SUPPORTING INFORMATION
Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen
Jun Chen1,2*, Susan M. Young1,2, Chris Allen1,2, Andrew Seeber7, Marie-Pierre Péli-Gulli9, Nicolas Panchaud9, Anna Waller1,2, Oleg Ursu1,2,4, Tuanli Yao10, Jennifer E. Golden10, J. Jacob Strouse1,2, Mark B. Carter1,2,Huining Kang2,6, Cristian G. Bologa1,2,4, Terry D. Foutz1,2, Bruce S.
Edwards1,2,3, Blake R. Peterson10,11, Jeffrey Aubé10,11, Margaret Werner-Washburne5, Robbie J.
Loewith8, Claudio De Virgilio9, Larry A. Sklar1,2,3*
1Center for Molecular Discovery, 2Cancer Research and Treatment Center, 3Department of Pathology, 4Division of Biocomputing, 5Department of Biology, 6Department of Internal Medicine, University of New Mexico, Albuquerque, NM 87131, United States; 7Swiss National Centre for Competence in Research: Frontiers in Genetics; 8Swiss National Centre for Competence in Research: Frontiers in Genetics and Chemical Biology; Dept. of Molecular Biology, Sciences III, University of Geneva, CH-1211, Geneva, Switzerland;9Department of Biology, Division of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland; 10University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS 66047, United States;
11Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, United States
Table S1. Rapamycin-responsive yeast GFP clones in YPD and SC media
Clones linked to TORC1 Clones not yet linked to TORC1
Clones FC_YPD* FC_SC* Branch Clones FC_YPD* FC_SC* Branch
CIT2 2.58 3.66 RTG GAS1 0.76 0.57 Unlinked
DLD3 2.96 1.27 RTG SER3 1.21 1.65 Unlinked
PYC1 2.96 1.47 RTG BDH1 1.56 2.20 Unlinked
MEP1 1.24 1.56 NDP BDH2 1.58 1.97 Unlinked
PUT1 1.84 1.40 NDP CAN1 1.67 1.59 Unlinked
UGA1 2.29 1.95 NDP ARG4 1.74 1.46 Unlinked
GDH1 2.37 1.33 NDP RTN2 1.75 1.49 Unlinked
DUR1_2 2.76 2.33 NDP MSC1 1.83 2.22 Unlinked
DIP5 2.89 2.47 NDP IDH2 1.85 1.23 Unlinked
CAR1 3.75 1.76 NDP HXK1 1.86 1.57 Unlinked
AGP1 5.09 1.91 NDP PBI2 1.94 1.88 Unlinked
MEP2 7.82 9.02 NDP PRB1 1.99 2.14 Unlinked
GAP1 11.76 7.65 NDP CHA1 2.00 1.26 Unlinked
SRX1 1.11 1.50 Stress CIT1 2.17 1.54 Unlinked
CTT1 1.16 1.55 Stress ASN1 2.45 1.52 Unlinked
DDR2 1.45 2.32 Stress UBI4 2.87 1.83 Unlinked
DCS2 1.54 1.21 Stress PTR2 2.88 2.63 Unlinked
AHP1 1.54 1.27 Stress BAT2 3.66 2.87 Unlinked
HSP26 1.54 5.11 Stress YJL144W 1.18 1.54 Uncharacterized
GDB1 1.59 1.24 Stress YHR087W 2.09 1.78 Uncharacterized
TSA2 1.70 1.33 Stress FMP12 2.34 2.17 Uncharacterized
HSP104 1.76 1.87 Stress FMP16 2.67 2.65 Uncharacterized
HOR7 1.97 1.35 stress
LAP4 1.97 2.42 Stress
PGM2 2.01 1.69 Stress
RPS24B 0.24 0.19 Translation RPL23B 0.42 0.34 Translation RPS18B 0.44 0.34 Translation RPS30B 0.44 0.53 Translation RPL23A 0.46 0.37 Translation RPS30A 0.47 0.56 Translation RPS23B 0.47 0.41 Translation RPL2A 0.47 0.50 Translation RPL40B 0.49 0.39 Translation
RPL5 0.57 0.57 Translation
RPL3 0.60 0.58 Translation
RPL26B 0.60 0.66 Translation RPL19A 0.61 0.65 Translation RPS1B 0.61 0.71 Translation RPS4B 0.62 0.66 Translation RPS22B 0.63 0.55 Translation RPL37A 0.64 0.69 Translation RPL34B 0.64 0.55 Translation
*FC_YPD or FC_SC: GFP Fold Changes in YPD or SC media. Clones with GFP fold change greater than 1.5 (red) or less than 0.67 (Green) were scored positive. Unlinked: gene function is known but is not yet linked to TORC1; Uncharacterized: gene function has not been studied.
Table S2. Strains used in this study
Strain Genotype Source
S288C 0$7Į68&PDOPHOJDO&83IORIOR-1, hap1 ATCC
BY4741 MATa;his3¨OHX¨XUD¨PHW¨ Euroscarf
BY4742 MATD;KLV¨OHX¨XUD¨O\V¨ Euroscarf
YL515 [BY4741/2] MATD;KLVǻOHXǻXUDǻ (1)
MP138-4C [YL515] MATaJOQ¨+<*5his3¨1, leu2¨0, ura3¨0 This study RL194-4C MATa; TCO89-TAP [KITRP1] 3HA-TOR1 [TB50] (2)
Table S3. Plasmids used in this study
Plasmid Description Source
pRS413 HIS3, CEN (3)
pRS416 URA3, CEN (3)
pJU1058 pRS415-Sch9T570A-5HA(LEU2, CEN) (1)
p1290 pHAC33-SCH9T723D, S726D, T737E, S758E, S765E
(URA3, CEN) This study
Reference
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2. Wanke, V., Cameroni, E., Uotila, A., Piccolis, M., Urban, J., Loewith, R., and De Virgilio, C. (2008) Caffeine extends yeast lifespan by targeting TORC1, Mol Microbiol 69, 277-285.
3. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications, Yeast 14, 115-132.
