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A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.

Iovine MK, Wente SR - J. Cell Biol. (1997)

Bottom Line: Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope.The protein A-tagged Nup116p complex also specifically contained Gle2p.These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

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Protein A–Nup116p  interacts with Kap95p and  Gle2p. (A and B) Nuclear lysates were prepared from the  diploid strain SWY960 (protein A–Nup116p) and incubated with IgG-Sepharose.  Lysate (L), unbound (U),  and acid eluted (E) fractions  were separated by electrophoresis in SDS polyacrylamide gels: 7.5% gels in A  and 10.5% gels in B. The gels  were either silver stained or  transferred to nitrocellulose.  Immunoblots were incubated  with anti-Kap95p antibody  (1:100) or anti-Gle2p antibody (1:25) as indicated. The  dashes along the silver stain  gel indicate the polypeptides  that align with the corresponding Kap95p and Gle2p  cross-reactive bands in the  immunoblots (single stars).  The double stars indicate  the position of the protein  A–Nup116. (C) A fraction of the protein A–Nup116p remains bound to the IgG-Sepharose. After elution with acidic buffer (yielding  the samples in lanes E), IgG beads were treated with SDS buffer, and the bound fraction was analyzed on 7.5% SDS polyacrylamide  gels. The immunoblot was developed with rabbit anti–IgG antibody (1:1,000): (lane 1) mock treated beads; (lane 2) beads incubated  with protein A–Nup116p nuclear lysate. (Lane 3) A silver stain of the same fraction analyzed in lane 2. The double star indicates the position of the bound protein A–Nup116p (the upper band of the doublet when compared with untreated resin). (D) The Kap95p and  Gle2p interactions with protein A–Nup116p are specific. Control experiments were conducted with nuclear lysates prepared from the  diploid strain W303a/α (WT nuclei) incubated with IgG-Sepharose (left), or from SWY960 (protein A–Nup116 nuclei) incubated with  protein A–Sepharose (right). Samples that eluted with acidic buffer from the IgG-Sepharose or that eluted with SDS buffer from the  protein A–Sepharose were separated by electrophoresis in 7.5% (for Kap95p) or 10.5% (for Gle2p) SDS polyacrylamide gels and  tested by immunoblotting. The respective positions expected for Kap95p and Gle2p bands are indicated by single stars. The bands by  the plus sign (+) are due to the protein A from the resin. For all the panels, the sizes of the molecular mass markers are indicated at the  right.
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Figure 1: Protein A–Nup116p interacts with Kap95p and Gle2p. (A and B) Nuclear lysates were prepared from the diploid strain SWY960 (protein A–Nup116p) and incubated with IgG-Sepharose. Lysate (L), unbound (U), and acid eluted (E) fractions were separated by electrophoresis in SDS polyacrylamide gels: 7.5% gels in A and 10.5% gels in B. The gels were either silver stained or transferred to nitrocellulose. Immunoblots were incubated with anti-Kap95p antibody (1:100) or anti-Gle2p antibody (1:25) as indicated. The dashes along the silver stain gel indicate the polypeptides that align with the corresponding Kap95p and Gle2p cross-reactive bands in the immunoblots (single stars). The double stars indicate the position of the protein A–Nup116. (C) A fraction of the protein A–Nup116p remains bound to the IgG-Sepharose. After elution with acidic buffer (yielding the samples in lanes E), IgG beads were treated with SDS buffer, and the bound fraction was analyzed on 7.5% SDS polyacrylamide gels. The immunoblot was developed with rabbit anti–IgG antibody (1:1,000): (lane 1) mock treated beads; (lane 2) beads incubated with protein A–Nup116p nuclear lysate. (Lane 3) A silver stain of the same fraction analyzed in lane 2. The double star indicates the position of the bound protein A–Nup116p (the upper band of the doublet when compared with untreated resin). (D) The Kap95p and Gle2p interactions with protein A–Nup116p are specific. Control experiments were conducted with nuclear lysates prepared from the diploid strain W303a/α (WT nuclei) incubated with IgG-Sepharose (left), or from SWY960 (protein A–Nup116 nuclei) incubated with protein A–Sepharose (right). Samples that eluted with acidic buffer from the IgG-Sepharose or that eluted with SDS buffer from the protein A–Sepharose were separated by electrophoresis in 7.5% (for Kap95p) or 10.5% (for Gle2p) SDS polyacrylamide gels and tested by immunoblotting. The respective positions expected for Kap95p and Gle2p bands are indicated by single stars. The bands by the plus sign (+) are due to the protein A from the resin. For all the panels, the sizes of the molecular mass markers are indicated at the right.

