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					                                       Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith       1



 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.     1C-D
                                                                              2005)
                        3E
AH035      MATa; SCH9        (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                      This study          3C, S4C
           yak1::NatMX4
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-                           This study          5E
           13myc::HIS3MX6 [TB50]
AH208      MATa; sch9::KanMX6 tap42::HphMX4 RPA190-TAP::HIS3MX6               This study          5F
           [TB50]
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


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                                     Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith    2

-          Deletion strains in the BY4741 background                        (Winzeler et al.    S3D
                                                                            1999)
W303α      MATα; ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1        wt                  S4A-B, S8B
                         as
AH091      MATα; sch9 (T492G) [W303]                                        This study          S4A-B, S8B
                     as              a                   as
Y3527      MATα; tpk1 (M164G) tpk2 s (M147G) tpk3 (M165G) [W303]            (Yorimitsu et al.   S4A-B, S8B
                                                                            2007)
                    as              as              as               as
Y3528      MATα; tpk (M164G) tpk2        (M147G) tpk3 (M165G) sch9          (Yorimitsu et al.   S4A-B, S8B-C
           (T492G) [W303]                                                   2007)
                         as
AH311-1b   MATα; sch9 ::TRP1 [TB50]                                         This study          S5
                         as
AH311-3a   MATα; sch9 ::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
                         as
AH478      MATα; sch9 (T492G) RPC82-TAP::HIS3MX6 [W303]                     This study          S8C
                     as              a                   as
AH479      MATα; tpk1 (M164G) tpk2 s (M147G) tpk3 (M165G) RPC82-            This study          S8C
           TAP::HIS3MX6 [W303]
                    as              as              as               as
AH480      MATα; tpk (M164G) tpk2 (M147G) tpk3 (M165G) sch9                 This study          S8C
           (T492G) RPC82-TAP::HIS3MX6 [W303]




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                                               Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith      3


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
                           as
pAH145        pRS414::sch9 (T492G)                          This study           3B, 4B-E, 5C-D, S6A, S7C-D, S8A, S9B
                               DE
pAH146        pRS414::SCH9 (T723D, S726D,                   This study           1A, 1D-E, 2B, 3D, 4A, 4F, 5A-B, 5E, 7A,
              T737E, S758E, S765E)                                               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
                                          kd
pAH107        pRS426::PGAL1-GST-SCH9                        (Urban et al.        3E
                                                            2007)
                                          3E
pAH108        pRS426::PGAL1-GST-SCH9                        This study           3E
pAH216        pGEX6P1::MAF1                                 This study           3E
                                    6A1
pAH229        pGEX6P1::MAF1 (S90A, S101A,                   This study           3E
              S177A, S178A, S209A, S210A)
                                    6A2
pAH237        pGEX6P1::MAF1 (S90A, S101A,                   This study           3E
              S178A, S179A, S209A, S210A)
                                    7A
pAH236        pGEX6P1::MAF1 (S90A, S101A,                   This study           3E
              S177A, S178A, S179A, S209A, S210A)
pAH095        pRS415::MAF1                                  This study           4B-E, 6C, S6A-B
                               7E
pAH238        pRS415::MAF1 (S90E, S101E,                    This study           4C-D, S6A-B
              S177E, S178E, S179E, S209E, S210E)
                               7A
pAH247        pRS415::MAF1 (S90A, S101A,                    This study           4C-D, S6A-B
              S177A, S178A, S179A, S209A, S210A)
                               7E
pAH240        pRS416::MAF1 -3HA (S90E, S101E,               This study           4F
              S177E, S178E, S179E, S209E, S210E)
pAH205        pRS416::RRN3-5HA                              This study           5F
pAH262        pRS416::TOD6-5HA                              This study           S1A
                               6A
pAH268        pRS416::TOD6 -5HA (S280A S298A                This study           S1A
              S308A S318A S333A S346A)
                               5A
pAH272        pRS416::DOT6 -5HA (S247A S282A                This study           S1A
              S313A S335A S368A)
pAH217        pRS416::MAF1-GFP                              This study           S7A-D
                               7A
pAH248        pRS416::MAF1 -3HA (S90A, S101A,               This study           S6A
              S177A, S178A, 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.

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                                                 Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   4


Table S3 Screens overview
                 Total          Number phosphorylation                Total distinct             Total distinct
                                                                                      d
                 detected       patterns / Common features            phosphopeptides            phosphoproteins
                 features       over all patterns
            a
Screen 1         47,515         6 / 7,832                             924                        521
            b
Screen 2         65,570         12 / 5,263                            1,156                      604
            c
Screen 3         67,150         12 / 5,216                            1,470                      684
  a
      wt cells vs. wt cells treated with cycloheximide
  b                                     3E
      wt cells +/- rapamycin vs. SCH9        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




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                                             Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   5


Table S4 Summary of the phosphoproteomic screens
             Total distinct        Downregulated           Upregulated            SCH9-dependent         TAP42-dependent
                                                   a                       b
             phosphopeptides       phosphopeptides         phosphopeptides        phosphopeptides        phosphopeptides
             (proteins)            (proteins)              (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




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                                                Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith           6


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




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                                     Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   7


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
                                            6A          5A
R[R/K]xS motifs in the two proteins (TOD6 and DOT6 ) 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.




