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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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Nup100p–GLFG  may also mediate Kap95p recycling. (A) Synthetic lethal  interactions between nup100ΔGLFG and nup 145Δ GLFG alleles. The heterozygous nup100ΔGLFG/NUP100 nup145ΔGLFG/NUP145  strain SWY585 was sporulated and dissected. The nup100ΔGLFG and nup145Δ GLFG alleles cosegregated  and resulted in the viable  haploid nup100ΔGLFG/nup145Δ GLFG strain SWY588.  To demonstrate viability of  cells, the double mutant haploid (SWY588), wild-type  (W303), and respective single  mutant parental strains (nup100ΔGLFG, SWY583; nup145Δ GLFG, SWY581) were  grown on YPD at 30°C for  2 d. (B) Synthetic lethal interactions between nup116ΔGLFG, nup100Δ GLFG, and  nup145ΔGLFG mutants.  Strains containing the respective double ΔGLFG mutation combinations are shown  on YPD and 5-FOA at 23°C.  Strains harboring double deletions of the GLFG regions  of NUP116, NUP100, and  NUP145 were obtained by dissecting the appropriate diploid strains (see Table I).  Confirmation of the nup116ΔGLFG chromosome segregation was determined by  immunoblotting with an anti116 carboxy-terminal antibody  (Iovine et al., 1995). On the YPD plate, the nup116Δ GLFG (SWY1407), nup100ΔGLFG/nup116ΔGLFG (SWY1406), and the  nup145ΔGLFG/nup116ΔGLFG (SWY1429) cells also carry pSW131 (NUP116/URA3); the nup100ΔGLFG (SWY1401) cells also carry  pSW132 (NUP100/URA3); and the nup145ΔGLFG (SWY656) cells also carry pSW190. (C) Protein A–Nup100p coimmunoprecipitates  Kap95p from isolated nuclei. Nuclear lysates were prepared from SWY1381 and processed as described for Fig. 1. Immunoblots of the  eluate fraction were incubated with either the anti-Kap95p antibody or an anti-IgG antibody. The latter antibody was used to detect the position of protein A–Nup100p (uppermost band) and any of its proteolytic fragments (asterisk).
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Figure 8: Nup100p–GLFG may also mediate Kap95p recycling. (A) Synthetic lethal interactions between nup100ΔGLFG and nup 145Δ GLFG alleles. The heterozygous nup100ΔGLFG/NUP100 nup145ΔGLFG/NUP145 strain SWY585 was sporulated and dissected. The nup100ΔGLFG and nup145Δ GLFG alleles cosegregated and resulted in the viable haploid nup100ΔGLFG/nup145Δ GLFG strain SWY588. To demonstrate viability of cells, the double mutant haploid (SWY588), wild-type (W303), and respective single mutant parental strains (nup100ΔGLFG, SWY583; nup145Δ GLFG, SWY581) were grown on YPD at 30°C for 2 d. (B) Synthetic lethal interactions between nup116ΔGLFG, nup100Δ GLFG, and nup145ΔGLFG mutants. Strains containing the respective double ΔGLFG mutation combinations are shown on YPD and 5-FOA at 23°C. Strains harboring double deletions of the GLFG regions of NUP116, NUP100, and NUP145 were obtained by dissecting the appropriate diploid strains (see Table I). Confirmation of the nup116ΔGLFG chromosome segregation was determined by immunoblotting with an anti116 carboxy-terminal antibody (Iovine et al., 1995). On the YPD plate, the nup116Δ GLFG (SWY1407), nup100ΔGLFG/nup116ΔGLFG (SWY1406), and the nup145ΔGLFG/nup116ΔGLFG (SWY1429) cells also carry pSW131 (NUP116/URA3); the nup100ΔGLFG (SWY1401) cells also carry pSW132 (NUP100/URA3); and the nup145ΔGLFG (SWY656) cells also carry pSW190. (C) Protein A–Nup100p coimmunoprecipitates Kap95p from isolated nuclei. Nuclear lysates were prepared from SWY1381 and processed as described for Fig. 1. Immunoblots of the eluate fraction were incubated with either the anti-Kap95p antibody or an anti-IgG antibody. The latter antibody was used to detect the position of protein A–Nup100p (uppermost band) and any of its proteolytic fragments (asterisk).

Mentions: To specifically test for functional overlap between the GLFG regions of Nup116p, Nup100p, and Nup145p, strains were constructed that expressed mutated versions of each nucleoporin in which only the respective GLFG region was internally deleted. As shown by the plasmid shuffle assay in Fig. 8 B, haploid cells harboring both the nup116ΔGLFG and nup100ΔGLFG alleles were inviable at all growth temperatures. This demonstrated that the GLFG region of Nup145p was not sufficient for viability in the absence of those from Nup116p and Nup100p. However, the GLFG region from Nup116p was adequate for function because nup100ΔGLFG/nup145ΔGLFG cells were viable (Fig. 8 A). At 23°C, nup116ΔGLFG/nup145ΔGLFG cells were also viable (Fig. 8 B). Overall, these genetic interactions suggested that the GLFG region of Nup100p was necessary in the absence of Nup116p–GLFG function.


