Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types

In this work, we have used novel mAbs against two proteins of the endoplasm ic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in additio n to a polyclonal antibody against the inner nuclear membrane protein LBR ( lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surf ace of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordere d one and that NEP-B78 defines a vesicle population involved in the earlies t events of reassembly in this system. Finally, we present evidence that NE P-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implicat ions for the understanding of the mechanisms of nuclear envelope disassembl y and reassembly during mitosis and for the development of systems to ident ify novel molecules that control these processes.