Nuclear Accumulation of the Small GTPase Gsp1p Depends on Nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the Acetyl-CoA Carboxylase Acc1p*
- Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the §University of Saarland, 66421 Homburg/Saar, Germany
- ¶ Supported by grants from National Science and Engineering Research Council of Canada, Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Quebec. A Chercheur National of Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Dept. of Physiology, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-2949, Fax: 514-398-7452; E-mail: ursula.stochaj{at}mcgill.ca.
Abstract
The small GTPase Ran/Gsp1p plays an essential role in nuclear trafficking of macromolecules, as Ran/Gsp1p regulates many transport processes across the nuclear pore complex (NPC). To determine the role of nucleoporins in the generation of the nucleocytoplasmic Gsp1p concentration gradient, mutations in various nucleoporin genes were analyzed in the yeast Saccharomyces cerevisiae. We show that the nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the enzyme acetyl-CoA carboxylase (MTR7) control the distribution and cellular concentration of Gsp1p. At the restrictive temperature the reporter protein GFP-Gsp1p, which is too large to diffuse across the nuclear envelope, fails to concentrate in nuclei of nup133Δ, rat2-1, nup85Δ, nic96ΔC, and mtr7-1 cells, demonstrating that GFP-Gsp1p nuclear import is deficient. In addition, the concentration of Gsp1p is severely reduced in mutants nup133Δ and mtr7-1 under these conditions. We have now identified the molecular mechanisms that contribute to the dissipation of the Gsp1p concentration gradient in these mutants. Loss of the Gsp1p gradient in nup133Δ and rat2-1 can be explained by reduced binding of the Gsp1p nuclear carrier Ntf2p to NPCs. Likewise, nup85Δ cells that mislocalize GFP-Gsp1p at the permissive as well as non-permissive temperature have a diminished association of Ntf2p-GFP with nuclear envelopes under both conditions. Moreover, under restrictive conditions Prp20p, the guanine nucleotide exchange factor for Gsp1p, mislocalizes to the cytoplasm in nup85Δ, nic96ΔC, and mtr7-1 cells, thereby contributing to a collapse of the Gsp1p gradient. Taken together, components of the NPC subcomplex containing Rat2p/Nup120p, Nup133p, and Nup85p, in addition to proteins Nic96p and Mtr7p, are shown to be crucial for the formation of a nucleocytoplasmic Gsp1p gradient.
Footnotes
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↵1 The abbreviations used are: NPC, nuclear pore complex; DAPI, 4′,6-diamidino-2-phenylindole; Nup, nucleoporin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; GFP, green fluorescent protein.
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↵2 S. M. Bailer and E. C. Hurt, unpublished data.
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at
http://www.jbc.org)
contains Fig. 1.
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↵‡ Both authors contributed equally to this work.
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- Received February 14, 2003.
- Revision received April 23, 2003.
- The American Society for Biochemistry and Molecular Biology, Inc.
