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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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GFP-nic96 and nup170-GFP genetically interact with ntf2-H104Y and rna1-S116F. Serial dilutions of the parental strain and Ran cycle mutants with integrated GFP-nic96 nup170-GFP (+) or lacking GFP-Nups (−) were pinned to plates and incubated for growth at 23, 34, or 37°C. GFP-nic96 nup170-GFP strains as in Fig. 1; SWY2517 (ntf2-H104Y) and SWY2518 (rna1-S116F) are NIC96 NUP170.
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fig5: GFP-nic96 and nup170-GFP genetically interact with ntf2-H104Y and rna1-S116F. Serial dilutions of the parental strain and Ran cycle mutants with integrated GFP-nic96 nup170-GFP (+) or lacking GFP-Nups (−) were pinned to plates and incubated for growth at 23, 34, or 37°C. GFP-nic96 nup170-GFP strains as in Fig. 1; SWY2517 (ntf2-H104Y) and SWY2518 (rna1-S116F) are NIC96 NUP170.

Mentions: Extensive studies suggest that Nic96 and Nup170 play structural roles in the mature NPC and serve as scaffolds for nups that interact with shuttling transport factors (Grandi et al., 1993; Aitchison et al., 1995; Zabel et al., 1996; Marelli et al., 1998). There is also direct evidence that Nic96 is required for NPC assembly in yeast (Zabel et al., 1996; Gomez-Ospina et al., 2000). Our strains expressing either GFP-Nic96 or Nup170-GFP showed GFP-Nup localization exclusively at the NPC/NE and had no growth defects compared with a wild-type strain at any temperature (unpublished data); however, combined expression of GFP-Nic96 and Nup170-GFP in the same haploid strain resulted in a ts lethal phenotype at 37°C (Fig. 5, parental+). This indicated that the GFP fusion proteins were not fully functional, although they were exclusively NPC localized. Originally, we predicted that using such a sensitized genetic background would bias our screen toward the isolation of NPC assembly mutants. To determine if expression of GFP-Nic96 and Nup170-GFP contributed to the mutant phenotypes, the ntf2-H104Y, prp20-G282S, and rna1-S116F alleles were crossed into a wild-type NIC96 NUP170 background. The absence of GFP-Nups had no effect on the growth of the RanGEF (prp20-G282S) mutant cells, and this strain remained ts at 34°C (unpublished data). In contrast, both ntf2-H104Y and RanGAP (rna1-S116F) mutants showed increased viability at 34°C in the absence of the GFP-Nups (Fig. 5, middle). However, these strains remained ts at 37°C (Fig. 5, right). The enhanced lethality was not due to either GFP-Nic96 or Nup170-GFP alone, but required expression of both GFP-Nups (unpublished data). These genetic interactions suggest that Nic96, Nup170, and the Ran GTPase cycle are required in a common pathway and provide a genetic link for Ran GTPase regulators to the NPC biogenesis mechanism.


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

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

GFP-nic96 and nup170-GFP genetically interact with ntf2-H104Y and rna1-S116F. Serial dilutions of the parental strain and Ran cycle mutants with integrated GFP-nic96 nup170-GFP (+) or lacking GFP-Nups (−) were pinned to plates and incubated for growth at 23, 34, or 37°C. GFP-nic96 nup170-GFP strains as in Fig. 1; SWY2517 (ntf2-H104Y) and SWY2518 (rna1-S116F) are NIC96 NUP170.
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Related In: Results  -  Collection

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

fig5: GFP-nic96 and nup170-GFP genetically interact with ntf2-H104Y and rna1-S116F. Serial dilutions of the parental strain and Ran cycle mutants with integrated GFP-nic96 nup170-GFP (+) or lacking GFP-Nups (−) were pinned to plates and incubated for growth at 23, 34, or 37°C. GFP-nic96 nup170-GFP strains as in Fig. 1; SWY2517 (ntf2-H104Y) and SWY2518 (rna1-S116F) are NIC96 NUP170.
Mentions: Extensive studies suggest that Nic96 and Nup170 play structural roles in the mature NPC and serve as scaffolds for nups that interact with shuttling transport factors (Grandi et al., 1993; Aitchison et al., 1995; Zabel et al., 1996; Marelli et al., 1998). There is also direct evidence that Nic96 is required for NPC assembly in yeast (Zabel et al., 1996; Gomez-Ospina et al., 2000). Our strains expressing either GFP-Nic96 or Nup170-GFP showed GFP-Nup localization exclusively at the NPC/NE and had no growth defects compared with a wild-type strain at any temperature (unpublished data); however, combined expression of GFP-Nic96 and Nup170-GFP in the same haploid strain resulted in a ts lethal phenotype at 37°C (Fig. 5, parental+). This indicated that the GFP fusion proteins were not fully functional, although they were exclusively NPC localized. Originally, we predicted that using such a sensitized genetic background would bias our screen toward the isolation of NPC assembly mutants. To determine if expression of GFP-Nic96 and Nup170-GFP contributed to the mutant phenotypes, the ntf2-H104Y, prp20-G282S, and rna1-S116F alleles were crossed into a wild-type NIC96 NUP170 background. The absence of GFP-Nups had no effect on the growth of the RanGEF (prp20-G282S) mutant cells, and this strain remained ts at 34°C (unpublished data). In contrast, both ntf2-H104Y and RanGAP (rna1-S116F) mutants showed increased viability at 34°C in the absence of the GFP-Nups (Fig. 5, middle). However, these strains remained ts at 37°C (Fig. 5, right). The enhanced lethality was not due to either GFP-Nic96 or Nup170-GFP alone, but required expression of both GFP-Nups (unpublished data). These genetic interactions suggest that Nic96, Nup170, and the Ran GTPase cycle are required in a common pathway and provide a genetic link for Ran GTPase regulators to the NPC biogenesis mechanism.

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