Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex

Abstract

In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.

Figure 1

Characterization of the mtr2-142 mutant. (A) mtr2-142 causes nuclear accumulation of Npl3-27. Localization of Npl3-27 in npl3-27 (PSY1031, a–c) and npl3-27 mtr2-142 (PSY1717, d–f) cells. Cells were shifted to 37°C for 30 min. Indirect immunofluorescence with polyclonal antibodies to Npl3 (left), DAPI (middle), and Nomarski images (right) are shown. (B) mtr2-142 cells display an mRNA export defect. Localization of poly (A)⁺ RNA in mtr2-142 cells (PSY1719) grown at 25°C (a–c) or shifted to 37°C for 10 min (d–f). In situ hybridization with an oligo (dT)₅₀ probe (left), DAPI (middle), and Nomarski images (right) are shown. (C) mtr2-142 cells mislocalize Mex67 to the cytoplasm. Localization of Mex67-GFP in mtr2-142 cells (PSY2857) grown at 25°C (a and b) or shifted to 37°C for 15 min (c and d). GFP fluorescence (left) and Nomarski images (right) are shown. (D) mtr2-142 cells display a large ribosome export defect. Localization of Nmd3-GFP in wild-type (FY23, a and b) and mtr2-142 cells (PSY1719, c and d). Localization of Rpl11b-GFP in wild-type (e and f) and mtr2-142 cells (g and h). Cells were grown at 25°C.

Figure 2

Sac3 functions primarily in mRNA export. A. sac3⁻ mutant cells display an mRNA export defect. Localization of poly (A)⁺ RNA in wild-type (FY23, a–c), sac3Δ139 cells (PSY2555, d–f), wild-type (PSY1930, g–i), and Δsac3-rg cells (PSY2844, j–l) grown at 30°C. In situ hybridization with an oligo (dT)₅₀ probe (left), DAPI (middle), and Nomarski images (right) are shown. (B) sac3Δ139 cells do not display a large ribosome export defect. Localization of Rpl11b-GFP in wild-type (FY23, a–d) and sac3Δ139 cells (PSY2555, e–h) grown at 25°C (a, b, e, and f) or shifted to 37°C for 1 h and back to 25°C for 1 h (c, d, g, and h). (C) sac3Δ139 cells do not have an NES-protein export defect. Localization of NLS-NES-GFP in wild-type (FY23) and sac3Δ139 cells (PSY2555) grown at 25°C.

Figure 3

Sac3 associates physically with mRNA export factors and nuclear pore associated proteins. (A) Sac3 coimmunoprecipitates with Mtr2. Lysates (left) and α-GFP immunoprecipitates (right) from Sac3-myc (PSY2451, lanes 1, 4, 7, and 10), EYFP-Mtr2 (PSY2729, lanes 2, 5, 8 and 11), and double tagged Sac3-myc EYFPMtr2 (PSY2729, lanes 2, 5, 8 and 11), and double tagged Sac3-myc EYFP-Mtr2 (PSY2747, lanes 3, 6, 9 and 12) strains. The asterisk denotes the heavy chain of the α-GFP antibody. Arrows point to EYFP-Mtr2. Lanes 7–9 were exposed ten times longer than lanes 10–12. B. Sac3 coimmunoprecipitates with Mex67. Lysates (left) and α-GFP immunoprecipitates (right) from Sac3-myc (PSY2451, lanes 1, 4, 7 and 10), Mex67-GFP (lanes 2, 5, 8 and 11), and double tagged Sac3-myc Mex67-GFP (PSY2691, lanes 3, 6, 9 and 12) strains. C. Sac3 coimmunoprecipitates with Mlp1. Lysates (left) and α-GFP immunoprecipitates (right) from Sac3-myc (PSY2451, lanes 1, 4, 7, and 10), Mlp1-EYFP (PSY2751, lanes 2, 5, 8, and 11), and double-tagged Sac3-myc Mlp1-EYFP (PSY2752, lanes 3, 6, 9, and 12) strains. (D) Sac3 coimmunoprecipitates with Nup116. Lysates (left) and α-GFP immunoprecipitates (right) from Sac3-myc (PSY2451, lanes 1, 4, 7, and 10), Nup116-EYFP (PSY1832, lanes 2, 5, 8, and 11), and double-tagged Sac3-myc Nup116-EYFP (PSY2856, lanes 3, 6, 9, and 12) strains. (E) Sac3 coimmunoprecipitates with Dbp5. Lysates (left) and α-GFP immunoprecipitates (right) from Sac3-myc (PSY2451, lanes 1, 4, 7, and 10), ECFP-Dbp5 (PSY2726, lanes 2, 5, 8, and 11), and double-tagged Sac3-myc ECFP-Dbp5 (PSY2755, lanes 3, 6, 9, and 12) strains. (F) Sac3 does not coimmunoprecipitate with NLS-NES-GFP. Lysates (left) and α-GFP immunoprecipitates (right) from a Sac3-myc strain containing empty vector (PSY2451 transformed with pPS703; lanes 1, 4, 7, and 10), a wild-type strain expressing NLS-NES-GFP (FY23 transformed with pPS1372; lanes 2, 5, 8, and 11), and a Sac3-myc strain expressing NLS-NES-GFP (PSY2451 transformed with pPS1372; lanes 3, 6, 9, and 12). The asterisk denotes the heavy chain of the α-GFP antibody. Arrows point to two bands corresponding to NLS-NES-GFP. Samples were run on an SDS-PAGE gel. Lysate (10 μg) and 1/20th of the IP (from a total of 1 mg of lysate) were blotted with α-GFP (lanes 7–12) and 10 μg of lysate and the remainder of the IP were blotted with α-myc (9E10, lanes 1–6).

Figure 4

Immunogold-localization of Sac3 in ECFP-Sac3 cells. (A) Triton X-100–extracted spheroplasts from ECFP-Sac3 cells were preimmunolabeled with a polyclonal anti-GFP antibody directly conjugated to 8-nm colloidal gold. Shown is a view along a cross-sectioned nuclear envelope stretch with labeled NPCs (arrows, top), and a gallery of selected samples of gold-labeled NPC cross sections (bottom). The anti-GFP antibody labeled exclusively the cytoplasmic periphery of the NPC. c, cytoplasm; n, nucleus. Bars, 100 nm. (B) Quantitative analysis of the gold particles associated with the NPC. Fifty-two gold particles were scored.

Figure 5

Mex67 mislocalizes in sac3Δ139 cells. (A) Localization of Mex67-GFP in wild-type (MEX67-GFP, a and b) and sac3Δ139 cells (c and d, PSY2843, top and PSY2842, bottom) grown at 30°C. GFP fluorescence (left) and Nomarski images (right) are shown. Arrows point to intense foci at the nuclear rim. (B) Localization of Nsp1 in wild-type (FY23, a and b), rat2-1 (Dat4-2, c and d), and sac3Δ139 cells (e and f) grown at 25°C. Indirect immunofluorescence with polyclonal antibodies to Nsp1 (left) and 4,6-diamidino-2-phenylindole (right) are shown.