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. 2003 Mar 31;160(7):1041-53.
doi: 10.1083/jcb.200209116. Epub 2003 Mar 24.

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

Kathryn J Ryan  1 , J Michael McCaffery, Susan R Wente
Affiliations

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

Kathryn J Ryan et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
GFP-Nups are mislocalized in Ran GTPase cycle mutants. Ran GTPase cycle mutant and parental strains were grown to early log phase at 23°C, and were then shifted to 34°C for 5 h. Localization of GFP-Nic96 and Nup170-GFP was visualized by direct fluorescence microscopy. Differential interference contrast images (DIC; columns 2 and 4) show cell morphology. Parental, SWY2090; ntf2-H104Y, SWY2514; prp20-G282S, SWY2515; rna1-S116F, SWY2516.
Figure 2.
Figure 2.
Ran GTPase cycle mutants result in mislocalization of Pom152. Cells in early log phase were incubated at 23°C or 34°C for 5 h and processed for indirect immunofluorescence microscopy. Pom152 was visualized using the antibody mAb118C3. Nuclei and mitochondria were detected with the DNA dye DAPI. Strains as in Fig. 1.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
Ran GTPase cycle mutants have massive membrane-based defects at the nonpermissive temperature. Cells in early log phase were shifted to 34°C for 5 h and processed for thin-section transmission electron microscopy. (A and B) RanGEF (prp20-G282S, SWY2515) mutant cells; (C–E) RanGAP (rna1-S116F, SWY2516) mutants cells; (F–H) ntf2-H104Y (SWY2514) mutant cells. Although NPCs (arrowheads) were present, mutants accumulated 80–100 nm vesicles (asterisk, vesicle clusters) or more extended, flattened membranes (arrows). The boxes in C and G correspond to the areas showing individual vesicles (D) and flattened membranes (H), respectively. (I) A cell from the parental strain showing wild-type morphology. N, nucleus; C, cytoplasm. Bars, 0.25 μm in D and H. All other bars, 1 μm.
Figure 7.
Figure 7.
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.
Figure 8.
Figure 8.
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.
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