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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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Ran GTPase cycle mutants have massive membrane-based defects at the nonpermissive temperature. Cells in early log phase were shifted to 34°C for 5 h and processed for thin-section transmission electron microscopy. (A and B) RanGEF (prp20-G282S, SWY2515) mutant cells; (C–E) RanGAP (rna1-S116F, SWY2516) mutants cells; (F–H) ntf2-H104Y (SWY2514) mutant cells. Although NPCs (arrowheads) were present, mutants accumulated 80–100 nm vesicles (asterisk, vesicle clusters) or more extended, flattened membranes (arrows). The boxes in C and G correspond to the areas showing individual vesicles (D) and flattened membranes (H), respectively. (I) A cell from the parental strain showing wild-type morphology. N, nucleus; C, cytoplasm. Bars, 0.25 μm in D and H. All other bars, 1 μm.
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fig6: Ran GTPase cycle mutants have massive membrane-based defects at the nonpermissive temperature. Cells in early log phase were shifted to 34°C for 5 h and processed for thin-section transmission electron microscopy. (A and B) RanGEF (prp20-G282S, SWY2515) mutant cells; (C–E) RanGAP (rna1-S116F, SWY2516) mutants cells; (F–H) ntf2-H104Y (SWY2514) mutant cells. Although NPCs (arrowheads) were present, mutants accumulated 80–100 nm vesicles (asterisk, vesicle clusters) or more extended, flattened membranes (arrows). The boxes in C and G correspond to the areas showing individual vesicles (D) and flattened membranes (H), respectively. (I) A cell from the parental strain showing wild-type morphology. N, nucleus; C, cytoplasm. Bars, 0.25 μm in D and H. All other bars, 1 μm.

Mentions: Conventional thin-section transmission electron microscopy was used to investigate possible ultrastructural changes associated with the Ran GTPase cycle mutants and their inability to assemble new NPCs. Wild-type NPCs appear as electron-dense structures spanning both the inner and outer nuclear membranes (Fig. 6 I, arrowheads). Mutant cells grown at the permissive temperature had only subtle alterations in cell and nuclear morphology (unpublished data). In contrast, the mutant strains showed massive membrane-based defects after growth at 34°C. Most striking was the accumulation of vesicles of 80–100 nm in diameter. Vesicle accumulation was most prevalent in the RanGEF (prp20-G282S) cells (Fig. 6 A). Often, the vesicles formed large aggregates that localized near the plasma membrane (Fig. 6, B and F, asterisk). In addition to vesicles, these mutants also showed increased amounts of extended cytoplasmic membrane structures. In RanGEF (prp20-G282S) and RanGAP (rna1-S116F) arrested mutant cells, the expanded membranes appeared as long ribbons stretching throughout the cell (Fig. 6, B and E, arrows). The ntf2-H104Y mutant cells had shorter stretches of membranes that were primarily localized near the cytoplasmic face of the NE and often arranged in loose stacks (Fig. 6, F–H, arrows). The general nuclear morphology was also dramatically changed in the temperature-arrested RanGAP (rna1-S116F) and RanGEF (prp20-G282S) mutant cells. The nuclear region was lobulated and the NE was invaginated (Fig. 6, B and C). In ntf2-H104Y mutant cells, the nuclear region was more typical of a wild-type sphere (Fig. 6, F and G). Finally, in RanGAP (rna1-S116F) arrested cells, the NE was discontinuous in many sections (Fig. 6 E). Overall, the Ran GTPase cycle mutants showed remarkable changes in NE, cytoplasmic, and vesicular membrane structures.


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

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

Ran GTPase cycle mutants have massive membrane-based defects at the nonpermissive temperature. Cells in early log phase were shifted to 34°C for 5 h and processed for thin-section transmission electron microscopy. (A and B) RanGEF (prp20-G282S, SWY2515) mutant cells; (C–E) RanGAP (rna1-S116F, SWY2516) mutants cells; (F–H) ntf2-H104Y (SWY2514) mutant cells. Although NPCs (arrowheads) were present, mutants accumulated 80–100 nm vesicles (asterisk, vesicle clusters) or more extended, flattened membranes (arrows). The boxes in C and G correspond to the areas showing individual vesicles (D) and flattened membranes (H), respectively. (I) A cell from the parental strain showing wild-type morphology. N, nucleus; C, cytoplasm. Bars, 0.25 μm in D and H. All other bars, 1 μm.
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Related In: Results  -  Collection

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fig6: Ran GTPase cycle mutants have massive membrane-based defects at the nonpermissive temperature. Cells in early log phase were shifted to 34°C for 5 h and processed for thin-section transmission electron microscopy. (A and B) RanGEF (prp20-G282S, SWY2515) mutant cells; (C–E) RanGAP (rna1-S116F, SWY2516) mutants cells; (F–H) ntf2-H104Y (SWY2514) mutant cells. Although NPCs (arrowheads) were present, mutants accumulated 80–100 nm vesicles (asterisk, vesicle clusters) or more extended, flattened membranes (arrows). The boxes in C and G correspond to the areas showing individual vesicles (D) and flattened membranes (H), respectively. (I) A cell from the parental strain showing wild-type morphology. N, nucleus; C, cytoplasm. Bars, 0.25 μm in D and H. All other bars, 1 μm.
Mentions: Conventional thin-section transmission electron microscopy was used to investigate possible ultrastructural changes associated with the Ran GTPase cycle mutants and their inability to assemble new NPCs. Wild-type NPCs appear as electron-dense structures spanning both the inner and outer nuclear membranes (Fig. 6 I, arrowheads). Mutant cells grown at the permissive temperature had only subtle alterations in cell and nuclear morphology (unpublished data). In contrast, the mutant strains showed massive membrane-based defects after growth at 34°C. Most striking was the accumulation of vesicles of 80–100 nm in diameter. Vesicle accumulation was most prevalent in the RanGEF (prp20-G282S) cells (Fig. 6 A). Often, the vesicles formed large aggregates that localized near the plasma membrane (Fig. 6, B and F, asterisk). In addition to vesicles, these mutants also showed increased amounts of extended cytoplasmic membrane structures. In RanGEF (prp20-G282S) and RanGAP (rna1-S116F) arrested mutant cells, the expanded membranes appeared as long ribbons stretching throughout the cell (Fig. 6, B and E, arrows). The ntf2-H104Y mutant cells had shorter stretches of membranes that were primarily localized near the cytoplasmic face of the NE and often arranged in loose stacks (Fig. 6, F–H, arrows). The general nuclear morphology was also dramatically changed in the temperature-arrested RanGAP (rna1-S116F) and RanGEF (prp20-G282S) mutant cells. The nuclear region was lobulated and the NE was invaginated (Fig. 6, B and C). In ntf2-H104Y mutant cells, the nuclear region was more typical of a wild-type sphere (Fig. 6, F and G). Finally, in RanGAP (rna1-S116F) arrested cells, the NE was discontinuous in many sections (Fig. 6 E). Overall, the Ran GTPase cycle mutants showed remarkable changes in NE, cytoplasmic, and vesicular membrane structures.

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