Binding of Mtr2p to the M domain of Mex67p. His₆-Mtr2p and untagged full-length Mex67p or the various truncation constructs (top) were expressed and purified from E. coli as described in Materials and Methods. The homogenate, soluble supernatant, insoluble pellet, wash, and His₆-Mtr2p eluate derived from the Ni-NTA agarose beads were analyzed by SDS-PAGE and Coomassie staining (bottom). Copurification of the full-length Mex67p and the M domain, respectively, with His₆-Mtr2p is seen. H, Homogenate; S, soluble supernatant; P, insoluble pellet; W, wash; E, eluate. The expression levels of the various Mex67p truncation constructs in E. coli were similar.

The C domain of Mex67p is not essential for cell growth. A, Domain organization of Mex67p and amino acid truncations/point mutations in the C domain. B, Growth of the mex67ΔC1 and mex67ΔC2 thermosensitive mutants at 23 and 37°C. The same amount of cells was diluted in 10⁻¹ steps, spotted onto YPD plates, and grown for 3 d at the indicated temperatures. The mutants were transformed with empty, MEX67, or MTR2 containing plasmids (pLEU2, either single or hc plasmid). C, The mex67(Δ544–559) allele is not dominant-lethal. The MEX67 shuffle strain (mex67::HIS3, pURA3-MEX67) was transformed with the pLEU2 and pTRP1 plasmids coding for the indicated gene constructs. Transformants were then streaked onto 5-FOA plates (+uracil, −leucine, −tryptophane) and grown for 5 d at 30°C. The nonviable mex67(Δ544–559) allele has been described recently (Segref et al. 1997). D, Western blot analysis of whole cell yeast lysates expressing mex67ΔC2 deletion constructs using anti-Mex67p antibodies. 1, full-length Mex67p; 2, mex67ΔC2 and mex67(Δ544–559) (low-copy); 3, mex67ΔC2 and mex67(L[552]>P) (low copy); 4, mex67ΔC2 and mex67(Δ544–559) (hc); 5, mex67ΔC2 and mex67(L[552]>P) (hc). The band marked by an asterisk is a protein cross-reacting with the anti-Mex67p immune serum. A molecular weight marker standard is also shown.

Inhibition of mRNA export in mex67ΔC cells. A, Subcellular localization of poly(A)⁺ RNA was analyzed by in situ hybridization, in MEX67 and mex67ΔC2 cells, either grown at 23°C or shifted for 1 h to 37°C. Nuclear DNA was stained with DAPI. B, Localization of GFP-tagged wild-type Mex67p and Mex67pΔC1 and Mex67pΔC2 mutants as revealed by fluorescence microscopy (left). Expression of Mex67p-GFP and Mex67pΔC2-GFP was analyzed by Western blotting using anti-GFP antibodies (right). C, Localization of GFP-tagged Mtr2p in MEX67, mex67ΔC1, and mex67ΔC2 strains as revealed by fluorescence microscopy. D, Localization of GFP-tagged Mex67pΔC2-GFP in a single copy (+ sc MTR2) and hc (+ hc MTR2) strain background at 23°C, as revealed by fluorescence microscopy.

A nuclear export activity in the C domain of Mex67p. A, The C domain of Mex67p (3) and its derived mutant forms mapping in the NES-like sequence (4 and 5) or the LRR domain (2) of Mex67p were fused to a GFP-NLS reporter construct (1) and expressed in wild-type yeast cells. Expression of the GFP constructs was tested by Western blotting using anti-GFP antibodies (top, lanes 1–5; note that in lane 2, five times as much as in the other lanes was loaded, which corresponds to the GFP signal for the fusion protein containing the LRR domain as compared with the other fusion proteins) and the intracellular localization determined by fluorescence microscopy (bottom, lanes 1–5). B, Nuclear export of GFP-Mex67p-C-NLS is impaired in xpo1-1 cells. Strain xpo1Δ (Stade et al. 1997) complemented either by plasmid-borne xpo1-1 or XPO1 were transformed with a plasmid harboring the GFP-Mex67-C-NLS reporter construct. Cells were grown in selective medium at 23°C before shift for 5, 10, and 20 min to the nonpermissive temperature (37°C). The intracellular localization of GFP-Mex67p-C-NLS was determined by fluorescence microscopy. C, Nuclear export of GFP-Mex67p-C-NLS is impaired in a S. cerevisiae strain, which is sensitive to LMB. LMB treatment of the LMB-sensitive (LMB-strain) and of a control strain (wild-type) was done as described (Neville and Rosbash 1999). Cells were incubated for 1 h in the presence of LMB.

