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Molecular and Cellular Biology, August 2001, p. 5396-5407, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5396-5407.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Catherina H. Bird,1 Elizabeth J. Blink,2 Claire E. Hirst,1 Marguerite S. Buzza,1 Pauline M. Steele,1 Jiuru Sun,1 David A. Jans,2 and Phillip I. Bird1,*

Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800,1 and Nuclear Signaling Laboratory, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601,2 Australia

Received 17 May 2001/Accepted 18 May 2001

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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, Victoria 3800, Australia. Phone: 61 3 9905 3771. Fax: 61 3 9905 4699. E-mail: Phil.Bird{at}med.monash.edu.au.

Molecular and Cellular Biology, August 2001, p. 5396-5407, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5396-5407.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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