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Published online 8 January 2001. doi:10.1083/jcb.152.1.127
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© The Rockefeller University Press, 0021-9525/2001//127 $5.00
The Journal of Cell Biology, Volume 152, Number 1, , 2001 127-140

Original Article

Calreticulin Is a Receptor for Nuclear Export



James M. Holaskaa,b, Ben E. Blacka,c, Dona C. Loved, John A. Hanoverd, John Leszyke, and Bryce M. Paschala,c

a Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
b Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
c Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
d Laboratory of Cell Biochemistry and Biology, National Institutes of Health, Bethesda, Maryland 20892
e Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01545
Center for Cell Signaling, Box 800577 Health Systems, University of Virginia School of Medicine, Charlottesville, VA 22908.(804) 924-1236(804) 243-6521

paschal{at}virginia.edu

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

Key Words: nucleocytoplasmic transport • Ran GTPase • nuclear pore complex • protein export • hormone receptor

© 2001 The Rockefeller University Press

Abbreviations used in this paper: BFP, blue fluorescent protein; bPKI, biotinylated PKI; CRT, calreticulin; DBD, DNA-binding domain; GFP, green fluorescent protein; GR, glucocorticoid receptor; GST, glutathione-S-transferase; LMB, leptomycin B; MUT, mutant; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; PKI, protein kinase inhibitor; RNP, ribonucleoprotein; STV, streptavidin; TB, transport buffer; WT, wild-type.


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