Mapping of CT-Mlp1 reveals a Nab2-binding region between residues 1586 and 1779. A, schematic depicting sizes of CT-Mlp1 truncation mutants (CT1–7) and the Nab2-binding domain of Mlp1 (Mlp1-NBD) constructed. B, CT-Mlp1 truncation mutant CT2 (residues 1490–1779) binds to Nab2 in vitro, but mutant CT5 (residues 1490–1585) does not bind to Nab2. Recombinant GST, GST-CT-Mlp1 (CT-Mlp1), or the GST-CT-Mlp1 truncation mutant (CT2, CT4, CT5) bound to glutathione-Sepharose beads were incubated with recombinant His-tagged Nab2 (Nab2) and unbound (U) and bound (B) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. C, CT-Mlp1 truncation mutant CT6 (residues 1586–1779) binds to Nab2 in vitro. Recombinant GST, GST-CT-Mlp1 (CT-Mlp1), or the GST-CT-Mlp1 truncation mutant (CT6, CT7) bound to glutathione-Sepharose beads was incubated with recombinant His-tagged Nab2 (Nab2) and unbound (U) and bound (B) fractions were analyzed by immunoblotting with anti-His antibody. The percentage of His-Nab2 band intensity in each bound fraction relative to the His-Nab2 band intensity in the bound fraction of GST-CT-Mlp1 is indicated below the immunoblot. D, expression of CT-Mlp1 truncation mutant CT2 (residues 1490–1779) causes relocalization of ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and CT-Mlp1 truncation mutants (CT1–5) were expressed in yeast cells expressing a Nab2 mutant fused to GFP (ΔRGG-Nab2-GFP), which displays localization throughout the cell (36), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector (Vec) was used as a control. Corresponding differential interference contrast (DIC) images are shown.

Mapping of CT-Mlp1 defines a region between residues 1490 and 1779 sufficient to cause nuclear poly(A) RNA accumulation. CT-Mlp1 and CT-Mlp1 truncation mutants (CT1–5) were expressed in yeast cells and poly(A) RNA (Poly(A)) was visualized by fluorescence in situ hybridization with an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast (DIC) images are shown.

The Nab2-binding domain of Mlp1 directly interacts with Nab2 and causes nuclear accumulation of poly(A) RNA. A, the Nab2-binding domain of Mlp1 (Mlp1-NBD; residues 1586–1768) binds to the N-terminal domain of Nab2 (Nab2-N; residues 1–97) in vitro. His-Nab2-N (Nab2-N) or, as a control, ovalbumin (Oval.) coupled to Sepharose beads was incubated with His-Mlp1-NBD and bound fractions were analyzed by SDS-PAGE and Coomassie staining. B, the Nab2-binding domain of Mlp1 (Mlp1-NBD) co-purifies with the N-terminal domain of Nab2 (Nab2-N). Cell lysates from E. coli expressing His-Mlp1-NBD and E. coli expressing GST-Nab2-N were combined, incubated with glutathione beads, and beads were washed three times. Input combined lysate (I), unbound (U) fraction, three wash fractions (W1, W2, W3), and bound (B) fraction were analyzed by SDS-PAGE and Coomassie staining. C, expression of the Nab2-binding domain of Mlp1 (Mlp1-NBD) relocalizes ΔRGG-Nab2-GFP to the nucleus. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells expressing ΔRGG-Nab2-GFP, which displays localization throughout the cell (36), and ΔRGG-Nab2-GFP was visualized by direct fluorescence microscopy. Empty vector (Vec) was used as control. Corresponding differential interference contrast (DIC) images are shown. D, expression of the Nab2-binding domain of Mlp1 (Mlp1-NBD) causes nuclear accumulation of poly(A) RNA. CT-Mlp1 and Mlp1-NBD were expressed in yeast cells and poly(A) RNA (Poly(A)) was visualized by fluorescence in situ hybridization with an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast images are shown.