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                                     Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   8


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.




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                                       Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   9


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.




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                                       Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   10


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.




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                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   11

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-
      Pro
tRNA were measured by quantitative RT-PCRs. Data are means of three independent experiments +/- s.d.
           Pro                         as
Pre-tRNA levels dropped upon Sch9 inhibition by 1NMPP1 in a Maf1-dependent manner. *** P < 0.001
               as
vs. the sch9 MAF1 control. ### P < 0.001 vs. the untreated isogenic control.




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                                     Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   12


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
                  7E        7A
expressing Maf1 or Maf1 display phenotypes intermediate between maf1 and MAF1 cells. B. Rapamycin
                                                                                                       7E
treatment results in a Maf1-dependent drop in the synthesis of tRNA and 5S rRNA. Cells expressing Maf1
        7A
or Maf1 display phenotypes intermediate between maf1 and MAF1 cells. The * indicates the migration of a
probable 5S degradation product.




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                                         Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   13


Figure S7 Sch9 regulates Maf1 localization
                                                                               DE
A. Rapamycin-induced Maf1 nuclear accumulation is not blocked by Sch9 . AH220 (sch9 HHF2-mCherry)
                                               wt                    DE
cells, complemented with pAH144 (SCH9 ) or pAH146 (SCH9 ) 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 <0.05, ### P <
0.001 vs. untreated isogenic control.




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                                       Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   14




Figure S8 Maf1 dephosphorylation controls its binding to RNA Pol III
            7A
A. The Maf1 mutant mimicking dephosphorylation constitutively interacts with RNA Pol III. Interaction of
Maf1-3HA with RNA Pol III was assessed by Rpc82-TAP-pulldowns followed by SDS-PAGE and western
blotting. Relevant genotypes (TB50 background) and rapamycin and 1NMPP1 treatments are indicated. B.
Cooling yeast cells induces a PKA-dependent hyperphosphorylation of Maf1. Cells of the indicated
genotypes (W303 background) and expressing Maf1-3HA were subjected to control or 200 nM 1NMPP1
treatment for 30 min. Where indicated (Cooling), cells were cooled in an ice/water bath for 15 minutes prior
to TCA addition, protein extraction and western blotting. Maf1 dephosphorylation triggered by Sch9 inhibition
is reversed by the cooling of cells prior to TCA addition and this effect is dependent on PKA activity. This
phenomenon probably explains why no (Fig. S8A) or little (Fig. S8C) Maf1-RNA Pol III interaction induction
was observed in co-purification assays upon Sch9 inhibition alone, and why Lee et al conclude that both
Sch9 and PKA each play prominent roles in Maf1 phosphorylation (Lee et al. 2009). C. Inhibition of Sch9 and
PKA co-operately induce Maf1 dephosphorylation and association to RNA Pol III in the W303 background.
Interaction of Maf1-3HA with RNA Pol III was assessed by Rpc82-TAP-pulldowns followed by SDS-PAGE
and western blotting. Relevant genotypes and 1NMPP1 treatments are indicated.




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                                     Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   15


Figure S9 Primer extension assays
A. AH090-7a (sch9 tap42) cells complemented with indicated alleles of SCH9 and TAP42, were grown in
YPD and subjected to rapamycin treatment. Total RNA was extracted at the indicated time points and
assayed for 35S vs. 18S rRNA levels by primer extension. B. RL215-4c (sch9) cells complemented with
indicated SCH9 alleles and made prototroph with pAH152 and pRS416 were grown in YPD and subjected to
1NM-PP1 treatment. Total RNA was extracted at the indicated time points and assayed for 35S vs. 18S rRNA
levels by primer extension.




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                                               Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith      16


Material and methods
Phosphopeptides isolation for phosphoproteomics

                                                                                                              o
  Disulfide bonds were reduced with tris(2-carboxyethyl)phosphine at a final concentration of 10 mM at 37 C for 1 h.

Free thiols were alkylated with 40 mM iodoacetamide at room temperature for 1 h. The solution was diluted with 20 mM

TrisHCl (pH 8.3) to a final concentration of 1.0 M urea and digested with sequencing-grade modified trypsin (Promega,
                                                                   o
Madison, Wisconsin) at 10 µg per mg of protein overnight at 37 C. Peptides were desalted on a C18 Sep-Pak cartridge

(Waters, Milford, Massachusetts) and dried in a speedvac.

