Poster Sessions

CB -36

Elvira Rafikova

E. Rafikova, K. Melikov, C. Ramos, C. Dye, L. Chernomordik

Cytosol-dependent Fusion of Protein-free Membranes into Nuclear Envelope and Endoplasmic Reticulum

Post-mitotic reassembly of nuclear envelope (NE) and endoplasmic reticulum (ER) has been reconstituted in the cell-free system based on interphase Xenopus egg extract. Mixing membrane vesicles and cytosol isolated from Xenopus laevis egg in the presence of sperm chromatin yields functional nuclei with an ability to actively transport substrates. The nucleus reassembly involves coordinated membrane area growth and nuclear pore complexes insertion. To evaluate relative contributions of cytosolic and transmembrane proteins in post-mitotic reassembly, we replaced a part of native membrane vesicles with those functionally impaired by trypsin or N-ethylmaleimide treatments and with protein-free liposomes. Although neither treated membrane vesicles nor liposomes form ER and functional nuclei on their own, in the presence of cytosol they support both assembly reactions by fusing with native membrane vesicles. At membrane vesicles concentrations insufficient to generate functional nuclei, addition of liposomes rescues nucleus assembly in cytosol-, GTP-, and active nuclear transport- dependent manner. This rescue involves growth of NE area by cytosol- and GTP-dependent fusion between membrane vesicles and liposomes, and restores normal function and spatial distribution of nuclear pores. These findings emphasize an unexpected tolerance of NE assembly to profound changes in the concentrations of transmembrane proteins and lipid composition, and suggest that a feedback mechanism couples NE expansion with nuclear pore assembly.