In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2

Nuclear envelope precursor vesicles were affinity purified from a Xenopus e gg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by d ifferential centrifugation, PV1 contained larger vesicles, When chromatin w as incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesic les bound to chromatin, fused with each other, formed a bilayered nuclear e nvelope, and assembled into spherical small nuclei. However, the thus assem bled nuclei did not grow to the normal size. Nuclear pore complexes were no t found on the thus assembled nuclei. On the other hand, PV2 contained smal ler vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV 1 supplemented with PV2 was used for the nuclear assembly reaction, the ass embled nuclei grew to the normal size. Nuclear pore complexes existed in th e thus assembled nuclear envelopes. These results suggested that 1) two ves icle populations, PV1 and PV2, are necessary for the assembly of normal siz ed nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane f usion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a mo lecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the a bility to assemble a nuclear membrane, and PV2 is necessary for the assembl y of nuclear pore complexes and for nuclei to grow to the normal size. an i n vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.