Nuclear-cytoplasmic Trafficking Research Articles

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

The human RAE1 gene is a functional homologue of Schizosaccharomyces pombe rae1 gene involved in nuclear export of Poly(A) + RNA

A Schizosaccharomyces pombe temperature-sensitive mutant, rae1-1, was previously identified by us as being defective in nuclear export of Poly(A) + RNA when grown at restrictive temperature. Here, we report the isolation of the human homologue of the S. pombe rae1 gene. The RAE1 genes are highly conserved in evolution in both structure and function. The human RAE1 cDNA, when expressed from the CMV-promoter, can suppress partially the temperature sensitivity of the rae1-1 mutant. This is also reflected by increased Poly(A) + RNA export at a restrictive temperature. An epitope tagged human Rae1p localizes to both the nucleus and the cytoplasm in transiently transfected HeLa cells. We discuss the potential role of Rae1p in nuclear cytoplasmic trafficking in yeast and higher eukaryotic cells.

Identification of Two Nuclear Proteins Which Bind to RNA CUG Repeats: Significance for Myotonic Dystrophy

Myotonic dystrophy is caused by a trinucleotide repeat expansion (CTG)n, located in the 3′ untranslated region of the DM-protein kinase gene. The cellular effects of the CTG expansion and how they lead to the diverse, multi-system clinical phenotype of DM are unknown. Studies on the expression of the DM gene in affected tissue have not yielded consistent results, leading to the suggestion that alterations of DM-PK may not be the sole molecular basis for DM. We explored the hypothesis that the expanded repeat in mutant DM RNA (CUG)n binds and titrates out nuclear RNA binding proteins. Alterations in the normal function of these proteins could result in the disruption of important cellular processes. We report here the identification and magnetic bead affinity purification of two nuclear proteins, 35 and 25 kD in size, which bind to RNA (CUG)n repeats and to a varying extent with other pyrimidine rich sequences. Sequence analysis of the 35 kD protein shows that it is a novel protein. Both these proteins are widely expressed, including human brain and skeletal muscle. We speculate that these proteins may play a role in DM pre-mRNA processing or nuclear cytoplasmic trafficking of RNA. Studies into the function of these proteins should yield important insights into the complex pathogenesis of myotonic dystrophy.

The hsp56 immunophilin component of untransformed steroid receptor complexes is localized both to microtubules in the cytoplasm and to the same nonrandom regions within the nucleus as the steroid receptor.

In their unliganded state, mouse glucocorticoid receptors (GR) that are overexpressed in the WCL2 line of Chinese hamster ovary cells are distributed in a nonrandom manner throughout all planes of the nucleus. These untransformed nuclear receptors exist in a heterocomplex containing three heat shock proteins, hsp90, hsp70, and hsp56, the latter being an immunophilin of the FK506 binding type whose cellular function is unknown. Because a knowledge of the cellular distribution of hsp56 could provide important clues to its function in steroid-receptor heterocomplexes, we have examined hsp56 localization in intact cells by indirect immunofluorescence using the UPJ56 antibody. The majority of hsp56 is located in the nucleus, with substantial amounts also visualized in the cytoplasm of intact cells. The cytoplasmic hsp56 was examined in rat pulmonary endothelial cells where the protein was found to colocalize with microtubules. The nuclear hsp56 was examined in the WCL2 cells, where the protein was found by confocal imaging to colocalize throughout all planes of the nucleus in the same mottled pattern as the overexpressed GR. Like the GR, the nuclear hsp56 is recovered largely in the cytosolic fraction after hypotonic rupture of WCL2 cells. An observation potentially related to the microtubule-associated fraction of hsp56 is that immunoadsorption of hsp56 from WCL2 cytosol is accompanied by coadsorption of the microtubule-associated protein-1C complex. These observations are discussed with respect to the possible biological functions of hsp56 in the folding and/or cytoplasmic-nuclear trafficking of the receptor.

Cytoplasmic-nuclear trafficking of progesterone receptor In vivo study of the mechanism of action of antiprogestins

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. Preliminary evidence suggests that the same mechanism exists for estradiol and glucocorticoid receptors. Experiments designed to study the traffic of steroid hormone receptors have been applied to the determination of the molecular mechanism of action of antisteroids. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same point in the cell as the hormone.