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. 1999 Feb;19(2):1547-57.
doi: 10.1128/MCB.19.2.1547.

Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase

M Seedorf  1 , M Damelin, J Kahana, T Taura, P A Silver
Affiliations

Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase

M Seedorf et al. Mol Cell Biol. 1999 Feb.

Abstract

Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.

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Figures

FIG. 1
FIG. 1
Expression of Pse1p, GFP-tagged Pse1p, and growth characteristics of wild-type, PSE1-GFP, and pse1-1–GFP strains. (A) Equal amounts of yeast lysates (20 μg of total protein) from a wild-type strain (PSY580) and a strain where PSE1 is replaced by PSE1-GFP were separated on an 8% gel by SDS-PAGE, transferred to nitrocellulose, and incubated with Pse1p-specific antibodies (74) (lanes 1 and 2) or with GFP-specific antibodies (lanes 3 and 4). (B) Growth of a wild-type yeast strain, a strain where PSE1 is replaced by PSE1-GFP, and a pse1-1 mutant strain where the C terminus of pse1-1 is replaced by the C-terminal portion of PSE1-GFP. Cells were streaked on YEPD plates and incubated for 60 h at 25 or 37°C.
FIG. 2
FIG. 2
Intracellular localization of importin-β homologues. (A) Cells with PSE1, SXM1, KAP95, or XPO1 replaced by GFP-tagged versions were grown in selective media at 25°C and analyzed by fluorescence microscopy. (B) Localization of GFP-tagged Pse1p in wild-type, rna1-1, and prp20-1 mutant cells. Cells were grown at 25°C in selective media and shifted for 60 min to 37°C. All of the cells were transferred onto slides and examined immediately.
FIG. 3
FIG. 3
Expression and immunoprecipitation of GFP-tagged importin-βs. (A) Equal amounts of yeast lysates (10 μg of total protein) from strains with PSE1 (lane 1), SXM1 (lane 2), XPO1 (lane 3), or KAP95 (lane 4) replaced with GFP-tagged versions and wild-type cells expressing LacZ-GFP (lane 5) were separated on an 8% gel by SDS-PAGE and transferred onto nitrocellulose. Anti-GFP beads were used to precipitate complexes from lysates. PSE1 (lane 6), SXM1 (lane 7), XPO1 (lane 8), KAP95 (lane 9), and LacZ-GFP (lane 10) containing complexes and corresponding to 250 μl of yeast lysate (approximately 1 mg/ml) were separated and transferred to membranes as well. The top row shows a blot probed with GFP-specific antibodies. A second blot with identical samples was cut in half, and the upper portion was probed with Kap95p-specific antibodies, and the bottom half was probed with Srp1p-specific antibodies. (B) Genomic PSE1 was replaced by PSE1-GFP in wild-type, rna1-1, and prp20-1 strains. Cells were grown at 25°C in liquid culture, and half of the cells were shifted for 90 min to 37°C. Pse1-GFP was immunoprecipitated with anti-GFP beads, and the bound fraction was probed with GFP-, Kap95p-, and Srp1p-specific antibodies.
FIG. 4
FIG. 4
Nucleoporins are present in importin-β–GFP complexes. (A) Yeast lysate from a strain expressing Pse1-GFP (lane 1) and precipitated proteins from strains where importin-β genes were replaced either by PSE1-GFP, SXM1-GFP, XPO1-GFP, or KAP95-GFP were separated by SDS-PAGE and analyzed by immunoblotting. Duplicates of identical blots were probed from top to bottom with GFP-specific antibodies, Nup159p-specific antibodies, Pom152p-specific antibodies, Nup116p-specific antibodies, and Nsp1p-specific antibodies. (B) Wild-type, rna1-1, and prp20-1 cells where genomic PSE1 was replaced by PSE1-GFP were grown at 25°C, and half of each culture was shifted for 90 min to 37°C. Identical samples with precipitated Pse1-GFP complexes were separated on an 8% gel by SDS-PAGE, transferred onto nitrocellulose, and incubated with GFP-, Nup159p-, Nup116p-, or Nsp1p-specific antibodies.