The Nab2-binding domain of Mlp1 binds to the N-terminal domain of Nab2 with micromolar affinity and Nab2 F73D substitution disrupts the Nab2/Mlp1 interaction. A, surface representation of the N-terminal domain of Nab2 (Nab2-N) showing the putative Mlp1 interaction site (gray) identified by Grant et al. (49). Phe⁷³ (dark gray) is central to this site and its hydrophobic aromatic side chain is completely exposed to the solvent. B, the binding affinity of the Nab2-binding domain of Mlp1 (Mlp1-NBD; residues 1586–1768) for Nab2-N wild-type and mutants F72D, F73D, and F73W (residues 1–97) was measured quantitatively in a solid-phase binding assay (54). Binding of S-Tag-Mlp1-NBD (Mlp1-NBD) to GST-Nab2-N wild-type (Nab2-N-WT), GST-Nab2-N-F72D (Nab2-N-F72D), GST-Nab2-N-F73D (Nab2-N-F73D), or GST-Nab2-N-F73W (Nab2-N-F73W) at increasing concentrations of S-Tag-Mlp1-NBD was measured at optical density of 450 nm (A₄₅₀) as described under “Experimental Procedures.” Measurements at A₄₅₀ (amount bound) were used to generate saturation binding curves by non-linear regression. Standard error bars for A₄₅₀ readings from triplicate samples of GST-Nab2-N-WT and mutants at each S-Tag-Mlp1-NBD concentration are indicated. These binding curves were used to calculate apparent dissociation constants for binding between Mlp1-NBD and Nab2-N wild-type (K_d ∼ 1.3 ± 0.4 μm), Nab2-N-F72D (K_d ∼ 2.3 ± 1.1 μm), or Nab2-N-F73W (K_d ∼ 0.8 ± 0.2 μm) (see Table 2). The weak binding between Mlp1-NBD and Nab2-N-F73D was indistinguishable from nonspecific binding, indicating that the F73D substitution essentially disrupts the Nab2/Mlp1 interaction.

nab2 F73D mutant cells grow similarly to wild-type cells but display nuclear accumulation of poly(A) RNA. A, nab2 F73D cells are viable and grow similarly to wild-type NAB2 cells. NAB2Δ cells maintained by a NAB2 URA3 plasmid and containing NAB2, nab2 ΔN, nab2 F72D, or nab2 F73D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions, and spotted on control and 5-FOA plates. Cells were grown at 30 °C. B, growth curve analysis of nab2 F73D cells confirms that they grow similarly to wild-type NAB2 cells. NAB2Δ cells carrying only NAB2, nab2 F72D, or nab2 F73D plasmids were grown to saturation, diluted, and their optical density was measured at A₆₀₀ for 40 h as described under “Experimental Procedures.” C, nab2 F73D cells display nuclear accumulation of poly(A) RNA. NAB2Δ cells carrying only NAB2, nab2 ΔN, nab2 F72D, or nab2 F73D plasmids were grown in liquid culture at 30 °C and poly(A) RNA (Poly(A)) was visualized by FISH using an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast (DIC) images are shown. FISH results were quantitated as described under “Experimental Procedures” to assess the mean percentage of cells with nuclear accumulation of poly(A) RNA within 10 fields of at least 195 cells each (indicated to the right of DIC images). Variation in the percentage of cells with nuclear poly(A) RNA in each field of cells from the mean was assessed by calculating the standard deviation: NAB2, 3.2 ± 1.0%; nab2 F72D, 3.9 ± 1.5%; and nab2 F73D, 8.2 ± 2.0%. Thus, nab2 F73D cells exhibit a 2.5-fold increase in nuclear poly(A) RNA accumulation relative to NAB2 cells, whereas nab2 F72D cells show only a modest 1.2-fold increase. The percentages of nuclear poly(A) RNA accumulation observed in nab2 F73D cells were typical of multiple independent experiments.

