title: Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import.
abstract: The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min). Cotransfection with epitope-tagged, wild-type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.
authors.affiliation: Department of Pathology, University of Vermont, Burlington, 05405-0068, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Carey
authors.forename: S A
authors.initials: SA
authors.lastname: Richards
authors.forename: K M
authors.initials: KM
authors.lastname: Lounsbury
authors.forename: I G
authors.initials: IG
authors.lastname: Macara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020931:ran GTP-Binding Protein
chemical_list: D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D020931:ran GTP-Binding Protein