The DNA fragments corresponding to Nup53, Nic96, Nup157, and Kap121 proteins (all from Saccharomyces cerevisiae) as well as the human Nup35 RRM domain were generated by polymerase chain reaction from genomic DNA and cloned into modified pGEX-4T1 or pNIC28-Bsa4 vectors (Addgene) using ligation-independent cloning (42, 43). The resulting fusion proteins contained N-terminal GST- or SUMO-His6 tags, cleavable with the tobacco etch virus (TEV) protease. Proteins were expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells (Stratagene) following induction at OD600nm (optical density at 600 nm) of 0.8 with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18°C for 12 to 16 hours in LB medium containing chloramphenicol (34 mg/liter) and ampicillin (100 mg/liter) or kanamycin (50 mg/liter). Cells were lysed using a cell disruptor (AVESTIN) in a buffer containing 20 mM Hepes (pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 5% (v/v) glycerol, and 3 mM dithiothreitol (DTT; lysis buffer for GST-tagged proteins) or 20 mM Hepes (pH 7.5), 300 mM NaCl, 20 mM imidazole, 5% (v/v) glycerol, and 5 mM β-mercaptoethanol (BME; lysis buffer for His-tagged proteins). All lysis buffers were complemented with 1 mM benzamidine and phenylmethylsulfonyl fluoride as well as bovine lung aprotinin and leupeptin (1 μg/ml; Sigma-Aldrich). After centrifugation at 30,000g for 45 min, cleared supernatants were purified by standard chromatography techniques described below. A dimerization-deficient Nup53mut fragment (W256E, W313E) was generated using the QuikChange Mutagenesis Kit (Stratagene). The details of the expression constructs are shown in table S1. HsKapβ1 and RnNup58 FG-repeat constructs were generated, expressed, and purified as previously described (34).

Purification of the yeast Nup53 and human Nup35 fragments. GST-tagged Nup53 lysates were loaded on a GSTrap column (GE Healthcare) and washed with the GST lysis buffer for ~75 column volumes. Proteins were eluted with 20 mM Hepes (pH 7.5), 100 mM NaCl, 5% glycerol, and 3 mM DTT supplemented with 15 mM reduced glutathione (Sigma-Aldrich). Following GST tag removal by TEV protease cleavage for 12 to 16 hours at 4°C, the Nup53 fragments were further purified over a HiTrap SP column (GE Healthcare) by gradually increasing ionic strength of the GST lysis buffer (corrected to pH 7.0) via a salt gradient from 100 to 600 mM NaCl. Last, proteins were purified over a HiLoad Superdex 200 26/60 gel filtration column (GE Healthcare) equilibrated with 20 mM Hepes (pH 7.5), 200 mM NaCl, and 3 mM DTT (protein storage buffer), concentrated to 5 to 15 mg/ml, flash-frozen in liquid nitrogen, and stored at −80°C.

Purification of Nic96 and Nup157. GST-tagged Nic96 and Nup157 fragments were purified using a previously described protocol (44). Briefly, proteins were affinity-purified over a GSTrap column, cleaved by TEV protease overnight at 4°C, and finally purified in the storage buffer using a Superdex 200 26/60 gel filtration column. As a last step, proteins were concentrated to 15 to 25 mg/ml, flash-frozen in liquid nitrogen, and stored at −80°C.

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