Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system

The molecular mechanism of nuclear envelope (NE) assembly is poorly underst ood, but in a cell-free system made from Xenopus eggs NE assembly is contro lled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1], Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the r oles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei th at actively transport proteins across the NE. Both RCC1 and the GTPase-acti vating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and R anGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precu rsor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by Ra nGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to f orm an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the re cruitment of vesicles and their fusion to form NEs,