//

// Tr resolution: used for to compute the peak area of an LC peak

// in the integration process

MS1 LC retention time resolution=0.01

//

// Peak detection absolute mass precision in Dalton (between isotopes) 0.01

Absolute isotope mass precision=0.05

//

// Peak detection relative mass precision in ppm (between isotopes) 10

Relative isotope mass precision=10

//

// Centroid is calculated in window of this size around local maxima

Centroid window width=5

//

// Coefficient of variance for intensities (also includes deviation from

IntensityCV=0.9

//

// Factor (f) to define which isotopic peaks are detectable relative to highest isotopic peak I_max: I_iso > I_max*f

Detectable isotope factor=0.2

//

// Minimal peak height (peaks smaller than this values are not considered as monoisotopic peaks)

Minimal peak height=0.0

//

// Intensity values below this value are considered as zero (before peak detection)

Intensity ground level=1.0

//

// Report all found monoisotopic peak to file mono_peaks.txt

Report mono peaks=0

//

// Directory where debug files are written

Debug directory=

//

// if "Report mono peaks"==1 the info about the peak detection at this scan number will be written to debug files

Report scan number=0

Supplementary references

Abramoff, M.D., Magalhaes, P.J. and Ram, S.J. 2004. Image processing with ImageJ. Biophoton Int 11(7): 36-41.

Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J. and Gygi, S.P. 2006. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24(10): 1285-1292.

Beckouet, F., Labarre-Mariotte, S., Albert, B., Imazawa, Y., Werner, M., Gadal, O., Nogi, Y. and Thuriaux, P. 2008. Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription. Mol Cell Biol 28(5): 1596-1605.

Bianchi, A., Negrini, S. and Shore, D. 2004. Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1. Mol Cell 16(1): 139-146.

Bodenmiller, B., Mueller, L.N., Mueller, M., Domon, B. and Aebersold, R. 2007. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4(3): 231-237.

Elias, J.E. and Gygi, S.P. 2007. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4(3): 207-214.

Eng, J.K., McCormack, A.L. and Yates, J.R. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal Of The American Society For Mass Spectrometry 5(11): 976-989.

Gruhler, A., Olsen, J.V., Mohammed, S., Mortensen, P., Faergeman, N.J., Mann, M. and Jensen, O.N. 2005. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol Cell Proteomics 4(3): 310-327.

Keller, A., Eng, J., Zhang, N., Li, X.J. and Aebersold, R. 2005. A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol Syst Biol 1: 2005 0017.

Keller, A., Nesvizhskii, A.I., Kolker, E. and Aebersold, R. 2002. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74(20): 5383-5392.

Laferte, A., Favry, E., Sentenac, A., Riva, M., Carles, C. and Chedin, S. 2006. The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev 20(15): 2030-2040.

Lee, J., Moir, R.D. and Willis, I.M. 2009. Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway. J Biol Chem 284(19): 12604-12608.

Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J.L., Bonenfant, D., Oppliger, W., Jenoe, P. and Hall, M.N. 2002. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 10(3): 457-468.

Pinkse, M.W.H., Uitto, P.M., Hilhorst, M.J., Ooms, B. and Heck, A.J.R. 2004. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76(14): 3935-3943.

Urban, J., Soulard, A., Huber, A., Lippman, S., Mukhopadhyay, D., Deloche, O., Wanke, V., Anrather, D., Ammerer, G., Riezman, H. et al. 2007. Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell 26(5): 663-674.

Winzeler, E.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J.D., Bussey, H. et al. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429): 901-906.

Yorimitsu, T., Zaman, S., Broach, J.R. and Klionsky, D.J. 2007. Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol Biol Cell 18(10): 4180-4189.

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