Limits...
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.

Show MeSH

Related in: MedlinePlus

Nups associate with cytoplasmic membranes and vesicles in vivo. (A) Parental (SWY2090) or (B–D) RanGEF (prp20-G282S, SWY2515) strains were grown for 5 h at 34°C and processed for immunoelectron microscopy with antibodies against GFP followed by a gold-conjugated secondary. m, mitochondria; pm, plasma membrane; ne, nuclear envelope; n, nucleus. Arrows indicate NPC labeling; arrowheads indicate label on cytoplasmic membranes/vesicles. Bars, 0.1 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172763&req=5

fig8: Nups associate with cytoplasmic membranes and vesicles in vivo. (A) Parental (SWY2090) or (B–D) RanGEF (prp20-G282S, SWY2515) strains were grown for 5 h at 34°C and processed for immunoelectron microscopy with antibodies against GFP followed by a gold-conjugated secondary. m, mitochondria; pm, plasma membrane; ne, nuclear envelope; n, nucleus. Arrows indicate NPC labeling; arrowheads indicate label on cytoplasmic membranes/vesicles. Bars, 0.1 μm.

Mentions: To confirm that nups were associated with vesicles in vivo, GFP-Nic96 and Nup170-GFP were localized after growth at 34°C using immunogold labeling of ultrathin cryosections with anti-GFP primary and gold-conjugated secondary antibodies. In the parental strain, GFP-Nup labeling was localized almost exclusively to NPCs in the NE (Fig. 8 A). Moreover, gold particles were absent from the nucleoplasm and cytoplasm even when additional membrane structures were present. In contrast, in each of the Ran GTPase cycle mutants grown at 34°C, a significant fraction of the GFP-Nup labeling was found in the cytoplasm associated with vesicles/membranes (Fig. 8, B–D; unpublished data). The vesicles were similar to those observed by conventional EM. This strongly supports the conclusion that nups associate with vesicles in vivo. Such nup-containing vesicles have not been previously reported in S. cerevisiae. They may result from the defect in NPC assembly and reflect accumulation of a novel vesicular NPC assembly intermediate.


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

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

Nups associate with cytoplasmic membranes and vesicles in vivo. (A) Parental (SWY2090) or (B–D) RanGEF (prp20-G282S, SWY2515) strains were grown for 5 h at 34°C and processed for immunoelectron microscopy with antibodies against GFP followed by a gold-conjugated secondary. m, mitochondria; pm, plasma membrane; ne, nuclear envelope; n, nucleus. Arrows indicate NPC labeling; arrowheads indicate label on cytoplasmic membranes/vesicles. Bars, 0.1 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Nups associate with cytoplasmic membranes and vesicles in vivo. (A) Parental (SWY2090) or (B–D) RanGEF (prp20-G282S, SWY2515) strains were grown for 5 h at 34°C and processed for immunoelectron microscopy with antibodies against GFP followed by a gold-conjugated secondary. m, mitochondria; pm, plasma membrane; ne, nuclear envelope; n, nucleus. Arrows indicate NPC labeling; arrowheads indicate label on cytoplasmic membranes/vesicles. Bars, 0.1 μm.
Mentions: To confirm that nups were associated with vesicles in vivo, GFP-Nic96 and Nup170-GFP were localized after growth at 34°C using immunogold labeling of ultrathin cryosections with anti-GFP primary and gold-conjugated secondary antibodies. In the parental strain, GFP-Nup labeling was localized almost exclusively to NPCs in the NE (Fig. 8 A). Moreover, gold particles were absent from the nucleoplasm and cytoplasm even when additional membrane structures were present. In contrast, in each of the Ran GTPase cycle mutants grown at 34°C, a significant fraction of the GFP-Nup labeling was found in the cytoplasm associated with vesicles/membranes (Fig. 8, B–D; unpublished data). The vesicles were similar to those observed by conventional EM. This strongly supports the conclusion that nups associate with vesicles in vivo. Such nup-containing vesicles have not been previously reported in S. cerevisiae. They may result from the defect in NPC assembly and reflect accumulation of a novel vesicular NPC assembly intermediate.

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