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Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway

Nature Chemical Biology volume 2, pages 103109 (2006) | Download Citation

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Abstract

Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.

  • Compound

    Rapamycin

  • Compound

    6-(phenylamino)-5,8-quinolinedione

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Acknowledgements

The authors thank M. Hall for the gift of the fpr1-8 strain, C.L. Liu for advice regarding the in vitro transcription amplification strategy and for help with data analysis, and A. Shamji for a careful reading of this manuscript. This work was supported by GM38627 (awarded to S.L.S.). The construction of the collection of yeast overexpression plasmids was supported by National Human Genome Research Institute R01-HG002923 (awarded to J.L.). S.L.S. is an Investigator at the Howard Hughes Medical Institute. R.A.B. was supported by a graduate fellowship from the National Science Foundation.

Author information

Author notes

    • Gerald Marsischky

    Present address: Codon Devices, 1 Kendall Square, Building 700, Cambridge, Massachusetts 02139, USA.

Affiliations

  1. Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

    • Rebecca A Butcher
    •  & Stuart L Schreiber
  2. Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02141, USA.

    • Rebecca A Butcher
    • , Ethan O Perlstein
    •  & Stuart L Schreiber
  3. Harvard Institute of Proteomics, 320 Charles Street, Cambridge, Massachusetts 02141, USA.

    • Bhupinder S Bhullar
    • , Gerald Marsischky
    •  & Joshua LaBaer
  4. Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.

    • Ethan O Perlstein
    •  & Stuart L Schreiber

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stuart L Schreiber.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Analysis of amplification method.

  2. 2.

    Supplementary Fig. 2

    Correlation plot for the two replicate microarray experiments of rapamycin treatment.

  3. 3.

    Supplementary Fig. 3

    Wild-type yeast are hypersensitive to LY-83583 on non-fermentable carbon sources.

  4. 4.

    Supplementary Fig. 4

    Inhibition of Guf1p GTPase activity by LY-83583.

  5. 5.

    Supplementary Table 1

    Overexpression strains that showed resistance or hypersensitivity to rapamycin.

  6. 6.

    Supplementary Table 3

    Retesting of the rapamycin sensitivity of strains that were enriched or depleted after rapamycin treatment.

  7. 7.

    Supplementary Table 4

    Retesting of the LY-83583 sensitivity of strains that were enriched or depleted after LY-83583 treatment.

Excel files

  1. 1.

    Supplementary Table 2

    Complete data set.

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DOI

https://doi.org/10.1038/nchembio762

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