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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 free Rch1 is not dependent on RCC1.  Rch133–529 was injected into the nuclei of tsBN2 cells preincubated  for 4 h at 33.5 or 39.5°C. Cells were fixed and stained immediately  after injection (a and c) or incubated for 30 min before fixing and  immunostaining (b and d). (B) Export of free Rch1 is independent of RCC1. tsBN2 and, as a control, BHK21 cells, were incubated for 4 h at 33.5 or 39.5°C before nuclear injection with  Rch133–529. For injections at 0°C, cells were first incubated at  33.5°C, but shifted to 0°C immediately before injection. After incubation at the indicated temperatures for the indicated time periods, cells were fixed and stained. Quantification was performed  as described in Fig. 1 B. Bar, 25 μm.
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Figure 8: (A) Export of free Rch1 is not dependent on RCC1. Rch133–529 was injected into the nuclei of tsBN2 cells preincubated for 4 h at 33.5 or 39.5°C. Cells were fixed and stained immediately after injection (a and c) or incubated for 30 min before fixing and immunostaining (b and d). (B) Export of free Rch1 is independent of RCC1. tsBN2 and, as a control, BHK21 cells, were incubated for 4 h at 33.5 or 39.5°C before nuclear injection with Rch133–529. For injections at 0°C, cells were first incubated at 33.5°C, but shifted to 0°C immediately before injection. After incubation at the indicated temperatures for the indicated time periods, cells were fixed and stained. Quantification was performed as described in Fig. 1 B. Bar, 25 μm.

Mentions: To differentiate between these two possibilities, we injected Rch133–529 into the nuclei of tsBN2 cells that had been incubated at the permissive and restrictive temperatures. The cells were fixed either immediately after injection (Fig. 8 A, a and c) or 30 min later (Fig. 8 A, b and d), and Rch133–529 was visualized by immunofluorescence. At both temperatures, Rch133–529 injected into the nuclei was found in the cytoplasm after a 30-min incubation. The time course (Fig. 8 B) shows clearly that nuclear export kinetics of Rch1 were identical in the presence or absence of RCC1. In a control experiment in BHK21 cells, Rch1 export kinetics were also identical at 33.5 and 39.5°C (Fig. 8 B). No export of Rch1 to the cytoplasm was observed at 0°C (data not shown), consistent with the temperature dependence of the Rch1 export reaction (Fig. 6). These results rule out a requirement for RCC1 in the export reaction itself and support the idea that it is required in an earlier step of receptor recycling.


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 free Rch1 is not dependent on RCC1.  Rch133–529 was injected into the nuclei of tsBN2 cells preincubated  for 4 h at 33.5 or 39.5°C. Cells were fixed and stained immediately  after injection (a and c) or incubated for 30 min before fixing and  immunostaining (b and d). (B) Export of free Rch1 is independent of RCC1. tsBN2 and, as a control, BHK21 cells, were incubated for 4 h at 33.5 or 39.5°C before nuclear injection with  Rch133–529. For injections at 0°C, cells were first incubated at  33.5°C, but shifted to 0°C immediately before injection. After incubation at the indicated temperatures for the indicated time periods, cells were fixed and stained. Quantification was performed  as described in Fig. 1 B. Bar, 25 μm.
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Figure 8: (A) Export of free Rch1 is not dependent on RCC1. Rch133–529 was injected into the nuclei of tsBN2 cells preincubated for 4 h at 33.5 or 39.5°C. Cells were fixed and stained immediately after injection (a and c) or incubated for 30 min before fixing and immunostaining (b and d). (B) Export of free Rch1 is independent of RCC1. tsBN2 and, as a control, BHK21 cells, were incubated for 4 h at 33.5 or 39.5°C before nuclear injection with Rch133–529. For injections at 0°C, cells were first incubated at 33.5°C, but shifted to 0°C immediately before injection. After incubation at the indicated temperatures for the indicated time periods, cells were fixed and stained. Quantification was performed as described in Fig. 1 B. Bar, 25 μm.
Mentions: To differentiate between these two possibilities, we injected Rch133–529 into the nuclei of tsBN2 cells that had been incubated at the permissive and restrictive temperatures. The cells were fixed either immediately after injection (Fig. 8 A, a and c) or 30 min later (Fig. 8 A, b and d), and Rch133–529 was visualized by immunofluorescence. At both temperatures, Rch133–529 injected into the nuclei was found in the cytoplasm after a 30-min incubation. The time course (Fig. 8 B) shows clearly that nuclear export kinetics of Rch1 were identical in the presence or absence of RCC1. In a control experiment in BHK21 cells, Rch1 export kinetics were also identical at 33.5 and 39.5°C (Fig. 8 B). No export of Rch1 to the cytoplasm was observed at 0°C (data not shown), consistent with the temperature dependence of the Rch1 export reaction (Fig. 6). These results rule out a requirement for RCC1 in the export reaction itself and support the idea that it is required in an earlier step of receptor recycling.

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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Related in: MedlinePlus