Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex
Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex
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DOI：
10.1006/jmbi.2001.5162
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发表时间：
2001-12-07
影响因子：
5.6
通讯作者：
Denis, CL
中科院分区：
文献类型：
--
作者：
Chen, JJ;Rappsilber, J;Denis, CL
CCR 4-NOT复合物是一种进化上保守的转录调控复合物，参与控制mRNA的起始、延伸和降解。来自酿酒酵母的CCR 4-NOT蛋白存在于两种复合物中，大小为1.9 × 10（6）Da和1.0 × 10（6）Da（1.0 MDa），并且这些复合物的各个组分显示诸如结合和限制TFIID功能、接触佐贺和促进mRNA去腺苷化的不同功能。作为表征1.0 Da复合物的功能作用的第一步，我们将其纯化至接近均一。随后使用质谱分析来鉴定复合物的所有组分。该复合物分子量为1.0 MDa，含有CCR 4、CAF 1、NOT 1 -5和两个新蛋白CAF 40和CAF 130。CAF 130和CAF 40是两种独特的酵母蛋白，CAF 40与其他真核生物的蛋白具有广泛的同源性。免疫沉淀和凝胶过滤实验证实CAF 130和CAF 40是1.9 MDa和1.0 MDa CCR 4-NOT复合物的组分。生物化学分析表明，CAF 40和CAF 130蛋白结合到NOT 1蛋白，并存在于与复合物中的其他两个蛋白亚群分离的位置：CCR 4和CAF 1蛋白，以及NOT 2，NOT 4和NOT 5蛋白。此外，CAF 40能够与人NOT 1相互作用，这表明人CAF 40也是最近鉴定的人CCR 4-NOT复合物的组分。caf 40和caf 130缺失的分析表明，它们引起的表型与其他CCR 4-NOT基因的缺陷相同。CAF 40和CAF 130的不同位置以及CAF 40的进化保守性暗示它们在CCR 4-NOT复合物的功能中具有新的作用。(C)北京：科学出版社.
The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation. The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9 x 10(6) Da and 1.0 x 10(6) Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation. As a first step in characterizing the functional roles of the 1.0 Da complex, we have purified it to near homogeneity. Mass spectrometric analysis was subsequently used to identify all the components of the complex. The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins. Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex. Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes. The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex. (C) 2001 Academic Press.