Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex.
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.
D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D022022:Nuclear Pore; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques
D008164:Luminescent Proteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C119239:nuclear pore complex protein 96; D049452:Green Fluorescent Proteins