Macaulay C, Forbes D J
Department of Biology, University of California San Diego, La Jolla 92093-0347, USA.
J Cell Biol. 1996 Jan;132(1-2):5-20. doi: 10.1083/jcb.132.1.5.
A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.
核形成中的一个关键事件是功能性核孔的组装。我们利用源自非洲爪蟾卵的核重建系统来体外研究核孔组装过程。借助该系统，我们鉴定出三种干扰核孔组装的试剂，即N - 乙基马来酰亚胺（NEM）、鸟苷 - 5'-三磷酸γ-硫酯（GTPγS）和钙离子螯合剂1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N'-四乙酸（BAPTA）。这些试剂使我们能够确定核孔的组装需要先形成双层核膜。用NEM预处理膜泡组分抑制核泡融合会阻碍孔复合体的组装。相反，用NEM处理已经融合的双层核膜不会阻碍孔的组装。这表明NEM抑制孔组装中的一个步骤——形成双层核膜所需的囊泡的初始融合。GTPγS的存在在两个不同步骤阻碍孔组装，首先是通过阻止核泡之间的融合，其次是通过阻碍在已经融合的双层核膜上发生的孔组装步骤。有趣的是，当将钙离子螯合剂BAPTA添加到核组装反应中时，它仅短暂地阻断核泡融合，但完全阻断核孔组装。这导致形成一个被双层核膜包围但没有核孔的细胞核。为了确定GTPγS和BAPTA干扰孔组装的位置顺序，开发了一种新型的锚定核组装测定法。该测定法表明，孔组装中对BAPTA敏感的步骤发生在对GTPγS敏感的第二个步骤之后。因此，通过使用体外核重建系统，有可能从生化角度定义和确定核孔组装中的多个步骤顺序。
