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Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Boche I, Fanning E - J. Cell Biol. (1997)

Bottom Line: Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm.After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export.However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

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(A) Export of Rch1244–529 is restored by a nuclear export signal. HIVNES-Rch1244–529 (a and b), Rch1NES-Rch1244–529  (c and d), and Rch1nes-Rch1244–529 (e and f) were injected into nuclei of Vero cells. Cells were fixed either at a 0-min time point (a,  c, and e) or 30 min later (b, d, and f). Immunofluorescence  was performed using an anti-T7 antibody. (B) Export kinetics  of Rch1244–529, HIVNES-Rch1244–529, Rch1NES-Rch1244–529, and  Rch1nes-Rch1244–529. Nuclear fluorescence at different time points  after injection was determined as described (Fig. 1 B). Bar, 25 μm.
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Figure 5: (A) Export of Rch1244–529 is restored by a nuclear export signal. HIVNES-Rch1244–529 (a and b), Rch1NES-Rch1244–529 (c and d), and Rch1nes-Rch1244–529 (e and f) were injected into nuclei of Vero cells. Cells were fixed either at a 0-min time point (a, c, and e) or 30 min later (b, d, and f). Immunofluorescence was performed using an anti-T7 antibody. (B) Export kinetics of Rch1244–529, HIVNES-Rch1244–529, Rch1NES-Rch1244–529, and Rch1nes-Rch1244–529. Nuclear fluorescence at different time points after injection was determined as described (Fig. 1 B). Bar, 25 μm.

Mentions: Nuclear export of Rch1NES-Rch1244–529, Rch1nes-Rch1244–529, and HIVNES-Rch1244–529 was then assayed after injection into nuclei of Vero cells. Cells were fixed either at a 0-min time point or 30 min later, followed by immunofluorescent staining of the exogenous protein. The fluorescent micrographs in Fig. 5 A demonstrate that cells injected with either Rch1NES-Rch1244–529 or HIVNES-Rch1244–529 showed nuclear staining immediately after injection (Fig. 5 A, a and c) and cytoplasmic staining after a 30-min incubation (Fig. 5 A, b and d), but cells injected with Rch1nes-Rch1244–529 showed nuclear staining at both time points (Fig. 5 A, e and f). To monitor the time course of the export reaction, cells were fixed at different time points after injection, and immunofluorescence was performed using the anti-T7 antibody. The amount of nuclear fluorescence was determined and plotted against time after microinjection. As a control, Rch1244–529 was also injected. The export kinetics in Fig. 5 B show that although Rch1244–529 and Rch1nes-Rch1244–529 were not exported, Rch1NES-Rch1244–529 was exported at a rate very similar to that of HIVNES-Rch1244–529. These results demonstrate that export of the deletion mutant was restored not only by the established nuclear export signal of HIV-Rev (Fischer et al., 1995), but also by amino acids 207–217 of Rch1, indicating that they function as a nuclear export signal. Leucines 110 and 112 of Rch1 appear to be necessary for this function.


Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Boche I, Fanning E - J. Cell Biol. (1997)

(A) Export of Rch1244–529 is restored by a nuclear export signal. HIVNES-Rch1244–529 (a and b), Rch1NES-Rch1244–529  (c and d), and Rch1nes-Rch1244–529 (e and f) were injected into nuclei of Vero cells. Cells were fixed either at a 0-min time point (a,  c, and e) or 30 min later (b, d, and f). Immunofluorescence  was performed using an anti-T7 antibody. (B) Export kinetics  of Rch1244–529, HIVNES-Rch1244–529, Rch1NES-Rch1244–529, and  Rch1nes-Rch1244–529. Nuclear fluorescence at different time points  after injection was determined as described (Fig. 1 B). Bar, 25 μm.
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Figure 5: (A) Export of Rch1244–529 is restored by a nuclear export signal. HIVNES-Rch1244–529 (a and b), Rch1NES-Rch1244–529 (c and d), and Rch1nes-Rch1244–529 (e and f) were injected into nuclei of Vero cells. Cells were fixed either at a 0-min time point (a, c, and e) or 30 min later (b, d, and f). Immunofluorescence was performed using an anti-T7 antibody. (B) Export kinetics of Rch1244–529, HIVNES-Rch1244–529, Rch1NES-Rch1244–529, and Rch1nes-Rch1244–529. Nuclear fluorescence at different time points after injection was determined as described (Fig. 1 B). Bar, 25 μm.
Mentions: Nuclear export of Rch1NES-Rch1244–529, Rch1nes-Rch1244–529, and HIVNES-Rch1244–529 was then assayed after injection into nuclei of Vero cells. Cells were fixed either at a 0-min time point or 30 min later, followed by immunofluorescent staining of the exogenous protein. The fluorescent micrographs in Fig. 5 A demonstrate that cells injected with either Rch1NES-Rch1244–529 or HIVNES-Rch1244–529 showed nuclear staining immediately after injection (Fig. 5 A, a and c) and cytoplasmic staining after a 30-min incubation (Fig. 5 A, b and d), but cells injected with Rch1nes-Rch1244–529 showed nuclear staining at both time points (Fig. 5 A, e and f). To monitor the time course of the export reaction, cells were fixed at different time points after injection, and immunofluorescence was performed using the anti-T7 antibody. The amount of nuclear fluorescence was determined and plotted against time after microinjection. As a control, Rch1244–529 was also injected. The export kinetics in Fig. 5 B show that although Rch1244–529 and Rch1nes-Rch1244–529 were not exported, Rch1NES-Rch1244–529 was exported at a rate very similar to that of HIVNES-Rch1244–529. These results demonstrate that export of the deletion mutant was restored not only by the established nuclear export signal of HIV-Rev (Fischer et al., 1995), but also by amino acids 207–217 of Rch1, indicating that they function as a nuclear export signal. Leucines 110 and 112 of Rch1 appear to be necessary for this function.

Bottom Line: Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm.After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export.However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Show MeSH
Related in: MedlinePlus