Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin.
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.
Takasuga
Y
Y
Department of Biology, Faculty of Science, Kwansei Gakuin University, Hyogo, Japan.
Yagura
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002474:Cell-Free System; D002843:Chromatin / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D000072200:Ku Autoantigen; D008297:Male; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000502:physiology*; D010063:Ovum; D013077:Sperm Head; D014981:Xenopus
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D005033:Ethylmaleimide