The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport

Abstract

Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1 coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640S temperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.

FIG. 1

In vitro reconstitution of two different heterotrimeric Nsp1p-containing complexes. (A) Schematic drawing of GST–Nsp1p-C constructs GST-Nsp1p coil 1 (positions 591 to 672), GST-Nsp1p coil 2 (673 to 738), GST-Nsp1p coils 2 and 3 (673 to 781), GST-Nsp1p coils 1 and 2 (591 to 738), and GST-Nsp1p coils 3 and 4 (728 to 823). (B) Purified GST–Nsp1p-C (lane 1) was mixed with urea-treated bacterial lysates containing His₆-Nup57p (lane 2). Proteins affinity purified via GSH-Sepharose (lane 4) were separated from unbound proteins (lane 3). (C) Purified GST–Nsp1p-C was mixed with urea-treated bacterial lysates containing His₆-Nup49p (lane 1), His₆-Nup57p (lane 2), or a mixture of both (lane 3). (D) Purified GST–Nsp1p-C (lanes 1 to 3) or GST alone (lane 4) was mixed with urea-treated bacterial lysate containing His₆-Nup82p (lane 1), His₆–Nup159p-C (lane 2 and 4), or a mixture of both (lane 3). (E) GST-tagged fragments of Nsp1p-C (as shown in panel A) were mixed with urea-treated bacterial lysate containing His₆-Nup57p or His₆-Nup82p. Open circles represent GST fusion proteins, filled triangles represent Nup57p, and open triangles represent Nup82p. (F) GST–Nsp1p-C (lane 1), GST-Nsp1p coils 2 and 3 (lane 2), GST-Nsp1p coil 2 (lane 3), or GST alone (lane 4) was mixed with urea-treated bacterial lysate containing His₆-Nup49p and His₆-Nup57p. (G) GST-Nsp1p coil 2 (lanes 1 to 3) was mixed with urea-treated bacterial lysate containing His₆-Nup82p (lane 1), His₆–Nup159p-C (lane 2), or a mixture of both (lane 3). For panels B to G, dialysis of the protein mixture was followed by affinity purification of the reconstituted GST-Nsp1p subcomplexes or of GST on glutathione-Sepharose. Proteins eluted from the column were analyzed by SDS-PAGE and Coomassie staining or Western blotting with anti-His, anti-Nsp1p, or anti-GST antibodies.

FIG. 2

Nup82p and Nup57p compete for binding to Nsp1p-C. (A) Schematic drawing of the competition assay. (B) GST-Nup57p (lanes 1 and 2, indicated by open circles) or GST–Nsp1p-C (lane 3, indicated by an open circle) was mixed with urea-treated bacterial lysate containing His₆-Nup82p and His₆–Nsp1p-C (lane 1) or only His₆-Nup82p (lane 2 and 3). In vitro reconstitution was performed as described in the legend to Fig. 1. Proteins eluted from the glutathione-Sepharose or unbound proteins were analyzed by SDS-PAGE, followed by silver staining or Western blotting with anti-Nup57p, anti-Nup82p, or anti-Nsp1p antibodies. GST-Nsp1p, used in lane 3, is not shown in the Western blot. The band seen in the anti-Nup57p Western blot (lane 3) results from the first incubation of this membrane with anti-Nup82p antibodies and thus represents His₆-Nup82p. (C) GST–Nsp1p-C was mixed with bacterial lysates containing His₆-Nup82p (lane 1), both His₆-Nup82p lysate and increasing amounts of purified His₆-Nup57p (lanes 2 to 6), or His₆-Nup57p alone (lane 7). In vitro reconstitution was performed as described in the legend to Fig. 1, and proteins eluted from glutathione-Sepharose were analyzed by SDS-PAGE and Coomassie staining or Western blotting with anti-His antibodies.

FIG. 3

Mutational analysis of the various coiled-coil regions within Nsp1p-C. (A) Schematic drawings of Nsp1p-C (591–823) (map 1), Nsp1p-C (630–823) (map 2), nsp1-(638–823) (map 3), nsp1-(642–823) (map 4), nsp1-L640S (map 5), nsp1 tsΔ4 (map 6), nsp1 ts18 (map 7), and nsp1-ala6 (map 8). (B) Growth properties of Nsp1p-C and nsp1 ts cells as indicated in panel A. In each case, the nsp1Δ strain was complemented by a plasmid expressing the corresponding mutant (see Table 2). Precultures were diluted in 10⁻¹ steps, and equivalent amounts of cells were dropped on YPD plates and incubated at 23, 30, or 37°C for 3 to 4 days.

FIG. 4

Roles of the various coiled-coil regions of Nsp1p-C in nucleocytoplasmic transport. (A) Nuclear poly(A)⁺ RNA export. NSP1-C or nsp1-L640S, nsp1-ala6, and nsp1 ts18 cells were grown at 23°C before a shift to 37°C for 0 min, 30 min, or 3 h. The localization of poly(A)⁺ RNA was analyzed by in situ hybridization with a Cy3-labeled oligo(dT) probe. DNA was visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. (B) NPC localization of GFP-Nup82p or GFP-Nup159p in nsp1-L640S cells as revealed by fluorescence microscopy. Cells were grown in selective media at 23°C or shifted to 37°C for 2 h. (C) Nuclear protein import. NSP1-C, nsp1-L640S, nsp1-ala6, and nsp1 ts18 cells expressing GFP-Npl3p were grown in selective media at 23°C or shifted to 37°C for 3 h and analyzed by fluorescence microscopy. In each case, the nsp1Δ strain was complemented by a plasmid expressing the corresponding mutant (see Table 2).

FIG. 5

Nsp1p coils 3 and 4 are required for docking of the Nsp1p-Nup57p-Nup49p complex to the NPC. (A) Schematic drawing of nsp1 ts18. (B) Localization of GFP-NSP1-C and GFP-nsp1 ts18 expressed in nsp1Δ cells. Bars, 4 μm. (C) Localization of GFP-Nic96p, GFP-Nup57p, GFP-Nup82p, and GFP-Nup159p in nsp1Δ/nupXΔ strains (see Table 1) complemented by two plasmids expressing NSP1-C or nsp1 ts18 cells and GFP-NupXp. Cells were grown in selective media at 23°C and analyzed by fluorescence microscopy or Nomarski optics. Bars, 4 μm. (D) Affinity purification of ProtA–Nsp1p-C and ProtA-nsp1 ts18 expressed in nsp1Δ cells. Whole-cell lysates (lanes 1 and 4) or proteins eluted from IgG-Sepharose (lanes 2 and 3) were analyzed by SDS-PAGE and Coomassie staining or by Western blotting with anti-Nup159p, anti-Nic96p, anti-Nup82p, anti-Nup57p, and anti-ProtA antibodies. The positions of ProtA fusion proteins are indicated by open circles, and the positions of copurifying proteins are indicated by asterisks.

FIG. 6

Model of interaction of Nup82p-Nup159p and Nup57p-Nup49p-Nic96p, respectively, with Nsp1p-C.