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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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Allele specificity of Ran GTPase cycle mutants. (A) Logarithmically growing parental, npa mutants (see Fig. 1 for strains) and previously isolated prp20–1 (SWY2541) and rna1–1 (SWY2542) cells in a GFP-nic96 nup170-GFP background were shifted to 34°C and monitored for growth over a period of 12 h by measuring OD600 nm. Starting densities were normalized to 1. (B) GFP-Nups were localized in prp20–1 and rna1–1 cells after 5 h at 34°C as in Fig. 1.
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fig4: Allele specificity of Ran GTPase cycle mutants. (A) Logarithmically growing parental, npa mutants (see Fig. 1 for strains) and previously isolated prp20–1 (SWY2541) and rna1–1 (SWY2542) cells in a GFP-nic96 nup170-GFP background were shifted to 34°C and monitored for growth over a period of 12 h by measuring OD600 nm. Starting densities were normalized to 1. (B) GFP-Nups were localized in prp20–1 and rna1–1 cells after 5 h at 34°C as in Fig. 1.

Mentions: To further analyze whether NPC assembly was altered, cell division was monitored after shifting the ntf2-H104Y, RanGEF (prp20-G282S), and RanGAP (rna1-S116F) strains to 34°C. The cells underwent roughly three doublings before ceasing to divide (Fig. 4 A; unpublished data). These results directly parallel the number of cell divisions observed before the arrest of a nic96 mutant strain that is blocked for new NPC assembly (Zabel et al., 1996; Gomez-Ospina et al., 2000). In the absence of new NPC assembly, the number of NPCs per nucleus would be diluted in half at every mitosis, and after three rounds of cell division, each resulting nucleus would harbor only one eighth of the original NPCs and GFP-Nup fluorescence at the NE. Thus, the time course and relative fluorescence level decrease of GFP-Nups at the NE (Fig. 1) for the Ran GTPase cycle mutant phenotypes correlate with a defect in new NPC assembly.


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

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

Allele specificity of Ran GTPase cycle mutants. (A) Logarithmically growing parental, npa mutants (see Fig. 1 for strains) and previously isolated prp20–1 (SWY2541) and rna1–1 (SWY2542) cells in a GFP-nic96 nup170-GFP background were shifted to 34°C and monitored for growth over a period of 12 h by measuring OD600 nm. Starting densities were normalized to 1. (B) GFP-Nups were localized in prp20–1 and rna1–1 cells after 5 h at 34°C as in Fig. 1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172763&req=5

fig4: Allele specificity of Ran GTPase cycle mutants. (A) Logarithmically growing parental, npa mutants (see Fig. 1 for strains) and previously isolated prp20–1 (SWY2541) and rna1–1 (SWY2542) cells in a GFP-nic96 nup170-GFP background were shifted to 34°C and monitored for growth over a period of 12 h by measuring OD600 nm. Starting densities were normalized to 1. (B) GFP-Nups were localized in prp20–1 and rna1–1 cells after 5 h at 34°C as in Fig. 1.
Mentions: To further analyze whether NPC assembly was altered, cell division was monitored after shifting the ntf2-H104Y, RanGEF (prp20-G282S), and RanGAP (rna1-S116F) strains to 34°C. The cells underwent roughly three doublings before ceasing to divide (Fig. 4 A; unpublished data). These results directly parallel the number of cell divisions observed before the arrest of a nic96 mutant strain that is blocked for new NPC assembly (Zabel et al., 1996; Gomez-Ospina et al., 2000). In the absence of new NPC assembly, the number of NPCs per nucleus would be diluted in half at every mitosis, and after three rounds of cell division, each resulting nucleus would harbor only one eighth of the original NPCs and GFP-Nup fluorescence at the NE. Thus, the time course and relative fluorescence level decrease of GFP-Nups at the NE (Fig. 1) for the Ran GTPase cycle mutant phenotypes correlate with a defect in new NPC assembly.

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