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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) Rch1244–529 is defective in nuclear export. Rch133–529 (a and b) and Rch1244–529 (c and d) proteins were injected into nuclei of  Vero cells. Fluorescence micrographs show cells fixed and stained 0 min (a and c) or 30 min (b and d) after injection. (B) Export kinetics  of Rch1. Vero cells were injected into the nucleus with either Rch133–529 or Rch1244–529 and incubated for the indicated time periods.  Quantification was performed as in Fig. 1 B. Bar, 25 μm.
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Figure 2: (A) Rch1244–529 is defective in nuclear export. Rch133–529 (a and b) and Rch1244–529 (c and d) proteins were injected into nuclei of Vero cells. Fluorescence micrographs show cells fixed and stained 0 min (a and c) or 30 min (b and d) after injection. (B) Export kinetics of Rch1. Vero cells were injected into the nucleus with either Rch133–529 or Rch1244–529 and incubated for the indicated time periods. Quantification was performed as in Fig. 1 B. Bar, 25 μm.

Mentions: To address the question of whether nuclear accumulation of Rch1244–529 could be due to a defect in recycling of this protein to the cytoplasm, export of Rch133–529 and Rch1244–529 was examined more closely. The purified proteins were microinjected into the nuclei of Vero cells, and the cells were fixed either immediately after injection or after a 30-min incubation at 37°C. Fig. 2 A shows that cells microinjected with Rch133–529 showed almost exclusively nuclear staining initially (Fig. 2 A, a) and strong cytoplasmic staining after a 30-min incubation (Fig. 2 A, b). In sharp contrast, the staining pattern of cells injected with Rch1244–529 was nuclear at both time points (Fig. 2 A, c and d). Analysis of the Rch1 export kinetics (Fig. 2 B) demonstrated a rapid decrease of nuclear fluorescence in cells injected with Rch133–529. However, the amount of nuclear Rch1244–529 fluorescence remained constant over the entire time period (Fig. 2 B). These observations indicate that Rch133–529 injected into the nuclei of Vero cells was rapidly exported, whereas Rch1244–529 was not. The results also suggest that a polypeptide sequence between aa 1 and 244 of Rch1 may be necessary to allow nuclear export of Rch1.


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) Rch1244–529 is defective in nuclear export. Rch133–529 (a and b) and Rch1244–529 (c and d) proteins were injected into nuclei of  Vero cells. Fluorescence micrographs show cells fixed and stained 0 min (a and c) or 30 min (b and d) after injection. (B) Export kinetics  of Rch1. Vero cells were injected into the nucleus with either Rch133–529 or Rch1244–529 and incubated for the indicated time periods.  Quantification was performed as in Fig. 1 B. Bar, 25 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139786&req=5

Figure 2: (A) Rch1244–529 is defective in nuclear export. Rch133–529 (a and b) and Rch1244–529 (c and d) proteins were injected into nuclei of Vero cells. Fluorescence micrographs show cells fixed and stained 0 min (a and c) or 30 min (b and d) after injection. (B) Export kinetics of Rch1. Vero cells were injected into the nucleus with either Rch133–529 or Rch1244–529 and incubated for the indicated time periods. Quantification was performed as in Fig. 1 B. Bar, 25 μm.
Mentions: To address the question of whether nuclear accumulation of Rch1244–529 could be due to a defect in recycling of this protein to the cytoplasm, export of Rch133–529 and Rch1244–529 was examined more closely. The purified proteins were microinjected into the nuclei of Vero cells, and the cells were fixed either immediately after injection or after a 30-min incubation at 37°C. Fig. 2 A shows that cells microinjected with Rch133–529 showed almost exclusively nuclear staining initially (Fig. 2 A, a) and strong cytoplasmic staining after a 30-min incubation (Fig. 2 A, b). In sharp contrast, the staining pattern of cells injected with Rch1244–529 was nuclear at both time points (Fig. 2 A, c and d). Analysis of the Rch1 export kinetics (Fig. 2 B) demonstrated a rapid decrease of nuclear fluorescence in cells injected with Rch133–529. However, the amount of nuclear Rch1244–529 fluorescence remained constant over the entire time period (Fig. 2 B). These observations indicate that Rch133–529 injected into the nuclei of Vero cells was rapidly exported, whereas Rch1244–529 was not. The results also suggest that a polypeptide sequence between aa 1 and 244 of Rch1 may be necessary to allow nuclear export of Rch1.

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