done as previously describe with slight modiﬁcations (Watanabe et al., 1997). Modiﬁed RIPA buffer (25 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet NP-40, 1% Na-deoxycholate) was used to break the cells, and 20 mg of total protein extract was used for the immunoprecipitation. The TB50a wild-type strain was used as mock control. A fraction of total lysate (20 g) was kept to check MPK1-HA expression and phosphorylation on Western blots. Zymolyase Sensitivity Assay Zymolyase sensitivity was tested as reported (Ho et al., 2005). Phosphoproteome Analysis: SILAC Labeling and Protein Extraction Two 200-ml cultures of YPJ2 cells (derived from TB50a) were grown at 30°C in SD medium supplemented with either 30 mg/l 12C6-arginine and 12C6- lysine (“light” culture) or 13C6 l-arginine and 13C6,15N2 l-lysine (“heavy” culture) to an optical density (600 nm) of 0.7. The heavy culture was treated for 15 min with 200 nM rapamycin, whereas the light culture was treated mock-treated with the vehicle for 15 min. To produce reliable quantitative data, four biological replicates were performed, always starting from a single yeast clone. In one of the four experiments the labeling was swapped to check that the labeling inﬂuences neither protein expression nor phosphorylation. After rapamycin treatment, both cell cultures were centrifuged at 3500 g for 10 min at 4°C, and the cell pellets were washed with ice-cold water. The cell pellets were individually resuspended in 2 ml ice-cold lysis buffer, containing 100 mM Tris-HCl, pH 7.5, 2.5% SDS, 10% glycerol, 1 protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN; dissolved in ddH2O), 1 phosphatase inhibitor cocktail 1 (Sigma-Aldrich, dissolved in 100% DMSO) and 1 mM PMSF (AppliChem, Darmstadt, Germany; dissolved in 100% DMSO). Total protein extraction from either light or heavy cultures was performed by bead-beating as described above. The lysates were cleared at 15,000 g for 10 min at 4°C. Protein concentrations in the extracts were measured with the bicinchonic acid assay (BCA, Sigma-Aldrich). About 2.5 mg of light- or heavy-labeled protein extracts were mixed and after addition of 6 sample buffer were incubated at 95°C for 5 min and subjected to preparative electrophoresis. Phosphoproteome Analysis: Protein Fractionation and In-Gel Digestion The mixed protein extracts were separated on a preparative 10% SDS slab gel. After electrophoresis, the gel was stained with SimplyBlue SafeStain (Invitro- gen). The gel was then sliced horizontally into 16 regions, and the individual slices were further diced into 1-mm3 cubes. The gel pieces were destained overnight in 1 ml 50% acetonitrile/50 mM NH4HCO3, dehydrated with 500 l 100% acetonitrile, and dried in a speed-vac. The proteins were in-gel reduced in 1 ml 50 mM NH4HCO3 containing 10 mM DTT at 55°C for 60 min. Alkylation was done in 1 ml 50 mM iodoacetamide (in 50 mM NH4HCO3) in the dark for 30 min. After the gel pieces were washed three times with 1 ml 50% acetonitrile/50 mM NH4HCO3, they were dehydrated with 500 l 100% acetonitrile, dried in a speed-vac, and then rehydrated on ice for 1 h in 1 ml 50 mM NH4HCO3, pH 8.0, containing 15 ng/ l trypsin (Sigma). Digestion was carried out overnight at 37°C. Supernatants were collected in fresh tubes and the gel pieces were extracted three times with 50% acetonitrile/5% formic acid, followed by a ﬁnal extraction with 100% acetonitrile. The volume of the individual digests was reduced in a speed-vac to 10 l to which 290 l 1% acetic acid was added. A small drop was spotted onto pH paper, and if necessary the pH was adjusted to 2.0 –2.5 with 10% acetic acid. Phosphoproteome Analysis: Peptide Desalting and Phosphopeptide Enrichment For phosphopeptide enrichment, the digests were desalted on C18 MacroSpin columns (500 l packed resin, The Nest Group, Southborough, MA) accord- ing to the manufacturer’s instructions. The peptides were eluted from the cartridge with 600 l 60% acetonitrile/1% acetic acid. The eluates were speed-vac dried to 10 l to which 90 l IMAC-buffer (30% acetonitrile/250 mM acetic acid) was added. One microliter of each digest was diluted 200-fold with 2% acetonitrile/0.1% acetic acid, and 10 l was analyzed by LC/MS/MS for expression analysis. For phosphopeptide enrichment, 40 l IMAC slurry (PHOS-Select, Sigma- Aldrich) was washed ﬁve times with 1 ml IMAC-buffer and then loaded into a constricted GELoader tip (Thingholm et al., 2006). The desalted digests were applied to the IMAC columns. The ﬂow-throughs were collected and re- loaded ﬁve times to ensure maximal binding. The resin was washed three times with 150 L IMAC-buffer. Bound phosphopeptides were eluted with three successive 70- l desorptions of 50 mM KH2PO4, pH 10.0, into Eppen- dorf tubes containing 30 l 10% formic acid. The IMAC eluates were desalted on disposable C18 MicroSpin columns (MA100 l packed volume, The Nest Group) according to the manufacturer’s instructions. After elution with 200 l 60% acetonitrile/1% acetic acid, the volume was reduced in a speed-vac to 10 l, and the phosphopeptides were diluted with 40 l 2% acetonitrile/ 0.1% formic acid for LC-MS/MS analysis. Vol. 21, October 1, 2010 TORC1 Regulates PKA through MPK1 Phosphoproteome Analysis: LC-MS/MS