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首页> 外文期刊>Journal of Molecular Biology >Cryoelectron Microscopy Imaging of Recombinant and Tissue Derived Vaults: Localization of the MVP N Termini and VPARP.
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Cryoelectron Microscopy Imaging of Recombinant and Tissue Derived Vaults: Localization of the MVP N Termini and VPARP.

机译：重组和组织衍生的保管库的冷冻电子显微镜成像：MVP N Termini和VPARP的定位。

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摘要

The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large beta-propeller fold.
机译：穹顶是在所有高级真核生物中发现的高度保守的核糖核蛋白颗粒。它具有桶形结构，由主要的穹顶蛋白（MVP）组成。穹顶聚（ADP-核糖）聚合酶（VPARP）;端粒酶相关蛋白1（TEP1）;和小的未翻译的RNA（vRNA）。尽管其强大的保守性和高丰度表明了重要的细胞作用，但穹顶的功能尚不清楚。在人类中，穹顶与化疗期间的多药耐药性有关。最近，已经在仅表达MVP的昆虫细胞中建立了重组穹顶的组装。在这里，我们证明了MVP与其他两种穹顶蛋白之一或两者的共表达会导致它们共同组装成规则形状的穹顶。比较了由具有不同长度的N末端肽标签的MVP组装而成的颗粒。使用低温电子显微镜（cryoEM）和单颗粒图像重建方法来确定9个不同组成的重组保管库以及野生型和TEP1缺陷型小鼠保管库的结构。具有MVP N端肽标签的重组穹顶显示出内部密度，其大小随标签的长度而变化。具有富含半胱氨酸标签的重组金库的重建显示该金库的48倍旋转对称性。提出了一个用于在保管库内组织MVP的模型，其中所有MVP N末端在保管库中段非共价相互作用，并且每个半保管库都有48个MVP副本。 CryoEM差异映射将VPARP定位到衬在保管库内表面的三个密度带。设计用于定位TEP1的差异图仅显示了瓶盖内部的较弱密度，这表明TEP1可能通过较小的相互作用区域与MVP相互作用。在没有针对VPARP或TEP1的原子分解结构的情况下，应用了折叠识别方法。预测TEP1 WD重复域共有21个重复，提示β-螺旋桨折叠异常大。

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