Members of the RSC chromatin-remodeling complex are required for maintaining proper nuclear envelope structure and pore complex localization

Abstract

The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO(7)-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO(7)-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.

Figure 1.

GFP-Nic96 mislocalizes in TetO₇-orf strains. (A) Pie chart representing the distribution between different classes of TetO₇-orf isolates with GFP-Nic96 perturbations. Genes linked to vesicular trafficking (Sec; blue), Ran/Kap (red), protein degradation (yellow), chromatin associated/chromatin remodeling (Chromatin; dark green), lipid biosynthesis (Lipid; purple), Nups (orange), others of defined function but unrelated to preceding (ND; brown), and GPI anchoring (GPI; light green). (B) Direct fluorescence microscopy of GFP-Nic96 localization in strains from the GFP-Nic96 TetO₇-orf collection is shown after growth in the presence of 10 μg/ml doxycycline for ∼14 h. Differential interference contrast (DIC) images reveal cell morphology. (C and D) Indirect immunofluorescence microscopy for Nup116 localization of TetO₇-orf strains (C) after culturing in doxycycline (as in B) and the rsc7Δ strain (D) at 23°C and after shifting to 34°C for 5 h. Bars, 5 μm.

Figure 2.

Nups mislocalize in the sth1-F793S temperature-sensitive strain. (A) Direct fluorescence microscopy of GFP-Nic96 and Nup170-GFP of logarithmically growing parental or sth1-F793S cells after growth at 23°C or after shifting to growth at 34°C for 5 h. Parental cells, SWY2089; sth1-F793S GFP-nic96 nup170-GFP cells, SWY3201. (B) Indirect immunofluorescence microscopy of sth1-F793S cells for Nup116 localization under the same growth conditions as described in A. Parental cells, SWY518; sth1-F793S, SWY3249. (C) STH1 expression rescues the GFP-Nic96 and Nup170-GFP mislocalization in the sth1-F793S mutant. Direct fluorescence microscopy was conducted with the sth1-F793S GFP-nic96 nup170-GFP strain (SWY3202) transformed with empty plasmid (pRS315) or the STH1 plasmid (pSW3051). Bars (A–C), 5 μm. (D) STH1 expression rescues the npa18-1 growth defect at 34°C. The sth1-F793S mutant strain (SWY3203) was transformed with empty plasmid (pRS315), plasmid harboring the STH1 ORF and its 5′ promoter region (pSW3051), or the YIL127C ORF and its 5′ promoter region (pSW3049). The resulting strains were streaked for growth on SM −Leu plates.

Figure 3.

The sth1-F793S allele is distinct from other sth1 alleles. (A) NPC mislocalization defect is specific to the sth1-F793S allele. Indirect immunofluorescence microscopy for Nup116 localization was conducted on logarithmically growing parental (WT) and designated sth1 mutant cells cultured at 30°C or 37°C for 4 h. Bar, 5 μm. (B) The growth phenotypes of the sth1-F793S allele are distinct from those for the sth1-3 allele. Serial diluted sth1-F793S and sth1-3 mutant cells and the corresponding WT strains, W303 (SWY518), and S288C (YOL183) respectively, were spotted onto YP agar plates with different carbon sources: TBZ (60 μg/ml) or HU (50 mM). The plates were incubated at semipermissive growth temperatures (30°C for sth1-F793S; 35°C for sth1-3) and monitored for growth after 2 d. EtOH, ethanol. (C) The sth1-F793S allele is an effective null at 34°C. The wild-type (SWY518) and sth1-F793S (SWY4143) strains were grown for 5 h at 23 or 34°C in the presence or absence of 0.4% BA. Total cell lysates were separated by SDS-PAGE and immunoblotted with a rabbit anti-Sth1 polyclonal antibody.

Figure 4.

The sth1-F793S and TetO₇-RSC mutant cells have severe NE perturbations at the nonpermissive or repressive conditions. (A–C) Logarithmically growing parental cells (A; SWY2089) or sth1-F793S mutant cells (B and C; SWY3202) were shifted to the 34°C for 5 h and then processed for TEM. (D–I) Logarithmically growing TetO₇-STH1 (D–F) and TetO₇-RSC58 (G–I) cells were cultured in the absence (D and G) or presence (E, F, H, and I) of 10 μg/ml doxycycline (dox) for 10 h and then processed for thin layer TEM. n, nucleus; c, cytoplasm; vac, vacuole; v, vesicle; arrowhead, NPC; *, NPC-like structure; arrow, membrane. Bars, 0.5 μm.

Figure 5.

Translation is required for RSC NE/NPC perturbations. (A) Indirect immunofluorescence microscopy for anti-Nup116 C-terminal antibody localization was conducted for sth1-F793S and rsc7Δ mutant cells. Logarithmically growing cells were cultured at 23 or 34°C for 5 h, in the presence or absence of 10 μg/ml cycloheximide. (B) Direct fluorescence microscopy was conducted for GFP-Nic96 and Nup170-GFP localization in logarithmically growing cells TetO₇-RSC8 cells cultured in the presence or absence of 10 μg/ml doxycycline (dox) and 10 μg/ml cycloheximide for 8 h. Corresponding DIC images are shown below each panel. Bars, 5 μm.

Figure 6.

Nup mislocalization in sth1-F793S cells requires ongoing transcription. The RPB4 deletion allele was integrated into the sth1-F793S strains expressing GFP-tagged Nic96 (SWY4243), Nup133 (SWY4245), or Nup60 (SWY4247). These strains and the corresponding parental sth1-F793S RPB4 strains (SWY4244, SWY4246, and SWY4248, respectively) were shifted to 34°C for 5 h. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. Bar, 5 μm.

Figure 7.

Benzyl alcohol treatment prevents GFP-Nup mislocalization in sth1-F793S cells. Logarithmically growing cultures of the sth1-F793S GFP-nic96 nup170-GFP (SWY3202) strain (A) and the sth1-F793S (SWY4143) strains with GFP-tagged Nic96, Nup60, Nup133, or Pom34 (B) were grown for 5 h at 23°C (left column) and then shifted to 34°C in the absence (middle column) or presence (right column) of 0.4% BA. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. For A, the corresponding DIC images are shown. Bars, 5 μm.

Figure 8.

The sth1-F793S NE and nuclear morphology perturbations are prevented by benzyl alcohol. Logarithmically growing wild type (WT, SWY518) (A) and sth1-F793S (SWY4143) (B–D) strains were incubated for 5 h at 23°C (B) or at 34°C (A, C, and D) in the absence (C) or presence (A and D) of 0.4% BA. Samples were processed for TEM. n, nucleus; c, cytoplasm; vac, vacuole; arrowhead, NPC; arrow, membrane. Bars, 0.5 μm.