Analysis LC-MS/MS analysis was performed on an LTQ-Orbitrap hybrid instrument (Thermo Scientiﬁc). Ten microliters of IMAC eluate was injected with an autosampler (CTC Analytics, Agilent Technologies, Basel, Switzerland) onto a C18 trapping column (300SB C-18 0.3 50 mm, Agilent Technologies) that was connected to a separation column (0.1 mm 10 cm) packed with Magic 300 Å C18 reverse-phase material (5- m particle size, Michrom Bioresources, Auburn, CA). A linear 80-min gradient from 2 to 50% solvent B (80% aceto- nitrile/0.1% acetic acid) in solvent A (2% acetonitrile/0.1% acetic acid) was delivered with a Rheos 2200 pump (Flux Instruments, Basel, Switzerland) at a ﬂow rate of 100 l/min. A precolumn split was used to reduce the ﬂow to 300 nl/min. The eluting peptides were ionized at 1.7 kV. The LTQ-Orbitrap was operated in data-dependent mode. A survey scan between m/z 375 and 1600 was acquired in proﬁle mode in the Orbitrap at 60,000 resolution, and the 10 most abundant ions were then selected for fragmentation in the LTQ part of the instrument at normalized collision energy of 35%. To facilitate fragmentation of phosphopeptides, a CID scan was followed by multistage activation. Singly charged ions were omitted from fragmentation, and previously selected ions were dynamically excluded for 25 s. Scan-to-scan calibration was allowed by setting the lock mass to m/z 445.120025 (Olsen et al., 2005). Phosphoproteome Analysis: Database Search, Statistics, and Quantitation To obtain statistically relevant quantitative phosphoproteomes, four indepen- dent SILAC experiments from either untreated or rapamycin-treated cell cultures were collected. The MS data were processed with MaxQuant (version 1.0.12.31;Max Plank Institute of Biochemistry, Martinsreid, Germany) and searched with the Mascot search engine (version 2.2.04, Matrix Science, Lon- don, United Kingdom) against a databank containing 31,426 protein se- quences (Perkins et al., 1999; Cox and Mann, 2008; Cox et al., 2009). The databank contained forward and reverse S. cerevisiae sequences as well as common contaminants (Swiss-Prot; from European Bioinformatics Institute; http://www.ebi.ac.uk/uniprot). Precursor ion and fragment ion mass toler- ances were set to 7 ppm and 0.6 Da, respectively. Two missed cleavages were allowed. Dynamic modiﬁcations were oxidation for methionine and phos- phorylation of serine, threonine, and tyrosine, whereas cysteine carbamidom- ethylation was set as ﬁxed modiﬁcation. To increase identiﬁcation stringency only peptides with a Mascot score above 20 were accepted. In addition, only MS/MS spectra with a posterior error probability (PEP) below 0.1 were accepted (Hilger et al., 2009). The MaxQuant output was simpliﬁed by replac- ing oxidized with unmodiﬁed methionines. In addition, proteins matching the same (phospho)peptides were merged into one hit. Gene products of the Ty retrotransposons and the S. cerevisiae virus L-A were manually removed. For expression analysis, all peptides belonging to the same protein were merged, whereas for phosphorylation analysis, all redundant phosphopep- tides containing the same phosphorylation site(s) were grouped together. To evaluate the biological reproducibility of the four experiments, the ratios of the heavy labels to light labels obtained in one experiment were compared with all other experiments. A high correlation was obtained for all four experiments, indicating good reproducibility throughout the entire study (Supplementary Figure 1A). To ﬁnd changes in expression or phosphoryla- tion that are above the experimental error, an experiment was done without rapamycin treatment. The SILAC ratios were log2 transformed, and the ex- perimental average and SDs were found to be 0.02 and 0.28 (SDcontrol), respectively. Then, the SILAC ratios of the four cell cultures were log2 transformed, and for those proteins or phosphopeptides that occurred in more than one experiment, a 95% conﬁdence interval was calculated along with the median of the log ratios to assess the inﬂuence of outliers. Up- regulation of expression or phosphorylation was considered signiﬁcant when the lower conﬁdence limits were more than or equal to 0.56 (twice the SDcontrol). Likewise, down-regulation of protein expression or the extent of phosphorylation was considered signiﬁcant when the upper conﬁdence limit was less than or equal to 0.56. Those proteins or phosphopeptides that were found in only one experiment but also that showed a large change in abun- dance or phosphorylation were accepted only if the log2-transformed ratios were more than 1.12 or less than 1.12 (four times SDcontrol). The choice of these two thresholds (two and four times the SD of the control, respectively) was made in a conservative way, in order to minimize the number of false positive we obtained. Notably, by using these two selection criteria, among the regulated hits we found many proteins that were already known to be controlled by TORC1 (Supplementary Figure 1, C and D). RESULTS PKA Sites Are Primary Targets for TORC1-dependent Phosphorylation Although it is known that TORC1 regulates many cellular processes, the molecular mechanisms by which TORC1 sig- nals to these diverse processes are often not well under- 3477