Schlaich N L, Häner M, Lustig A, Aebi U, Hurt E C
Institut für Biochemie I, University of Heidelberg, Germany.
Mol Biol Cell. 1997 Jan;8(1):33-46. doi: 10.1091/mbc.8.1.33.
The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.
酵母核孔蛋白Nsp1p、Nup49p和Nup57p在核孔处形成一个复合体，该复合体参与核质运输。为了研究复合体形成的分子基础，在大肠杆菌中表达了重组全长Nup49p和Nup57p以及Nsp1p的羧基末端结构域（其缺乏FXFG重复结构域）。当将这三种纯化的蛋白混合在一起时，它们自发缔合形成一个化学计量比为1:1:1的150 kDa复合体。在这个三聚体复合体中，Nup57p充当组织中心的角色，Nup49p和Nsp1p分别与之结合。为了发生这种相互作用，仅需要Nsp1p羧基末端结构域的两个七肽重复区域，每个区域长度约为50个氨基酸。最后，重构的复合体能够结合全长Nic96p，但不能结合在体内也不相互作用的突变形式。当添加到透化的酵母细胞中时，该复合体与核膜和核孔结合。我们得出结论，Nsp1p、Nup49p和Nup57p能够在体外重构一个复合体，该复合体能够与核孔的其他组分进一步组装。