The nab2 F73D mutant displays genetic interactions with mRNA export mutants mex67-5 and yra1–8 and mex67-5 nab2 F73D cells show nuclear poly(A) RNA accumulation. A, mex67-5 nab2 F73D cells or yra1–8 nab2 F73D cells have a slow growth phenotype. NAB2Δ mex67-5 or NAB2Δ yra1–8 cells maintained by a NAB2 URA3 plasmid and containing NAB2, nab2 ΔN, nab2 F72D, or nab2 F73D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions and spotted on control and 5-FOA plates. Cells were grown at 30 °C. B, growth curve analysis of mex67-5 nab2 F73D cells confirms that they grow more slowly than mex67-5 NAB2 cells. mex67-5 NAB2Δ cells carrying only NAB2, nab2 F72D, or nab2 F73D plasmids were grown to saturation, diluted, and their optical density was measured at A₆₀₀ for 40 h as described under “Experimental Procedures.” C, mex67-5 nab2 F73D cells show nuclear accumulation of poly(A) RNA. NAB2Δ mex67-5 cells carrying only NAB2, nab2 F72D, or nab2 F73D plasmids were grown in liquid culture at 30 °C and poly(A) RNA (Poly(A)) was visualized by FISH using an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast (DIC) images are shown. FISH results were quantitated as described under “Experimental Procedures” to assess the mean percentage of cells with nuclear accumulation of poly(A) RNA within 10 fields of at least 43 cells each (indicated to the right of the DIC images). Variation in the percentage of cells with nuclear poly(A) RNA in each field of cells from the mean was assessed by calculating the standard deviation: mex67-5 NAB2, 18.6 ± 4.8%; mex67-5 nab2 F72D, 20.3 ± 6.0%; and mex67-5 nab2 F73D, 30.8 ± 6.9. Thus, mex67-5 nab2 F73D cells exhibit a 1.7-fold increase in nuclear poly(A) RNA accumulation relative to mex67-5 NAB2 cells, whereas mex67-5 nab2 F72D cells show only a slight 1.1-fold increase. The percentages of nuclear poly(A) RNA accumulation observed in mex67-5 nab2 F73D cells were typical of multiple independent experiments.

Models for Nab2 function in mRNA export and the observed growth and mRNA export defects in mex67-5 nab2 F73D cells at the permissive temperature. A, model for Nab2 function in mRNA export in which Nab2 helps to target mRNA to the nuclear pore for export. Initially, mRNA is co-transcriptionally loaded with the mRNA export adaptor, Yra1 (21, 62), and bound by Nab2 at the poly(A) tail (34–36). Yra1 then binds to the mRNA export protein, Mex67 (18, 19), recruiting the Mex67/Mtr2 heterodimeric receptor to the mRNA (16) and Mex67/Mtr2 interacts with FG-nucleoporins at the pore to translocate the mRNA to the cytoplasm (17). Alongside Yra1 and Mex67, Nab2 binds to Mlp1 at the pore (38), via the Nab2-N/Mlp1-NBD interaction. The assembled mRNA complex containing Nab2 is then translocated through the NPC channel by Mex67/Mtr2 (17, 63). Upon reaching the cytoplasmic face of the nuclear pore, the mRNA export complex encounters the DEAD-box helicase, Dbp5 (64, 65), tethered and activated by the nucleoporin, Gle1 (66–69), which remodels the mRNP to trigger dissociation of Nab2 from the RNA (70), release of Mex67, and disassembly of the complex (71). B, model to explain the observed growth and mRNA export defects in mex67-5 nab2 F73D cells at the permissive temperature in which combined impairment of Mex67 and Nab2 reduces mRNA export. Here, the Mex67-5 mutant, which cannot interact with Mtr2 and FG-nucleoporins at the non-permissive temperature (8, 16), may also be impaired in binding to FG-nucleoporins at the permissive temperature, potentially resulting in a moderate reduction in the rate of translocation. Similarly, the Nab2 F73D mutant, which cannot interact with Mlp1, may reduce the rate of mRNA concentration at the pore. Combination of the reduction in mRNA translocation rate produced by Mex67-5 and reduction in the mRNA concentration rate caused by Nab2 F73D could become rate-limiting and result in a decreased rate of mRNA export coupled with nuclear accumulation of poly(A) RNA.