Mentions: To further characterize the physiological significance of a Kap95p–GLFG nucleoporin interaction, coimmunoprecipitation experiments were conducted from yeast cell fractions. These studies initially focused on Nup116p because its GLFG region is uniquely required for cell growth (Iovine et al., 1995). For these experiments, full-length Nup116p was tagged in its amino-terminal region with five tandem IgG-binding domains from Staphylococcus aureus (protein A) and expressed from the NUP116 promoter in an nup116 strain. The protein A–Nup116p completely complemented the nup116 phenotype, and it localized exclusively at NPCs as determined by indirect immunofluorescence microscopy (data not shown). Lysates of purified yeast nuclei containing protein A–Nup116p were incubated with IgG-Sepharose. Copurifying proteins were eluted with acidic buffer and analyzed by SDS-PAGE. Multiple polypeptides were detected by silver staining (Fig. 1, A and B). Immunoblotting with affinity-purified polyclonal anti-Kap95p antibodies revealed that the polypeptide of ∼95 kD corresponded to Kap95p (Fig. 1 A). The majority of the protein A–Nup116p remained associated with the IgG-Sepharose and was only eluted with SDS treatment (Fig. 1 C). Therefore, Kap95p was isolated from yeast cells in a complex containing protein A–Nup116p.


A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.

Iovine MK, Wente SR - J. Cell Biol. (1997)

Protein A–Nup116p  interacts with Kap95p and  Gle2p. (A and B) Nuclear lysates were prepared from the  diploid strain SWY960 (protein A–Nup116p) and incubated with IgG-Sepharose.  Lysate (L), unbound (U),  and acid eluted (E) fractions  were separated by electrophoresis in SDS polyacrylamide gels: 7.5% gels in A  and 10.5% gels in B. The gels  were either silver stained or  transferred to nitrocellulose.  Immunoblots were incubated  with anti-Kap95p antibody  (1:100) or anti-Gle2p antibody (1:25) as indicated. The  dashes along the silver stain  gel indicate the polypeptides  that align with the corresponding Kap95p and Gle2p  cross-reactive bands in the  immunoblots (single stars).  The double stars indicate  the position of the protein  A–Nup116. (C) A fraction of the protein A–Nup116p remains bound to the IgG-Sepharose. After elution with acidic buffer (yielding  the samples in lanes E), IgG beads were treated with SDS buffer, and the bound fraction was analyzed on 7.5% SDS polyacrylamide  gels. The immunoblot was developed with rabbit anti–IgG antibody (1:1,000): (lane 1) mock treated beads; (lane 2) beads incubated  with protein A–Nup116p nuclear lysate. (Lane 3) A silver stain of the same fraction analyzed in lane 2. The double star indicates the position of the bound protein A–Nup116p (the upper band of the doublet when compared with untreated resin). (D) The Kap95p and  Gle2p interactions with protein A–Nup116p are specific. Control experiments were conducted with nuclear lysates prepared from the  diploid strain W303a/α (WT nuclei) incubated with IgG-Sepharose (left), or from SWY960 (protein A–Nup116 nuclei) incubated with  protein A–Sepharose (right). Samples that eluted with acidic buffer from the IgG-Sepharose or that eluted with SDS buffer from the  protein A–Sepharose were separated by electrophoresis in 7.5% (for Kap95p) or 10.5% (for Gle2p) SDS polyacrylamide gels and  tested by immunoblotting. The respective positions expected for Kap95p and Gle2p bands are indicated by single stars. The bands by  the plus sign (+) are due to the protein A from the resin. For all the panels, the sizes of the molecular mass markers are indicated at the  right.
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Related In: Results  -  Collection