Recombinant Mex67p/Mtr2p complex binds to different Nup repeat sequences. A, Nup159p-derived FG repeat sequences were expressed as a GST-fusion protein in E. coli and immobilized on GSH-beads, before incubation with partially purified Mex67p/His₆-Mtr2p complex, also derived from E. coli. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and Coomassie staining (top) or Western blotting (bottom) using anti-Mex67p and anti-Mtr2p antibodies, respectively. The pull-down experiment was carried out under different salt concentrations. Partially purified Mex67p/His₆-Mtr2p complex (lane 1; L, load); GST-FG_Nup159 immobilized on GSH-beads (lane 2; buffer); GST immobilized on GSH-beads (lane 3; buffer); immobilized GST-FG_Nup159 incubated with Mex67p/His₆-Mtr2p complex (lanes 4, 6, and 8) at the indicated salt concentrations; immobilized GST incubated with Mex67p/His₆-Mtr2p complex (lanes 5, 7, and 9) at the indicated salt concentrations. The asterisks indicate GST-FG_Nup159 and its breakdown products. B, Repeat sequences derived from Nup116p, Nup159p, Nsp1p, and Rip1p were expressed as GST-fusion proteins in E. coli and immobilized on GSH-beads, before incubation with E. coli whole cell lysates containing the recombinant Mex67p/His₆-Mtr2p complex. After the binding reaction, GST-beads were eluted with SDS-sample buffer and analyzed by SDS-PAGE and Coomassie staining or Western blotting using anti-Mex67p and anti-Mtr2p antibodies. L, E. coli lysate containing the Mex67p/His₆-Mtr2p complex; + and −, incubation of beads with lysate or buffer alone, respectively. Soluble E. coli lysate (L) containing the Mex67p/His₆-Mtr2p complex (lane 1); GST-GLFG_Nup116 immobilized on GSH-beads incubated with lysate (lane 2) or buffer (lane3); GST-FG_Nup159 immobilized on GSH-beads incubated with lysate (lane 4) or buffer (lane 5); GST-FXFG_Nsp1 immobilized on GSH-beads incubated with lysate (lane 6) or buffer (lane 7); GST-FG_Rip1 immobilized on GSH-beads incubated with lysate (lane 8) or buffer (lane 9); GST immobilized on GSH-beads incubated with lysate (lane 10) or buffer (lane 11). The positions of Mex67p and His-Mtr2p are indicated. C, Comparison of the highly purified Mex67p/Mtr2p complex and Mex67p/Mtr2p bound to GST-Nup116p repeat sequences. 1, Protein standard; 2, recombinant Mex67p/His-Mtr2p complex, expressed in E. coli, and purified by Ni-NTA affinity, FPLC-MonoS, and gel filtration chromatography; 3, Mex67p and Mtr2p bound to GST-Nup116p repeats. In lane 2 and 3, similar amounts of Mex67p were loaded and analyzed by SDS-PAGE and Coomassie staining. D, Concentration-dependent binding of Mex67p/Mtr2p to Nup repeat sequences. An E. coli lysate with Mex67p/His-Mtr2p complex was used undiluted or diluted 1:5, 1:10, or 1:50 with cold lysate lacking Mex67p/Mtr2p, and the derived lysates were incubated with GST-Nup116p or GST-Nup159p repeat sequences. The amount of bound Mex67p/Mtr2p was analyzed by SDS-PAGE and Coomassie staining. 1–4, GST-Nup116p repeats; 5–8, GST-Nup159p repeats; 1 and 5, undiluted lysate; 2 and 6, 1:5 diluted lysate; 3 and 7, 1:10 diluted lysate; 4 and 8, 1:50 diluted lysate. A protein standard is also shown. Note that a protein from the E. coli lysate binds to GST-Nup16p repeats when little Mex67p/Mtr2p complex is present. E, GST-FG_Nup159 containing GSH-beads (4–7; B, bound) were incubated with soluble E. coli cell lysates (1–3; L, load) that contain the recombinant Mex67p/His₆-Mtr2p complex (lanes 1 and 4), recombinant His₆-Mex67p (lanes 2 and 5), and recombinant His₆-Mtr2p (lanes 3 and 6) or buffer (lane 7). After a 1-h incubation, proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and Coomassie staining (top) or Western blotting (bottom) using anti-Mex67p and anti-Mtr2p antibodies.

