Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymp hocyte granule proteinase granzyme B (graB) and is thought to protect cytot oxic lymphocytes and bystander cells from graB-mediated apoptosis. Followin g uptake into cells, graB promotes DNA degradation, rapidly translocating t o the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cyto toxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8 , plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhi bit similar nucleocytoplasmic distributions. Because these serpins lack cla ssical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively . Large (similar to 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleo cytoplasmic distributions to the parent proteins, indicating that nuclear i mport is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion. that it re quires cytosolic factors but not ATP, and that it does not bind an intranuc lear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We co nclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.