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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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Related in: MedlinePlus

Cycloheximide treatment prevents GFP-Nup mislocalization. Cells in early log phase were shifted to 34°C for 6 h in the absence or presence of 10 μg/ml cycloheximide. GFP-Nic96 and Nup170-GFP localization was visualized by direct fluorescence microscopy. Strains as in Fig. 1.
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fig3: Cycloheximide treatment prevents GFP-Nup mislocalization. Cells in early log phase were shifted to 34°C for 6 h in the absence or presence of 10 μg/ml cycloheximide. GFP-Nic96 and Nup170-GFP localization was visualized by direct fluorescence microscopy. Strains as in Fig. 1.

Mentions: The mislocalization of nups in the Ran cycle mutants could reflect a defect in either new NPC assembly or the stability of existing NPCs. To distinguish between these two possibilities, cycloheximide treatment was used to inhibit protein synthesis (Fig. 3). In cells treated with cycloheximide, the GFP-Nups remained associated with the NE after 6 h at 34°C (Fig. 3, right columns), whereas untreated cells showed characteristic GFP-Nup mislocalization (Fig. 3, left columns). Thus, preexisting NPCs in these mutants were stable at the nonpermissive temperature and mislocalization was dependent on new protein synthesis. This suggests that the Ran GTPase cycle mutants are defective in the assembly of new NPCs.


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

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

Cycloheximide treatment prevents GFP-Nup mislocalization. Cells in early log phase were shifted to 34°C for 6 h in the absence or presence of 10 μg/ml cycloheximide. GFP-Nic96 and Nup170-GFP localization was visualized by direct fluorescence microscopy. Strains as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Cycloheximide treatment prevents GFP-Nup mislocalization. Cells in early log phase were shifted to 34°C for 6 h in the absence or presence of 10 μg/ml cycloheximide. GFP-Nic96 and Nup170-GFP localization was visualized by direct fluorescence microscopy. Strains as in Fig. 1.
Mentions: The mislocalization of nups in the Ran cycle mutants could reflect a defect in either new NPC assembly or the stability of existing NPCs. To distinguish between these two possibilities, cycloheximide treatment was used to inhibit protein synthesis (Fig. 3). In cells treated with cycloheximide, the GFP-Nups remained associated with the NE after 6 h at 34°C (Fig. 3, right columns), whereas untreated cells showed characteristic GFP-Nup mislocalization (Fig. 3, left columns). Thus, preexisting NPCs in these mutants were stable at the nonpermissive temperature and mislocalization was dependent on new protein synthesis. This suggests that the Ran GTPase cycle mutants are defective in the assembly of new NPCs.

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