Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.
Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.
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DOI：
10.1091/mbc.e10-04-0309
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发表时间：
2010-12
影响因子：
3.3
通讯作者：
Forbes DJ
中科院分区：
文献类型：
--
作者：
Fichtman B;Ramos C;Rasala B;Harel A;Forbes DJ
核孔复合体（NPC）的特征在于连接内外核膜的长寿命膜内衬通道。这种稳定的细胞膜通道（核孔就建在其中）在进化上几乎没有先例。在这份报告中，我们证明和地图的内/外核膜融合在NPC大会。核孔复合物（Nuclear pore complex，NPCs）是一种位于双核膜上的蛋白质通道，可进行核质交换。核孔组装的机制涉及一个独特的挑战，因为它需要创建一个长寿命的膜内衬通道连接内部和外部核膜。这种稳定的膜通道几乎没有进化的先例。在这里，我们映射内/外核膜融合在NPC组装生化使用新的组装中间体和膜融合抑制剂。在14°C下孵育非洲爪蟾体外核组装系统揭示了早期孔中间体，其中核孔蛋白亚基POM 121和Nup 107 -160复合物在内核膜上以点状模式组织。随着时间的推移，这种中间体进展到扩散通道的形成，并最终完成核孔组装。正确的通道形成被半融合抑制剂溶血磷脂酰胆碱（LPC）阻断，但如果同时加入互补形状的脂质，油酸（OA），则不会被阻断，如用新型荧光葡聚糖淬灭测定法所确定的。重要的是，在扩散通道形成之前未观察到FG核孔蛋白（成熟核孔的特征）的大量募集，并且LPC或OA阻止了该募集，但LPC+OA未阻止该募集。这些结果映射的关键内/外核膜融合事件的NPC组件下游的POM 121/NUP 107 -160复杂的相互作用和上游或在FG核孔蛋白招聘的时间。
The nuclear pore complex (NPC) is characterized by a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel, within which the nuclear pore is built, has little evolutionary precedent. In this report we demonstrate and map the inner/outer nuclear membrane fusion in NPC assembly. Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.