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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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RCC1 inhibits formation of the NLS receptor/substrate  complex. (A) Karyopherin β was immobilized on an ELISA plate  before addition of different amounts of Rch133–529, as indicated  on the x-axis of the graph. Plates were washed and Rch1 bound to  the solid phase was detected in a colorimetric reaction. (B)  Karyopherin β was immobilized on ELISA plates. T-antigen was  added in the amounts indicated on the x-axis of the graph, either  alone or together with Rch133–529. Plates were washed, and the  amount of T-antigen bound to the solid phase was determined in  a colorimetric reaction. (C) Karyopherin β was immobilized on  ELISA immunoplates before addition of either Rch133–529 and  T-antigen (columns 1 and 3–7) or T-antigen alone (column 2).  The binding reactions were then supplemented with RanGDP  and GDP (column 3); RanGDP, GDP, and RCC1 (column 4);  RanGTP and GTP (column 5); RanGTP-γS and GTP-γS (column 6); or RanGTP, GTP, and RCC1 (column 7). MgCl2 was  present in all reactions at 5 mM. The T-antigen bound to each  solid phase was determined in a colorimetric reaction. The columns  depict the mean values from at least two separate experiments;  the standard deviation of the mean is indicated by error bars.
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Figure 9: RCC1 inhibits formation of the NLS receptor/substrate complex. (A) Karyopherin β was immobilized on an ELISA plate before addition of different amounts of Rch133–529, as indicated on the x-axis of the graph. Plates were washed and Rch1 bound to the solid phase was detected in a colorimetric reaction. (B) Karyopherin β was immobilized on ELISA plates. T-antigen was added in the amounts indicated on the x-axis of the graph, either alone or together with Rch133–529. Plates were washed, and the amount of T-antigen bound to the solid phase was determined in a colorimetric reaction. (C) Karyopherin β was immobilized on ELISA immunoplates before addition of either Rch133–529 and T-antigen (columns 1 and 3–7) or T-antigen alone (column 2). The binding reactions were then supplemented with RanGDP and GDP (column 3); RanGDP, GDP, and RCC1 (column 4); RanGTP and GTP (column 5); RanGTP-γS and GTP-γS (column 6); or RanGTP, GTP, and RCC1 (column 7). MgCl2 was present in all reactions at 5 mM. The T-antigen bound to each solid phase was determined in a colorimetric reaction. The columns depict the mean values from at least two separate experiments; the standard deviation of the mean is indicated by error bars.

Mentions: To develop a method to test this possibility, we first assembled NLS receptor/substrate complexes using purified proteins in a solid phase assay. Karyopherin β and, as a control, BSA were immobilized on ELISA plates and soluble Rch133–529 was added. After washing, bound Rch133–529 was detected using the anti-T7 antibody and a peroxidase-coupled secondary antibody. Fig. 9 A shows that Rch133–529 was bound in the presence of karyopherin β, but not in the presence of BSA, indicating that Rch133–529 bound specifically to karyopherin β. To test whether the immobilized Rch133–529/karyopherin β complexes were able to bind T-antigen as an NLS-bearing protein, we immobilized karyopherin β in the ELISA plate and then added either T-antigen alone or T-antigen together with Rch133–529. Bound T-antigen was detected using a specific monoclonal antibody and a peroxidase-coupled second antibody. Fig. 9 B shows that bound T-antigen was detectable in the presence of karyopherin β and Rch133–529 but not with karyopherin β alone, indicating the assembly of an αβ NLS receptor complex with the transport substrate T-antigen.


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)

RCC1 inhibits formation of the NLS receptor/substrate  complex. (A) Karyopherin β was immobilized on an ELISA plate  before addition of different amounts of Rch133–529, as indicated  on the x-axis of the graph. Plates were washed and Rch1 bound to  the solid phase was detected in a colorimetric reaction. (B)  Karyopherin β was immobilized on ELISA plates. T-antigen was  added in the amounts indicated on the x-axis of the graph, either  alone or together with Rch133–529. Plates were washed, and the  amount of T-antigen bound to the solid phase was determined in  a colorimetric reaction. (C) Karyopherin β was immobilized on  ELISA immunoplates before addition of either Rch133–529 and  T-antigen (columns 1 and 3–7) or T-antigen alone (column 2).  The binding reactions were then supplemented with RanGDP  and GDP (column 3); RanGDP, GDP, and RCC1 (column 4);  RanGTP and GTP (column 5); RanGTP-γS and GTP-γS (column 6); or RanGTP, GTP, and RCC1 (column 7). MgCl2 was  present in all reactions at 5 mM. The T-antigen bound to each  solid phase was determined in a colorimetric reaction. The columns  depict the mean values from at least two separate experiments;  the standard deviation of the mean is indicated by error bars.
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Related In: Results  -  Collection

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Figure 9: RCC1 inhibits formation of the NLS receptor/substrate complex. (A) Karyopherin β was immobilized on an ELISA plate before addition of different amounts of Rch133–529, as indicated on the x-axis of the graph. Plates were washed and Rch1 bound to the solid phase was detected in a colorimetric reaction. (B) Karyopherin β was immobilized on ELISA plates. T-antigen was added in the amounts indicated on the x-axis of the graph, either alone or together with Rch133–529. Plates were washed, and the amount of T-antigen bound to the solid phase was determined in a colorimetric reaction. (C) Karyopherin β was immobilized on ELISA immunoplates before addition of either Rch133–529 and T-antigen (columns 1 and 3–7) or T-antigen alone (column 2). The binding reactions were then supplemented with RanGDP and GDP (column 3); RanGDP, GDP, and RCC1 (column 4); RanGTP and GTP (column 5); RanGTP-γS and GTP-γS (column 6); or RanGTP, GTP, and RCC1 (column 7). MgCl2 was present in all reactions at 5 mM. The T-antigen bound to each solid phase was determined in a colorimetric reaction. The columns depict the mean values from at least two separate experiments; the standard deviation of the mean is indicated by error bars.
Mentions: To develop a method to test this possibility, we first assembled NLS receptor/substrate complexes using purified proteins in a solid phase assay. Karyopherin β and, as a control, BSA were immobilized on ELISA plates and soluble Rch133–529 was added. After washing, bound Rch133–529 was detected using the anti-T7 antibody and a peroxidase-coupled secondary antibody. Fig. 9 A shows that Rch133–529 was bound in the presence of karyopherin β, but not in the presence of BSA, indicating that Rch133–529 bound specifically to karyopherin β. To test whether the immobilized Rch133–529/karyopherin β complexes were able to bind T-antigen as an NLS-bearing protein, we immobilized karyopherin β in the ELISA plate and then added either T-antigen alone or T-antigen together with Rch133–529. Bound T-antigen was detected using a specific monoclonal antibody and a peroxidase-coupled second antibody. Fig. 9 B shows that bound T-antigen was detectable in the presence of karyopherin β and Rch133–529 but not with karyopherin β alone, indicating the assembly of an αβ NLS receptor complex with the transport substrate T-antigen.

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