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© The Rockefeller University Press, 0021-9525/2000//835 $5.00
The Journal of Cell Biology, Volume 149, Number 4, , 2000 835-850

Original Article

Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms



Michael E. Nemerguta and Ian G. Macarab

a Department of Microbiology, Markey Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908
b Department of Pharmacology, Markey Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908
University of Virginia, Hospital West RM 7191, Box 800577 HSC, Charlottesville, VA 22908.(804) 924-1236(804) 982-0083

men5w{at}virginia.edu

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an ~45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH2-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-{alpha}3 isoform of importin-{alpha}. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-{alpha} or importin-β or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.

Key Words: nuclear transport • nuclear pore complex • importin • DNA-binding protein • permeabilized cells

© 2000 The Rockefeller University Press

Abbreviations used in this paper: BIB: β-like import receptor binding domain; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; GGNLS, GST-GFP-NLS; GST, glutathione-S-transferase; NLS, nuclear localization signal; NPC, nucleoplasmin core-domain; NTD, NH2-terminal domain; PFA, paraformaldehyde; RCC1, regulator of chromosome condensation protein; TMRM, tetramethylrhodamine-5-maleimide.


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