NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes.
In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.
Salpingidou
Georgia
G
Integrative Cell Biology Laboratories, School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK.
Rzepecki
Ryszard
R
Kiseleva
Elena
E
Lyon
Carol
C
Lane
Birgit
B
Fusiek
Kasia
K
Golebiewska
Anja
A
Drummond
Shoena
S
Allen
Terry
T
Ellis
Juliet A
JA
Smythe
Carl
C
Goldberg
Martin W
MW
Hutchison
Christopher J
CJ
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D022022:Nuclear Pore / Q000378:metabolism* / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
D046911:Macromolecular Substances; D008565:Membrane Proteins; C510250:NDC1 protein, Xenopus; C512903:NUP210 protein, Xenopus; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C082264:Pom121 protein, rat; D029867:Xenopus Proteins