Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Mol Cell Proteomics. 2010 Feb;9(2):271-84. doi: 10.1074/mcp.M900415-MCP200. Epub 2009 Nov 10.
To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Deltaggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.
为了鉴定雷帕霉素（一种抗癌和免疫抑制剂）的新分子靶点，我们在转录组和蛋白质组水平上分析了酵母在 6 小时内对雷帕霉素的反应的时间变化，并将表达模式与功能谱整合在一起。我们表明，转录组学、蛋白质组学和功能数据集的整合为雷帕霉素作用的分子机制提供了新的见解。我们首先观察到 mRNA 和蛋白质表达之间存在时间延迟，其中在 1 小时和 2 小时时的 mRNA 表达与雷帕霉素处理 6 小时后蛋白质表达变化的相关性最佳。这种情况尤其适用于核糖体生物发生的抑制和热休克和自噬的诱导，这对于促进细胞对雷帕霉素的敏感性至关重要。然而，液泡蛋白酶水平的增加可以增强对雷帕霉素的抗性。在 85 个被鉴定为丰度发生统计学显著变化的蛋白质中，大多数丰度降低的蛋白质与 mRNA 表达降低相关。然而，在 56 个丰度增加的蛋白质中，有 26 个与 mRNA 表达增加不相关。这些蛋白质变化与 mRNA 表达不变或下调相关。这些参与线粒体基因组维护、内吞作用或药物外排的蛋白质代表了影响雷帕霉素作用的新候选物，其表达可能受到转录后或翻译后调控。我们鉴定了 GGC1，一种线粒体 GTP/GDP 载体，作为雷帕霉素/雷帕霉素靶蛋白（TOR）信号通路的新组成部分。我们确定 GGC1 的蛋白质产物在雷帕霉素存在下稳定，并且 GGC1 的缺失增强了雷帕霉素存在下的生长适应性。对用雷帕霉素处理的 Deltaggc1 和野生型细胞的动态 mRNA 表达分析表明，Ggc1p 在 TOR 控制下核糖体生物发生和细胞周期进程的调节中起着关键作用。