Recombinant Mex67p/Mtr2p complex lacking the C domain of Mex67p binds to Nup repeat sequences. Partially purified Mex67p/His₆-Mtr2p complex, containing full-length Mex67p (lane 2) or Mex67pΔC1 (lane 3) was incubated with GST-Nup159p, GST-Rip1p, or GST-Nup116p repeats. After binding, GST-beads were eluted with SDS-sample buffer and analyzed by SDS-PAGE and Coomassie staining. GST-FG_Nup159 incubated with Mex67p/His₆-Mtr2p (lane 4), Mex67pΔC1/His₆-Mtr2p (lane 5), or buffer (lane 6); GST-FG_Rip1 incubated with Mex67p/His₆-Mtr2p (lane 7), Mex67pΔC1/His₆-Mtr2p (lane 8), or buffer (lane 9); GST-GLFG_Nup116 incubated with Mex67p/His₆-Mtr2p (lane 10), Mex67pΔC1/His₆-Mtr2p (lane 11), or buffer (lane 12). The positions of Mex67p, Mex67pΔC1, and His₆-Mtr2p are indicated. Molecular weight protein standard (lane 1).

Genetic interactions between MEX67 and Nups carrying repeat sequences. A, Schematic drawing of wild-type and mutant MEX67 alleles. mex67-5 and mex67-6 are impaired in the interaction with MTR2 (Segref et al. 1997) and YRA1 (Sträßer and Hurt 2000), respectively. The mex67(LL>EE) maps in the C domain (Segref et al. 1997). B, Genetic interaction between nup116 null (nup116::HIS3) and different mex67 ts alleles. The nup116::HIS3/mex67::HIS3 shuffle strain containing pURA3-MEX67 (Table ) was transformed as indicated with plasmids containing NUP116, mex67-5, mex67-6, and mex67ΔC1 gene constructs. The synthetic lethal relationship was tested by spotting transformants on 5-FOA–containing plates in 10⁻¹ dilution steps. Plates were incubated at 23°C for 6 d. Growth inhibition on 5-FOA plates indicates synthetic lethality. C, Genetic interaction between nup159, rip1, and nsp1 mutant alleles with different mex67 ts alleles. The corresponding mutant alleles are described in Materials and Methods. nupX::HIS3/mex67::HIS3 strains containing the complementing pURA3 plasmids (Table ) were transformed as indicated with plasmids containing nup159 or nsp1-ala6 mutants and the mex67-5, mex67-6, mex67ΔC1, or mex67(LL>EE) constructs. The synthetic lethal relationship was tested by spotting transformants on 5-FOA containing plates in 10⁻¹ dilution steps. Plates were incubated at 23°C for 6 d.