Chromatin-Independent Nuclear Envelope Assembly Induced by Ran GTPase in Xenopus Egg Extracts

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Abstract
The nuclear envelope (NE) forms a controlled boundary between the cytoplasm and the nucleus of eukaryotic cells. To facilitate investigation of mechanisms controlling NE assembly, we developed a cell-free system made from Xenopus laevis eggs to study the process in the absence of chromatin. NEs incorporating nuclear pores were assembled around beads coated with the guanosine triphosphatase Ran, forming pseudo-nuclei that actively imported nuclear proteins. NE assembly required the cycling of guanine nucleotides on Ran and was promoted by RCC1, a nucleotide exchange factor recruited to beads by Ran–guanosine diphosphate (Ran-GDP). Thus, concentration of Ran-GDP followed by generation of Ran-GTP is sufficient to induce NE assembly.