  Phosphopeptides were enriched from 1 mg of peptides using titanium dioxide (TiO2) as described previously (Pinkse

et al. 2004; Bodenmiller et al. 2007). In short: The peptides were resuspended in an 80 % acetonitrile (ACN), 3.5 %

trifluoroacetic acid (TFA) solution saturated with phthalic acid. Peptides were added to 1.2 mg equilibrated TiO2 (5 m

bead size, GL Science, Saitama, Japan) in a blocked mobicol spin column (MoBiTec, Göttingen, Germany) and was

incubated for 30 min with end-over-end rotation. The column was washed two times with the saturated phthalic acid

solution, two times with 80 % ACN, 0.1 % TFA and finally two times with 0.1 % TFA. The peptides were eluted with a 0.3

M NH4OH solution. After elution the pH was adjusted to 2.7 using a 10 % TFA solution and phosphopeptides were

purified using an appropriate C18 cartridge.


LC-MS(/MS) analysis

  Chromatographic separation of peptides was achieved on an Eksigent nano LC system (Eksigent Technologies,

Dublin, CA, USA), equipped with a 11 cm fused silica emitter, 75 µm inner diameter (BGB Analytik, Böckten,

Switzerland), packed in-house with a Magic C18 AQ 5 µm resin (Michrom BioResources, Auburn, CA, USA). Peptides

were loaded from a cooled (4°C) Spark Holland auto sampler and separated using ACN/water solvent system containing

0.2% formic acid with a flow rate of 200 nl/min. Peptide mixtures were separated with a gradient from 3 to 28 % ACN in
                                        2
90 minutes. Up to 3 data-dependent MS spectra were acquired in the linear ion trap for each Orbitrap-MS spectral

acquisition range, the latter acquired at 60,000 FWHM nominal resolution. Charge state screening was employed to

either select over a complete LC-MS/MS analysis for ions with two charges and rejecting ion with one, three or higher

and undetermined charge state, or to select over a complete run ions with three charges and higher (excluding 1, 2 and
                                                                                                      5           4
undetermined charge state). For injection control the automatic gain control (AGC) was set to 5 · 10 and 3 · 10 for full
                                   2
Orbitrap-MS and linear ion trap MS respectively. The instrument was calibrated externally according to manufacturer

instructions and used the lock masses at m/z 429.0887 and m/z 445.12 to recalibrate each spectrum internally.


Mass spectrometric data analysis

  The fragment ion spectra a were queried against a decoy version of the SGD non redundant database containing

13,590 (6,795 forward protein entries and 6,795 reversed protein entries) (Elias and Gygi 2007) using the SORCERER-

SEQUEST(TM) v3.0.3 (Eng et al. 1994) which was run on the SageN Sorcerer2 (Thermo Electron, San Jose, CA, USA).

                                                                                                                          16
                                             Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith           17

For the in silico digest, trypsin was defined as protease, cleaving after K and R (if followed by P the cleavage was not

allowed). Two missed cleavages and one non-tryptic terminus were allowed for the peptides which had a maximum mass

of 6,000 Da. The precursor ion tolerance was set to 15 ppm and fragment ion tolerance was set to 0.5 Da. The data were

searched allowing phosphorylation (+79.9663 Da) of serine, threonine and tyrosine as a variable modification and

carboxyamidomethylation of cysteine (+57.0214 Da) residues as a fixed modification. In the end, the database search

results obtained by Sequest were subjected to statistical filtering in order to estimate the false positive rate of peptide

assignment. For this purpose the PeptideProphet (V3.0) (Keller et al. 2005) and decoy database approach were

exploited (Elias and Gygi 2007). Of note, the dCn between the first and second ranked hit for a tandem mass spectrum

as determined by Sequest was used to compute the dCn, allowing to estimate the accuracy of phosphorylation site

location (Supplemental File F1) (Beausoleil et al. 2006). A phosphopeptide with a dCn > 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.


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.


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

                                                                                                                              17
                                            Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith       18

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.


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.

       3
  For H-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
                                                                             Pro
PCR reactions (Applied biosystems) using primers for ACT1 and pre-tRNA             (ACT1 Fw 5’-GAATT GAGAG TTGCC
                                                                  Pro
CCAGA-3’; Rev 5’-AGAAG GCTGG AACGT TGAAA-3; pre-tRNA                    Fw 5’-GCTTTGGGCGACTTCCTG-3’; Rev 5’-

GGGGCGAGCTGGGAATTGAA-3’).


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).


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.

                                                                                                                        18
                                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   19

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
                                                       32
dGTP, 500 μM dTTP, 5 μM dATP and 5 μCi α- P-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.


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

                                                                                                                             19
                                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   20

//                               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:

                                                                                                                             20
                                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   21

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

                                                                                                                             21
                                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith       22

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
                                                                                                                               22
                                                    Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   23

//                                                   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:
//
                                                                                                                             23
                                          Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   24

// 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
//

                                                                                                                   24
                                        Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   25

//      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-

                                                                                                                 25
                                       Huber, Bodenmiller, Uotila, Stahl, Wanka, Gerrits, Aebersold & Loewith   26

         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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