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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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The NES region of  Kap95p mediates nuclear export. (A) Alignment of a region of Kap95p with the NES  sequences of HIV-1 Rev, PKI,  TFIIIA, and Gle1p is shown.  (B) GST-Kap95-(1-77) and  GST-Kap95-(1-77)ΔNES fusion proteins were purified  and conjugated to FITC. The  fluorescent conjugates were  individually coinjected into  the nuclei of COS-1 cells with  Texas red–HSA. After a 45min incubation at either 37°C  or 4°C, the cells were fixed  and examined by fluorescence  microscopy. The Texas red– HSA remained nuclear localized (bottom row); however,  the FITC-GST-Kap95-(1-77)  moved to the cytoplasm (top,  left) when incubated at 37°C.  The FITC-GST-Kap95-(177) was confined to the nucleus at 4°C incubation (top,  middle). The fusion lacking  the NES motif (FITC-GSTKap95-(1-77)ΔNES) remained  nuclear even at 37°C (top,  right). Bar, 10 μm.
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Figure 3: The NES region of Kap95p mediates nuclear export. (A) Alignment of a region of Kap95p with the NES sequences of HIV-1 Rev, PKI, TFIIIA, and Gle1p is shown. (B) GST-Kap95-(1-77) and GST-Kap95-(1-77)ΔNES fusion proteins were purified and conjugated to FITC. The fluorescent conjugates were individually coinjected into the nuclei of COS-1 cells with Texas red–HSA. After a 45min incubation at either 37°C or 4°C, the cells were fixed and examined by fluorescence microscopy. The Texas red– HSA remained nuclear localized (bottom row); however, the FITC-GST-Kap95-(1-77) moved to the cytoplasm (top, left) when incubated at 37°C. The FITC-GST-Kap95-(177) was confined to the nucleus at 4°C incubation (top, middle). The fusion lacking the NES motif (FITC-GSTKap95-(1-77)ΔNES) remained nuclear even at 37°C (top, right). Bar, 10 μm.

Mentions: The goal of our continued studies was to determine when a Kap95p–Nup116p interaction might be required during nuclear transport. Previous studies showed that the first 130 amino acids of Kap95p are sufficient for a strong twohybrid interaction with the GLFG region of Nup116p (Iovine et al., 1995). We inspected this region of Kap95p amino acid sequence for motifs that may be facilitating binding to Nup116p–GLFG and observed a region from residues 55–65 with striking similarity to leucine-rich NES motifs (Fig. 3 A). Interestingly, NES motifs are necessary for the interaction of Rev and Gle1p with FG repeat–containing nucleoporins (Bogerd et al., 1995; Fritz et al., 1995; Stutz et al., 1995; Fritz and Green, 1996; Murphy and Wente, 1996).


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)

The NES region of  Kap95p mediates nuclear export. (A) Alignment of a region of Kap95p with the NES  sequences of HIV-1 Rev, PKI,  TFIIIA, and Gle1p is shown.  (B) GST-Kap95-(1-77) and  GST-Kap95-(1-77)ΔNES fusion proteins were purified  and conjugated to FITC. The  fluorescent conjugates were  individually coinjected into  the nuclei of COS-1 cells with  Texas red–HSA. After a 45min incubation at either 37°C  or 4°C, the cells were fixed  and examined by fluorescence  microscopy. The Texas red– HSA remained nuclear localized (bottom row); however,  the FITC-GST-Kap95-(1-77)  moved to the cytoplasm (top,  left) when incubated at 37°C.  The FITC-GST-Kap95-(177) was confined to the nucleus at 4°C incubation (top,  middle). The fusion lacking  the NES motif (FITC-GSTKap95-(1-77)ΔNES) remained  nuclear even at 37°C (top,  right). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139834&req=5

Figure 3: The NES region of Kap95p mediates nuclear export. (A) Alignment of a region of Kap95p with the NES sequences of HIV-1 Rev, PKI, TFIIIA, and Gle1p is shown. (B) GST-Kap95-(1-77) and GST-Kap95-(1-77)ΔNES fusion proteins were purified and conjugated to FITC. The fluorescent conjugates were individually coinjected into the nuclei of COS-1 cells with Texas red–HSA. After a 45min incubation at either 37°C or 4°C, the cells were fixed and examined by fluorescence microscopy. The Texas red– HSA remained nuclear localized (bottom row); however, the FITC-GST-Kap95-(1-77) moved to the cytoplasm (top, left) when incubated at 37°C. The FITC-GST-Kap95-(177) was confined to the nucleus at 4°C incubation (top, middle). The fusion lacking the NES motif (FITC-GSTKap95-(1-77)ΔNES) remained nuclear even at 37°C (top, right). Bar, 10 μm.
Mentions: The goal of our continued studies was to determine when a Kap95p–Nup116p interaction might be required during nuclear transport. Previous studies showed that the first 130 amino acids of Kap95p are sufficient for a strong twohybrid interaction with the GLFG region of Nup116p (Iovine et al., 1995). We inspected this region of Kap95p amino acid sequence for motifs that may be facilitating binding to Nup116p–GLFG and observed a region from residues 55–65 with striking similarity to leucine-rich NES motifs (Fig. 3 A). Interestingly, NES motifs are necessary for the interaction of Rev and Gle1p with FG repeat–containing nucleoporins (Bogerd et al., 1995; Fritz et al., 1995; Stutz et al., 1995; Fritz and Green, 1996; Murphy and Wente, 1996).

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