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 are associated with vesicles in mutant cell lysates. Parental (SWY2090) or RanGEF mutant (prp20-G282S, SWY2515) strains were incubated at 34°C for 5 h, and were then fractionated into a nuclear and large organelle fraction (Nuc.), a high speed membrane fraction (HSM), and a cytoplasmic fraction (Cyto.) by differential centrifugation. An equal number of cell equivalents from each fraction were separated by SDS-PAGE and analyzed for GFP-Nic96 by immunoblotting with an antibody against GFP. As a control, the distribution of the nuclear protein Nop1 was also analyzed. Strains are indicated on the left, and antibodies on the right.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172763&req=5

fig7: Nups are associated with vesicles in mutant cell lysates. Parental (SWY2090) or RanGEF mutant (prp20-G282S, SWY2515) strains were incubated at 34°C for 5 h, and were then fractionated into a nuclear and large organelle fraction (Nuc.), a high speed membrane fraction (HSM), and a cytoplasmic fraction (Cyto.) by differential centrifugation. An equal number of cell equivalents from each fraction were separated by SDS-PAGE and analyzed for GFP-Nic96 by immunoblotting with an antibody against GFP. As a control, the distribution of the nuclear protein Nop1 was also analyzed. Strains are indicated on the left, and antibodies on the right.

Mentions: We speculated that the vesicles reflected the accumulation of an NPC assembly intermediate. To test this hypothesis, we biochemically analyzed whether nups were associated with a vesicular fraction. Using RanGEF (prp20-G282S) mutant cells, lysates were separated into a nuclear/large organelle fraction, a cytoplasmic fraction, and a high speed membrane fraction by differential centrifugation. The fractions were analyzed by immunoblotting for GFP-Nic96 (Fig. 7). In the wild-type parental cells shifted to 34°C, GFP-Nic96 fractionated with nuclei, indicating that the majority of the protein was assembled into NPCs in the NE (Fig. 7, top row). In contrast, in lysate from prp20-G282S cells shifted to 34°C, a significant portion of GFP-Nic96 was isolated in the high speed membrane fraction (Fig. 7, middle row). The presence of GFP-Nic96 in the high-speed membrane fraction is consistent with vesicle association. The nucleolar protein Nop1 remained in the nuclear fraction, indicating that the nuclei remained intact in mutant cells and during the fractionation (Fig. 7, bottom row).


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

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

Nups are associated with vesicles in mutant cell lysates. Parental (SWY2090) or RanGEF mutant (prp20-G282S, SWY2515) strains were incubated at 34°C for 5 h, and were then fractionated into a nuclear and large organelle fraction (Nuc.), a high speed membrane fraction (HSM), and a cytoplasmic fraction (Cyto.) by differential centrifugation. An equal number of cell equivalents from each fraction were separated by SDS-PAGE and analyzed for GFP-Nic96 by immunoblotting with an antibody against GFP. As a control, the distribution of the nuclear protein Nop1 was also analyzed. Strains are indicated on the left, and antibodies on the right.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Nups are associated with vesicles in mutant cell lysates. Parental (SWY2090) or RanGEF mutant (prp20-G282S, SWY2515) strains were incubated at 34°C for 5 h, and were then fractionated into a nuclear and large organelle fraction (Nuc.), a high speed membrane fraction (HSM), and a cytoplasmic fraction (Cyto.) by differential centrifugation. An equal number of cell equivalents from each fraction were separated by SDS-PAGE and analyzed for GFP-Nic96 by immunoblotting with an antibody against GFP. As a control, the distribution of the nuclear protein Nop1 was also analyzed. Strains are indicated on the left, and antibodies on the right.
Mentions: We speculated that the vesicles reflected the accumulation of an NPC assembly intermediate. To test this hypothesis, we biochemically analyzed whether nups were associated with a vesicular fraction. Using RanGEF (prp20-G282S) mutant cells, lysates were separated into a nuclear/large organelle fraction, a cytoplasmic fraction, and a high speed membrane fraction by differential centrifugation. The fractions were analyzed by immunoblotting for GFP-Nic96 (Fig. 7). In the wild-type parental cells shifted to 34°C, GFP-Nic96 fractionated with nuclei, indicating that the majority of the protein was assembled into NPCs in the NE (Fig. 7, top row). In contrast, in lysate from prp20-G282S cells shifted to 34°C, a significant portion of GFP-Nic96 was isolated in the high speed membrane fraction (Fig. 7, middle row). The presence of GFP-Nic96 in the high-speed membrane fraction is consistent with vesicle association. The nucleolar protein Nop1 remained in the nuclear fraction, indicating that the nuclei remained intact in mutant cells and during the fractionation (Fig. 7, bottom row).

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