Lakshminarasimhan Mahadevan, Boanca Gina, Banks Charles A S, Hattem Gaye L, Gabriel Ana E, Groppe Brad D, Smoyer Christine, Malanowski Kate E, Peak Allison, Florens Laurence, Washburn Michael P
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110 and.
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110 and §Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160
Mol Cell Proteomics. 2016 Mar;15(3):960-74. doi: 10.1074/mcp.M115.053165. Epub 2016 Feb 1.
The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.
高度保守的酵母R2TP复合物由Rvb1、Rvb2、Pih1和Tah1组成,参与从蛋白质复合物组装到细胞凋亡等多种细胞过程。Rvb1和Rvb2是属于AAA+超家族的密切相关蛋白,对细胞存活至关重要。尽管已证明Rvbs与包括Ino80和Swr1染色质重塑复合物在内的各种蛋白质复合物相关,但我们对其相关蛋白进行了系统的定量蛋白质组分析,并鉴定出另外两种与Rvb1和Rvb2相关的复合物:含伴侣蛋白的T复合物和蛋白酶体复合物的19S调节颗粒。我们还分析了从缺乏PIH1和TAH1的酵母菌株中纯化的Rvb1和Rvb2。这些分析表明,Rvb1和Rvb2仍与Hsp90相关,并且高度富集RNA聚合酶II复合物成分。我们的分析还表明,即使在没有Pih1和Tah1蛋白的情况下,Rvb1和Rvb2也会被招募到Ino80和Swr1染色质重塑复合物中。通过进一步的生化分析,我们表明Rvb1和Rvb2直接与Hsp90以及RNA聚合酶II复合物相互作用。与野生型菌株相比,缺失菌株的RNA-Seq分析显示核糖体生物合成和核糖核蛋白复合物生物合成基因上调,对非生物刺激基因的反应下调,对温度刺激基因的反应下调。对PIH1或TAH1缺失菌株中蛋白质关联发生改变的80种蛋白质进行的基因本体分析发现,核糖核蛋白复合物蛋白是最富集的类别。这表明R2TP复合物在蛋白质组和基因组水平的核糖核蛋白复合物生物合成中具有重要功能。最后,这些结果表明缺失网络分析可以为细胞系统提供新的见解。