| Autor: |
Rafikova ER; Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892-1855, USA., Melikov K, Ramos C, Dye L, Chernomordik LV |
| Abstrakt: |
Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5'-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly. |
