Ramos Corinne, Rafikova Elvira R, Melikov Kamran, Chernomordik Leonid V
Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA.
Biochem J. 2006 Dec 15;400(3):393-400. doi: 10.1042/BJ20061218.
All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.
所有已鉴定的膜融合蛋白都是跨膜蛋白。在本研究中，我们探索了有丝分裂后核膜（NE）的重新组装。驱动核膜组装过程中膜重排的蛋白质仍然未知。为了确定跨膜蛋白是否是这种融合机制的必备组分，我们聚焦于无细胞体系中的核重建。将两栖类卵的可溶性间期胞质提取物和膜泡（MV）与染色质混合，会形成功能性细胞核。我们用预先与间期胞质共同孵育的无蛋白磷脂酰胆碱脂质体（LS）取代了MV和胞质溶胶。虽然没有MV的整合蛋白就无法进行产生功能性细胞核的核膜组装后期阶段，但与LS相关的胞质蛋白足以重建膜与染色质的靶向结合以及GTP依赖的脂质混合。结合涉及与LS相关的A型核纤层蛋白，融合涉及Ran GTP酶。因此，与融合后阶段不同，核膜组装中的融合起始，如同出芽和裂变过程中的膜重塑一样，并不需要跨膜蛋白。
