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. 2000 Apr 3;149(1):41–54. doi: 10.1083/jcb.149.1.41

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9910076.f4ab.jpg — Distinct domains within Nup85p are required for interaction with the various members of the Nup84p complex. (a) Growth of nup85ΔC and nup85ΔN strains at 23, 30, and 35°C. A nup85::HIS3 null deletion mutant was transformed with the two nup85 truncation constructs and the corresponding strains were spotted onto yeast extract–peptone–d-glucose plates at the indicated temperatures. (b) Affinity-purification ProtA–tagged Nup85p full-length (FL) and of the Nup85pΔN (Δ1–182) and Nup85pΔC (Δ453–745) constructs. Shown is a Coomassie-stained gel of the purified complexes (the fusion proteins are indicated by asterisks, and the components of the Nup84p complex by lines), and a Western blot (right panel, only the relevant area from each blot is shown) using anti–ProtA, anti-Nup84p, anti-Nup145Cp, anti-Seh1p, and anti-Sec13p antibodies. (c) Deletion of the Nup85p COOH-terminal domain causes NPC clustering. The corresponding strains were transformed with a plasmid expressing GFP-Nup49p, shifted for 3 h to 37°C and the distribution of this NPC reporter was analyzed in the fluorescence microscope. (d) Both the COOH and NH₂ domains of Nup85p are required for efficient nuclear mRNA export. The nup85ΔC and nup85ΔN mutants as well as a wild-type control (NUP85 ⁺) were grown for 3 h at 37°C, fixed, and hybridized with a oligo(dT)-FITC probe to reveal poly(A)⁺ RNA distribution. Cells were also stained with Hoechst 33258 (DNA).

graphic file with name JCB9910076.f4cd.jpg — Distinct domains within Nup85p are required for interaction with the various members of the Nup84p complex. (a) Growth of nup85ΔC and nup85ΔN strains at 23, 30, and 35°C. A nup85::HIS3 null deletion mutant was transformed with the two nup85 truncation constructs and the corresponding strains were spotted onto yeast extract–peptone–d-glucose plates at the indicated temperatures. (b) Affinity-purification ProtA–tagged Nup85p full-length (FL) and of the Nup85pΔN (Δ1–182) and Nup85pΔC (Δ453–745) constructs. Shown is a Coomassie-stained gel of the purified complexes (the fusion proteins are indicated by asterisks, and the components of the Nup84p complex by lines), and a Western blot (right panel, only the relevant area from each blot is shown) using anti–ProtA, anti-Nup84p, anti-Nup145Cp, anti-Seh1p, and anti-Sec13p antibodies. (c) Deletion of the Nup85p COOH-terminal domain causes NPC clustering. The corresponding strains were transformed with a plasmid expressing GFP-Nup49p, shifted for 3 h to 37°C and the distribution of this NPC reporter was analyzed in the fluorescence microscope. (d) Both the COOH and NH₂ domains of Nup85p are required for efficient nuclear mRNA export. The nup85ΔC and nup85ΔN mutants as well as a wild-type control (NUP85 ⁺) were grown for 3 h at 37°C, fixed, and hybridized with a oligo(dT)-FITC probe to reveal poly(A)⁺ RNA distribution. Cells were also stained with Hoechst 33258 (DNA).