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)

Nup100p–GLFG  may also mediate Kap95p recycling. (A) Synthetic lethal  interactions between nup100ΔGLFG and nup 145Δ GLFG alleles. The heterozygous nup100ΔGLFG/NUP100 nup145ΔGLFG/NUP145  strain SWY585 was sporulated and dissected. The nup100ΔGLFG and nup145Δ GLFG alleles cosegregated  and resulted in the viable  haploid nup100ΔGLFG/nup145Δ GLFG strain SWY588.  To demonstrate viability of  cells, the double mutant haploid (SWY588), wild-type  (W303), and respective single  mutant parental strains (nup100ΔGLFG, SWY583; nup145Δ GLFG, SWY581) were  grown on YPD at 30°C for  2 d. (B) Synthetic lethal interactions between nup116ΔGLFG, nup100Δ GLFG, and  nup145ΔGLFG mutants.  Strains containing the respective double ΔGLFG mutation combinations are shown  on YPD and 5-FOA at 23°C.  Strains harboring double deletions of the GLFG regions  of NUP116, NUP100, and  NUP145 were obtained by dissecting the appropriate diploid strains (see Table I).  Confirmation of the nup116ΔGLFG chromosome segregation was determined by  immunoblotting with an anti116 carboxy-terminal antibody  (Iovine et al., 1995). On the YPD plate, the nup116Δ GLFG (SWY1407), nup100ΔGLFG/nup116ΔGLFG (SWY1406), and the  nup145ΔGLFG/nup116ΔGLFG (SWY1429) cells also carry pSW131 (NUP116/URA3); the nup100ΔGLFG (SWY1401) cells also carry  pSW132 (NUP100/URA3); and the nup145ΔGLFG (SWY656) cells also carry pSW190. (C) Protein A–Nup100p coimmunoprecipitates  Kap95p from isolated nuclei. Nuclear lysates were prepared from SWY1381 and processed as described for Fig. 1. Immunoblots of the  eluate fraction were incubated with either the anti-Kap95p antibody or an anti-IgG antibody. The latter antibody was used to detect the position of protein A–Nup100p (uppermost band) and any of its proteolytic fragments (asterisk).
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Figure 8: Nup100p–GLFG may also mediate Kap95p recycling. (A) Synthetic lethal interactions between nup100ΔGLFG and nup 145Δ GLFG alleles. The heterozygous nup100ΔGLFG/NUP100 nup145ΔGLFG/NUP145 strain SWY585 was sporulated and dissected. The nup100ΔGLFG and nup145Δ GLFG alleles cosegregated and resulted in the viable haploid nup100ΔGLFG/nup145Δ GLFG strain SWY588. To demonstrate viability of cells, the double mutant haploid (SWY588), wild-type (W303), and respective single mutant parental strains (nup100ΔGLFG, SWY583; nup145Δ GLFG, SWY581) were grown on YPD at 30°C for 2 d. (B) Synthetic lethal interactions between nup116ΔGLFG, nup100Δ GLFG, and nup145ΔGLFG mutants. Strains containing the respective double ΔGLFG mutation combinations are shown on YPD and 5-FOA at 23°C. Strains harboring double deletions of the GLFG regions of NUP116, NUP100, and NUP145 were obtained by dissecting the appropriate diploid strains (see Table I). Confirmation of the nup116ΔGLFG chromosome segregation was determined by immunoblotting with an anti116 carboxy-terminal antibody (Iovine et al., 1995). On the YPD plate, the nup116Δ GLFG (SWY1407), nup100ΔGLFG/nup116ΔGLFG (SWY1406), and the nup145ΔGLFG/nup116ΔGLFG (SWY1429) cells also carry pSW131 (NUP116/URA3); the nup100ΔGLFG (SWY1401) cells also carry pSW132 (NUP100/URA3); and the nup145ΔGLFG (SWY656) cells also carry pSW190. (C) Protein A–Nup100p coimmunoprecipitates Kap95p from isolated nuclei. Nuclear lysates were prepared from SWY1381 and processed as described for Fig. 1. Immunoblots of the eluate fraction were incubated with either the anti-Kap95p antibody or an anti-IgG antibody. The latter antibody was used to detect the position of protein A–Nup100p (uppermost band) and any of its proteolytic fragments (asterisk).
Mentions: To specifically test for functional overlap between the GLFG regions of Nup116p, Nup100p, and Nup145p, strains were constructed that expressed mutated versions of each nucleoporin in which only the respective GLFG region was internally deleted. As shown by the plasmid shuffle assay in Fig. 8 B, haploid cells harboring both the nup116ΔGLFG and nup100ΔGLFG alleles were inviable at all growth temperatures. This demonstrated that the GLFG region of Nup145p was not sufficient for viability in the absence of those from Nup116p and Nup100p. However, the GLFG region from Nup116p was adequate for function because nup100ΔGLFG/nup145ΔGLFG cells were viable (Fig. 8 A). At 23°C, nup116ΔGLFG/nup145ΔGLFG cells were also viable (Fig. 8 B). Overall, these genetic interactions suggested that the GLFG region of Nup100p was necessary in the absence of Nup116p–GLFG function.

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