Hsf 1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis

TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/ oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections. Citation: Bandhakavi S, Xie H, O’Callaghan B, Sakurai H, Kim D-H, et al (2008) Hsf1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis. PLoS ONE 3(2): e1598. doi:10.1371/journal.pone. 0001598 Editor: Jürg Bähler, Wellcome Trust Sanger Institute, United Kingdom Received July 13, 2007; Accepted January 18, 2008; Published February 13, 2008 Copyright: ! 2008 Bandhakavi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by a proteomics seed grant from the Biomedical Genomics Center at the University of Minnesota, American Heart Association grant 0755679Z, NIH grant DK073731, and a grant to TJG from Eli Lili & Company. Competing Interests: The authors have declared that no competing interests exist. *E-mail: tgriffin@umn.edu