Takasuga Y, Yagura T
Department of Biology, Faculty of Science, Kwansei Gakuin University, Hyogo, Japan.
Cell Struct Funct. 1993 Aug;18(4):205-10. doi: 10.1247/csf.18.205.
To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly. The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract. Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex. Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide. Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly. The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein. However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure.
为研究有丝分裂末期核膜装配的过程，我们开发了一种无染色质的体外系统，用于在非洲爪蟾间期卵提取物中装配包膜结构，并检测了Ku同源蛋白在该装配过程中的参与情况。在使提取物流动产生最小化的条件下，包膜结构在玻璃板之间无染色质或DNA的情况下自发装配。使用电子显微镜进行的形态学研究表明，围绕包膜结构的膜是双层膜，其中包含类似核孔复合体的间隙。它们的装配依赖于ATP，并受到GTP-γ-S或N-乙基马来酰亚胺添加的抑制。通过将间期卵提取物与大量过量的精子头部染色质预孵育来耗尽核前囊泡，会损害装配过程。与非洲爪蟾的Ku同源蛋白相关的膜囊泡，通过与针对Ku p70蛋白制备的单克隆抗体反应证明参与了装配。然而，该抗体仅轻微抑制了包膜结构的装配过程，这表明Ku同源蛋白不参与囊泡融合形成包膜结构的过程。
