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

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.

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Figure 1: Microarray-based method for identifying overexpression plasmids that affect sensitivity to a small molecule.
Figure 2: Amplification method.
Figure 3: Identification of overexpression strains that affect sensitivity to rapamycin.
Figure 4: Identification of overexpression strains that affect sensitivity to LY-83583.
Figure 5: Several overexpression strains with altered sensitivity to the rapamycin suppressor LY-83583 and to rapamycin have enrichment and depletion characteristics that are anticorrelated.

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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.

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Correspondence to Stuart L Schreiber.

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Supplementary information

Supplementary Fig. 1

Analysis of amplification method. (PDF 256 kb)

Supplementary Fig. 2

Correlation plot for the two replicate microarray experiments of rapamycin treatment. (PDF 428 kb)

Supplementary Fig. 3

Wild-type yeast are hypersensitive to LY-83583 on non-fermentable carbon sources. (PDF 172 kb)

Supplementary Fig. 4

Inhibition of Guf1p GTPase activity by LY-83583. (PDF 204 kb)

Supplementary Table 1

Overexpression strains that showed resistance or hypersensitivity to rapamycin. (PDF 47 kb)

Supplementary Table 2

Complete data set. (XLS 865 kb)

Supplementary Table 3

Retesting of the rapamycin sensitivity of strains that were enriched or depleted after rapamycin treatment. (PDF 14 kb)

Supplementary Table 4

Retesting of the LY-83583 sensitivity of strains that were enriched or depleted after LY-83583 treatment. (PDF 15 kb)

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Butcher, R., Bhullar, B., Perlstein, E. et al. Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway. Nat Chem Biol 2, 103–109 (2006). https://doi.org/10.1038/nchembio762

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