Figure 3.

(A) Yrb1p but not an NES protein requires the acidic C-terminus of Gsp1p for complex formation. GST-Gsp1p-GTP (lanes 1–6) and GST-Gsp1ΔCp-GTP (lanes 7–11)(4 μg per reaction) were immobilized on glutathione Sepharose and incubated for 30 min at 4°C with 8 μg of PKI-NES-2GFP (NESp), 8 μg of P12-NES-2GFP (mutant NESp), and/or 12 μg of Xpo1p, as indicated. Bound material was washed three times and either eluted with SDS sample buffer (lanes 1–3 and 6–9) or incubated further for 15 min at 4°C with 4 μg of Yrb1p (lanes 4 and 10) or 1 μg of Rna1p (lanes 5 and 11) and then washed again three times. All samples were analyzed by SDS PAGE and Coomassie blue staining. (B) Xpo1p, Yrb1p, and Gsp1p-GTP form a stable 1:1:1 complex. Xpo1p (125 kDa), Yrb1p (23 kDa), and 6His-Gsp1pQ71L-GTP (26 kDa) were mixed as indicated and incubated for 30 min at 4°C. The reactions were gelfiltrated with a Superose 6 column (Pharmacia) in PBSKMT buffer (see Materials and Methods) at a flow rate of 0.4 ml/min. Absorption units at 280 nm were recorded using the Äkta Explorer 100 software (Pharmacia) and plotted against the fraction numbers and the volume after sample injection. Molecular weight markers (Sigma): apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa).