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PMID:12730220

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Citation

Gao, H, Sumanaweera, N, Bailer, SM and Stochaj, U (2003) Nuclear accumulation of the small GTPase Gsp1p depends on nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the acetyl-CoA carboxylase Acc1p. J. Biol. Chem. 278:25331-40

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 nup133delta, rat2-1, nup85delta, nic96deltaC, and mtr7-1 cells, demonstrating that GFP-Gsp1p nuclear import is deficient. In addition, the concentration of Gsp1p is severely reduced in mutants nup133Delta 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 nup133delta and rat2-1 can be explained by reduced binding of the Gsp1p nuclear carrier Ntf2p to NPCs. Likewise, nup85delta 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 nup85delta, nic96deltaC, 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.

Links

PubMed Online version:10.1074/jbc.M301607200

Keywords

Acetyltransferases/physiology; Cell Division; Cell Nucleus/metabolism; Cytoplasm/metabolism; Green Fluorescent Proteins; Hot Temperature; Luminescent Proteins/metabolism; Membrane Proteins/physiology; Microscopy, Fluorescence; Monomeric GTP-Binding Proteins/metabolism; Mutation; Nuclear Pore Complex Proteins/physiology; Nuclear Proteins/metabolism; Nuclear Proteins/physiology; Plasmids/metabolism; Protein Structure, Tertiary; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/physiology; Temperature; Time Factors

Significance

Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status


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References

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