CHARACTERIZATION OF THE BINDING OF NUCLEAR-ENVELOPE PRECURSOR VESICLES AND CHROMATIN, AND PURIFICATION OF THE VESICLES

The binding of nuclear envelope precursor vesicles and chromatin was c haracterized by using an in vitro system constituted from a Xenopus eg g extract and demembranated Xenopus sperm chromatin. The results of bi nding studies in the presence of salts, urea, and a chelator showed th at the binding involves an ionic interaction. Chemical modification st udies suggested that a protein(s) in the vesicles, which is responsibl e for the binding with chromatin, has essential lysine, histidine, and methionine residues. The vesicle protein could not be extracted from vesicles with 1M KCl, 2 M urea, or 0.1 M Na2CO2, suggesting that it is an intrinsic membrane protein. The protein was denatured with 8 M ure a and 0.1 M Na2CO3, and could be renatured by incubation at 23 degrees C, suggesting that the native conformation of the protein is importan t for the binding. Affinity purification of nuclear envelope precursor vesicles was achieved by binding to chromatin and dissociation with 0 .24 M NaCl. The vesicle fraction thus obtained exhibited the ability t o form nuclear envelope on incubation with chromatin in Xenopus egg cy tosol without any other membrane fraction. These results suggested tha t there is a nuclear envelope precursor vesicle population containing both a chromatin targeting protein and vesicle fusion machinery.