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.

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.

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).

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.

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.