FIG. 5
FIG. 5
Intracellular localization of Pse1-1 protein. (A) The schematic (not drawn to scale) depicts pPS1538 containing a C-terminal fragment of PSE1 fused to GFP and the resulting arrangement after the replacement of the pse1-1 allele with the C-terminal portion of PSE1-GFP. Amino acid changes due to mutations are indicated. (B) Cells expressing Pse1-GFP or Pse1-1–GFP were grown at 25°C in selective media, and half of the cultures were shifted for 30 min to 37°C. Cells were transferred to slides and immediately subjected to microscopic analysis. The GFP signal from Pse1-1 cells was detected by a threefold longer exposure time than that from wild-type cells. (C) Cells expressing Pse1-1–GFP were probed with MAb 414 to visualize the nuclear envelope and with anti-Nop1p to visualize the nucleolus and then stained with DAPI to visualize their DNA after a shift to 37°C. Arrowheads indicate Pse1-1–GFP, and the smaller arrow indicates the nucleolus, as determined by localization of Nop1p.
FIG. 5
FIG. 5
Intracellular localization of Pse1-1 protein. (A) The schematic (not drawn to scale) depicts pPS1538 containing a C-terminal fragment of PSE1 fused to GFP and the resulting arrangement after the replacement of the pse1-1 allele with the C-terminal portion of PSE1-GFP. Amino acid changes due to mutations are indicated. (B) Cells expressing Pse1-GFP or Pse1-1–GFP were grown at 25°C in selective media, and half of the cultures were shifted for 30 min to 37°C. Cells were transferred to slides and immediately subjected to microscopic analysis. The GFP signal from Pse1-1 cells was detected by a threefold longer exposure time than that from wild-type cells. (C) Cells expressing Pse1-1–GFP were probed with MAb 414 to visualize the nuclear envelope and with anti-Nop1p to visualize the nucleolus and then stained with DAPI to visualize their DNA after a shift to 37°C. Arrowheads indicate Pse1-1–GFP, and the smaller arrow indicates the nucleolus, as determined by localization of Nop1p.
FIG. 6
FIG. 6
Mutant Pse1-1p does not interact with Ran/Gsp1p. (A) Cells expressing Pse1-GFP or Pse1-1–GFP were grown at 25°C. Lysates were split, and half of each lysate was incubated with 1 mM GMP-PNP. Pse1-GFP and pse1-1–GFP were immunoprecipitated, bound proteins were separated on a 12% gel by SDS-PAGE and transferred to nitrocellulose, and the membrane was cut in half. The top portion was probed with GFP-specific antibodies, and the bottom half was probed with Ran/Gsp1p-specific antibodies. (B) Lysates from Pse1-1–GFP and Pse1-GFP expressing cells were treated with GMP-PNP and subjected to immunoprecipitation. To match equal amounts of Pse1-GFP, 10-fold more pse1-1–GFP lysate was used for immunoprecipitation. The bound proteins were analyzed as described for panel A.
FIG. 7
FIG. 7
Nucleoporins and importins present in Pse1-GFP and pse1-1–GFP complexes. Cells were grown at 25°C, and half of each culture was shifted for 1 h to 37°C. Pse1-GFP- and pse1-1–GFP-containing complexes were precipitated, and identical samples were separated on an 8% gel by SDS-PAGE and transferred to nitrocellulose. Blots were incubated with GFP-, Nsp1p-, Nup116p-, or Rat7/Nup159p-specific antibodies. One blot was cut in half; the upper half was incubated with Kap95p, and the bottom half was incubated with Srp1p-specific antibodies.
FIG. 8
FIG. 8
Diagram summarizing Pse1p interactions. In wild-type cells or when Prp20p is inactive, Pse1p is at the NPC complexed to Nup159, Nup116, and Nsp1p.
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