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© The Rockefeller University Press,
0021-9525/1999//225 $5.00
The Journal of Cell Biology, Volume 144, Number 2,
, 1999 225-240
Regular Articles |
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
Department of Biochemistry, and Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom; and || Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX, United Kingdom
In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition 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 surface 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 ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.
Key Words: Xenopus • nuclear envelope • NEP-B78 • LBR • cell cycle
Abbreviations used in this paper: BRB, blot rinse buffer; DHCC, 3,3'- dihexyloxocarbocyanine; EGS, ethylene glycol bis-(succinic acid N-hydroxysuccinimide ester); INM, inner nuclear membrane; LAPS, lamin-associated proteins; LBR, lamin B receptor; LSS, low speed supernatant; MP1, membrane pellet 1; MPF, maturation promoting factor; NCS, newborn calf serum; NE, nuclear envelope; NEM, N-ethylmaleimide; NEP-B, nuclear envelope precursor binding factor; NSF, NEM-sensitive factor; ONM, outer nuclear membrane; SNAPS, soluble NSF attachment proteins.
Address correspondence to Carl Smythe, MRC Protein Phosphorylation Unit, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, Scotland, UK. Tel.: 44 1382 345095. Fax: 44 1382 223778. E-mail: cgwsmythe{at}bad.dundee.ac.uk or Christopher Hutchison, Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland, UK. Tel.: 44 1382 344728. E-mail: c.j.hutchison{at}dundee.ac.uk
C.J. Hutchison and C. Smythe are members of the Dundee Eukaryotic Membrane Dynamics MRC Cooperative Group at the University of Dundee. This work was supported by the Medical Research Council, Biotechnology and Biological Sciences Research Council, and the Wellcome Trust. C.J. Hutchison is the recipient of a Wellcome Trust Research Leave Fellowship. S. Drummond is the recipient of a BBSRC-CASE quota studentship.
Dr. Ferrigno's current address is SM922, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115.
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