Show All Figures
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Figure 1: Protein A–Nup116p interacts with Kap95p and Gle2p. (A and B) Nuclear lysates were prepared from the diploid strain SWY960 (protein A–Nup116p) and incubated with IgG-Sepharose. Lysate (L), unbound (U), and acid eluted (E) fractions were separated by electrophoresis in SDS polyacrylamide gels: 7.5% gels in A and 10.5% gels in B. The gels were either silver stained or transferred to nitrocellulose. Immunoblots were incubated with anti-Kap95p antibody (1:100) or anti-Gle2p antibody (1:25) as indicated. The dashes along the silver stain gel indicate the polypeptides that align with the corresponding Kap95p and Gle2p cross-reactive bands in the immunoblots (single stars). The double stars indicate the position of the protein A–Nup116. (C) A fraction of the protein A–Nup116p remains bound to the IgG-Sepharose. After elution with acidic buffer (yielding the samples in lanes E), IgG beads were treated with SDS buffer, and the bound fraction was analyzed on 7.5% SDS polyacrylamide gels. The immunoblot was developed with rabbit anti–IgG antibody (1:1,000): (lane 1) mock treated beads; (lane 2) beads incubated with protein A–Nup116p nuclear lysate. (Lane 3) A silver stain of the same fraction analyzed in lane 2. The double star indicates the position of the bound protein A–Nup116p (the upper band of the doublet when compared with untreated resin). (D) The Kap95p and Gle2p interactions with protein A–Nup116p are specific. Control experiments were conducted with nuclear lysates prepared from the diploid strain W303a/α (WT nuclei) incubated with IgG-Sepharose (left), or from SWY960 (protein A–Nup116 nuclei) incubated with protein A–Sepharose (right). Samples that eluted with acidic buffer from the IgG-Sepharose or that eluted with SDS buffer from the protein A–Sepharose were separated by electrophoresis in 7.5% (for Kap95p) or 10.5% (for Gle2p) SDS polyacrylamide gels and tested by immunoblotting. The respective positions expected for Kap95p and Gle2p bands are indicated by single stars. The bands by the plus sign (+) are due to the protein A from the resin. For all the panels, the sizes of the molecular mass markers are indicated at the right.
Mentions: To further characterize the physiological significance of a Kap95p–GLFG nucleoporin interaction, coimmunoprecipitation experiments were conducted from yeast cell fractions. These studies initially focused on Nup116p because its GLFG region is uniquely required for cell growth (Iovine et al., 1995). For these experiments, full-length Nup116p was tagged in its amino-terminal region with five tandem IgG-binding domains from Staphylococcus aureus (protein A) and expressed from the NUP116 promoter in an nup116 strain. The protein A–Nup116p completely complemented the nup116 phenotype, and it localized exclusively at NPCs as determined by indirect immunofluorescence microscopy (data not shown). Lysates of purified yeast nuclei containing protein A–Nup116p were incubated with IgG-Sepharose. Copurifying proteins were eluted with acidic buffer and analyzed by SDS-PAGE. Multiple polypeptides were detected by silver staining (Fig. 1, A and B). Immunoblotting with affinity-purified polyclonal anti-Kap95p antibodies revealed that the polypeptide of ∼95 kD corresponded to Kap95p (Fig. 1 A). The majority of the protein A–Nup116p remained associated with the IgG-Sepharose and was only eluted with SDS treatment (Fig. 1 C). Therefore, Kap95p was isolated from yeast cells in a complex containing protein A–Nup116p.

Bottom Line: Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope.The protein A-tagged Nup116p complex also specifically contained Gle2p.These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

Show MeSH
Related in: MedlinePlus