Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins
Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins
复制标题
DOI：
10.1093/emboj/21.3.387
复制
发表时间：
2002-02-01
期刊：
影响因子：
11.4
通讯作者：
Hurt, E
中科院分区：
文献类型：
--
作者：
Lutzmann, M;Kunze, R;Hurt, E
现在很可能所有的酵母核孔蛋白都是已知的，最终的目标之一是在体外组装整个核孔复合体，从它的类似的30个单独的成分。在这里，我们报道了七种蛋白质(Nup133p、Nup145p-C、Nup120p、Nup85p、Nup84p、Seh1p和Sec13p)重组为一个七聚体0.5MDA核孔亚基的过程。我们发现，双质粒转化结合双顺反子mRNA翻译允许在单个大肠杆菌细胞中表达和组装多达五个核孔蛋白的不同亚复合体。在Nup84P复合体的顺序重组过程中，可以分离出较小的组装中间体，通过电子显微镜确定这些中间体呈现模块化结构，最终形成整个Y形Nup84P复合体。重要的是，第七个亚基Nup133p通过与Nup84p的相互作用被整合到该复合体中，从而将Y形组装的一条臂延长到类似于40 nm长的茎。综上所述，我们的数据证明，Nup84P-Nup133P复合体是在一个模块化的概念中自组装而成的，它来自不同的较小的核孔蛋白结构集。
Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its similar to30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an similar to40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.