Sec13 Shuttles between the Nucleus and the Cytoplasm and Stably Interacts with Nup96 at the Nuclear Pore Complex

Abstract

Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

ACKNOWLEDGMENTS

We thank Gunter Blobel for helpful discussions and support. We thank Carlos Arana, Papia Chakraborty, and Helene Valentine for assistance in some of the experiments. We thank Helen Shio for technical support of EM samples. We thank Pier P. Pandolfi for the generous gift of the PML-EGFP plasmid. We thank Aurelian Radu, Michel C. Nussenzweig, and Victor Nussenzweig for critical reading of the manuscript and Jurgen Helmers for image processing.

This work was supported by Florida biomedical research grant BM025 (to B.F.), National Institutes of Health grant RO1 GM67159-01 (to B.F.), American Cancer Society institutional grant G98-277 (to B.F.), and by Stanley J. Glaser biomedical research grant 700393 (to B.F.).