Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cell phenotypes.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. Here, we decided to test the significance of the hetero-oligomeric nature of CCT for its function by introducing, in each of the eight subunits in turn, an identical mutation at a position involved in ATP binding and conserved in all the subunits, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, cells with the mutation in CCT2 have an excess of actin patches and are the only pseudo-diploid strain. By contrast, cells with the mutation in CCT7 are the only ones to accumulate juxta-nuclear protein aggregates that may reflect the absence of stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks including ribosome biogenesis and TOR2 that help to explain the phenotypic variability observed We used microarrays to reveal the differences in mRNA expression caused by the different mutations. All yeast strains were grown at 30 °C to OD(600)=0.5. Their total RNA was extracted and reverse transcribed to cDNA and transcribed back to RNA in the presence of biotinylated nucleotide analog. The biotinylated RNA was fragmented and hybridized to GenCHip Yeast Genome 2.0 array.

真核胞质含TCP-1的伴侣蛋白复合体（chaperonin-containing TCP-1, CCT）是由两个背对背堆叠的异源八聚体环组成的复合物，以ATP依赖的方式辅助肌动蛋白、微管蛋白及其他蛋白质的折叠。本研究旨在通过在CCT的八个亚基中依次各引入一处位于ATP结合位点且在所有亚基中均保守的相同突变，以此探究该复合体的异源寡聚化特性对其功能的意义，进而明确各亚基的“个体特异性”程度。研究结果表明，这些相同的突变会引发截然不同的表型。例如，携带CCT2亚基突变的细胞会出现肌动蛋白斑过量的现象，且是唯一的假二倍体菌株；与之相反，仅携带CCT7亚基突变的细胞会积累核旁蛋白质聚集体，这或许反映了该菌株不存在应激反应。通过RNA微阵列对各菌株开展系统级分析，本研究揭示了CCT与核糖体生物发生、TOR2等多个细胞网络之间的关联，这有助于解释本次研究中观察到的表型差异。本研究利用微阵列技术，旨在揭示不同突变所导致的mRNA表达差异。所有酵母菌株均在30℃下培养至光密度OD(600)=0.5，随后提取其总RNA并反转录为cDNA，再以cDNA为模板在生物素标记的核苷酸类似物存在下转录为RNA。将生物素标记的RNA片段化后，与GenCHip酵母基因组2.0芯片进行杂交。