Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.
Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.
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DOI：
10.1007/s00438-012-0709-5
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发表时间：
2012-09
期刊：
影响因子：
--
通讯作者：
Yao G
中科院分区：
文献类型：
--
作者：
Richardson R;Denis CL;Zhang C;Nielsen MEO;Chiang YC;Kierkegaard M;Wang X;Lee DJ;Andersen JS;Yao G
多聚腺苷酸结合蛋白（Poly（A）binding protein，PAB 1）参与真核细胞的多种RNA代谢功能，并与多种蛋白质结合。我们已经使用质谱分析，以确定55个非核糖体蛋白质，特异性相互作用的PAB 1从酿酒酵母。由于这些因子中的许多因子可能仅通过PAB 1-mRNP结构的组分与PAB 1间接相关，因此我们还对7种代谢定义的PAB 1缺失衍生物进行了质谱分析，以界定这些蛋白质与PAB 1之间的相互作用。这些后者的分析确定了13种蛋白质，其与PAB 1的关联通过删除PAB 1的一个或另一个定义的结构域而减少。在这13种蛋白质的列表中包括翻译起始因子eIF 4G 1和eIF 4G 2，翻译终止因子eRF 3和PBP 2，所有这些先前已知的与特定PAB 1结构域的直接相互作用都得到了证实，划界或扩展。通过特异性PAB 1结构域相互作用的其余9种蛋白质是CBF 5、SLF 1、UPF 1、CBC 1、SSD 1、N 0 P77、yGR 250 c、NAB 6和GBP 2。在进一步的研究中，参与无义介导的衰变的UPF 1被证实通过RRM 1结构域与PAB 1相互作用。我们还确定，虽然PAB 1的RRM 1结构域是在无义介导的衰变过程中UPF 1诱导的去腺苷化加速所必需的，但它不是加速mRNA去帽的更关键步骤所必需的。这些结果开始鉴定最有可能与PAB 1相互作用的蛋白质和PAB 1的结构域，通过这些结构域进行接触。
Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1’s defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.
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