Nucleocytoplasmic Distribution of the Ovalbumin Serpin PI-9 Requires a Nonconventional Nuclear Import Pathway and the Export Factor Crm1

Abstract

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to 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 cytotoxic 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 exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (∼70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import 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 requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

ACKNOWLEDGMENTS

We thank B. Henderson (Westmead Hospital, Sydney, Australia) for advice and the RevMAPKK/GFP plasmid, A. Calderone for construction of the GFP–PAI-2 plasmid, and J. Whisstock (Monash University) for molecular modeling. We also thank D. Huang (Walter and Eliza Hall Institute) for Apaf-1 antibodies, E. Remold-O'Donnell (Center for Blood Research, Boston, Mass.) for MNEI antibodies, J. Wilson and J. Wang (Michigan State University) for LDH antiserum, and D. Pickup (Duke University Medical Center) for the CrmA cDNA and antiserum.

This work was supported by the National Health and Medical Research Council of Australia.