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The Ran GTPase cycle is required for yeast nuclear pore complex assembly.

Ryan KJ, McCaffery JM, Wente SR - J. Cell Biol. (2003)

Bottom Line: A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci.The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth.We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

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GFP-Nups are mislocalized in Ran GTPase cycle mutants. Ran GTPase cycle mutant and parental strains were grown to early log phase at 23°C, and were then shifted to 34°C for 5 h. Localization of GFP-Nic96 and Nup170-GFP was visualized by direct fluorescence microscopy. Differential interference contrast images (DIC; columns 2 and 4) show cell morphology. Parental, SWY2090; ntf2-H104Y, SWY2514; prp20-G282S, SWY2515; rna1-S116F, SWY2516.
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fig1: GFP-Nups are mislocalized in Ran GTPase cycle mutants. Ran GTPase cycle mutant and parental strains were grown to early log phase at 23°C, and were then shifted to 34°C for 5 h. Localization of GFP-Nic96 and Nup170-GFP was visualized by direct fluorescence microscopy. Differential interference contrast images (DIC; columns 2 and 4) show cell morphology. Parental, SWY2090; ntf2-H104Y, SWY2514; prp20-G282S, SWY2515; rna1-S116F, SWY2516.

Mentions: To further investigate the defect in these mutants, the localization of GFP-Nic96 and Nup170-GFP was analyzed in cells shifted to 34°C for 5 h (Fig. 1). In the parental strain, GFP-Nic96 and Nup170-GFP localized in a punctate nuclear rim pattern, typical for NPC localization. The localization was maintained when the parental cells were grown at 34°C (Fig. 1, parental). In contrast, after growth at 34°C, all three Ran GTPase cycle mutants showed dramatic perturbations in GFP-Nup localization. Although nuclear rim localization was still detected in most cells, there was a marked decrease in the relative GFP fluorescence level associated with the NE along with a coincident increase in the level of diffuse, cytoplasmic GFP signal. Mutant cells also had foci or clusters of GFP-Nups that were not observed in the parental strain (Fig. 1). Mutant cells were also processed for indirect immunofluorescence microscopy to localize the integral membrane pore–associated protein, Pom152 (Fig. 2). Although previous studies have localized Pom152 exclusively to NPCs (Wozniak et al., 1994; Strambio-de-Castillia et al., 1995), our parental and mutant strains showed a diffuse non-NE pool of Pom152 at the permissive growth temperature (Fig. 2, left column). This could be due to the epitope tagging of both Nic96 and Nup170 in these strains (see section on genetic interactions), as both NIC96 and NUP170 are genetically and functionally linked to POM152 (Aitchison et al., 1995; Tcheperegine et al., 1999). Despite the slight mislocalization of Pom152 in our parental strain, the phenotype was not exacerbated when cells were shifted to 34°C (Fig. 2, top row). In contrast, the localization of Pom152 in the mutants was altered at 34°C with levels at the NE rim diminished, cytoplasmic/reticular levels increased, and concentrated foci or clusters present. Thus, the GFP-Nic96, Nup170-GFP, and Pom152 localizations were coincidentally altered in similar manners. This suggests that multiple (if not all) components of the NPC were mislocalized in the Ran GTPase cycle mutants.


The Ran GTPase cycle is required for yeast nuclear pore complex assembly.

Ryan KJ, McCaffery JM, Wente SR - J. Cell Biol. (2003)

GFP-Nups are mislocalized in Ran GTPase cycle mutants. Ran GTPase cycle mutant and parental strains were grown to early log phase at 23°C, and were then shifted to 34°C for 5 h. Localization of GFP-Nic96 and Nup170-GFP was visualized by direct fluorescence microscopy. Differential interference contrast images (DIC; columns 2 and 4) show cell morphology. Parental, SWY2090; ntf2-H104Y, SWY2514; prp20-G282S, SWY2515; rna1-S116F, SWY2516.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172763&req=5

fig1: GFP-Nups are mislocalized in Ran GTPase cycle mutants. Ran GTPase cycle mutant and parental strains were grown to early log phase at 23°C, and were then shifted to 34°C for 5 h. Localization of GFP-Nic96 and Nup170-GFP was visualized by direct fluorescence microscopy. Differential interference contrast images (DIC; columns 2 and 4) show cell morphology. Parental, SWY2090; ntf2-H104Y, SWY2514; prp20-G282S, SWY2515; rna1-S116F, SWY2516.
Mentions: To further investigate the defect in these mutants, the localization of GFP-Nic96 and Nup170-GFP was analyzed in cells shifted to 34°C for 5 h (Fig. 1). In the parental strain, GFP-Nic96 and Nup170-GFP localized in a punctate nuclear rim pattern, typical for NPC localization. The localization was maintained when the parental cells were grown at 34°C (Fig. 1, parental). In contrast, after growth at 34°C, all three Ran GTPase cycle mutants showed dramatic perturbations in GFP-Nup localization. Although nuclear rim localization was still detected in most cells, there was a marked decrease in the relative GFP fluorescence level associated with the NE along with a coincident increase in the level of diffuse, cytoplasmic GFP signal. Mutant cells also had foci or clusters of GFP-Nups that were not observed in the parental strain (Fig. 1). Mutant cells were also processed for indirect immunofluorescence microscopy to localize the integral membrane pore–associated protein, Pom152 (Fig. 2). Although previous studies have localized Pom152 exclusively to NPCs (Wozniak et al., 1994; Strambio-de-Castillia et al., 1995), our parental and mutant strains showed a diffuse non-NE pool of Pom152 at the permissive growth temperature (Fig. 2, left column). This could be due to the epitope tagging of both Nic96 and Nup170 in these strains (see section on genetic interactions), as both NIC96 and NUP170 are genetically and functionally linked to POM152 (Aitchison et al., 1995; Tcheperegine et al., 1999). Despite the slight mislocalization of Pom152 in our parental strain, the phenotype was not exacerbated when cells were shifted to 34°C (Fig. 2, top row). In contrast, the localization of Pom152 in the mutants was altered at 34°C with levels at the NE rim diminished, cytoplasmic/reticular levels increased, and concentrated foci or clusters present. Thus, the GFP-Nic96, Nup170-GFP, and Pom152 localizations were coincidentally altered in similar manners. This suggests that multiple (if not all) components of the NPC were mislocalized in the Ran GTPase cycle mutants.

Bottom Line: A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci.The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth.We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

Show MeSH
Related in: MedlinePlus