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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) Rch1185–529 is efficiently exported from the nucleus.  Rch1185–529 was injected into the nuclei of Vero cells, which were  fixed and immunostained 0 min (a) or 30 min (b) later. (B) Cells  were injected into the nucleus with either Rch1185–529 or Rch133–529  to compare export kinetics of the two proteins. After injection,  cells were fixed and stained. Nuclear fluorescence was determined and plotted against time after microinjection as in Fig. 1 B.  Bar, 25 μm.
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Figure 3: (A) Rch1185–529 is efficiently exported from the nucleus. Rch1185–529 was injected into the nuclei of Vero cells, which were fixed and immunostained 0 min (a) or 30 min (b) later. (B) Cells were injected into the nucleus with either Rch1185–529 or Rch133–529 to compare export kinetics of the two proteins. After injection, cells were fixed and stained. Nuclear fluorescence was determined and plotted against time after microinjection as in Fig. 1 B. Bar, 25 μm.

Mentions: Another Rch1 deletion mutant, Rch1185–529, was used to narrow down the region that might contain the information necessary for nuclear export of the NLS receptor subunit. Rch1185–529 was injected into the nuclei of Vero cells, and the cells were fixed and stained either immediately after injection or after a 30-min incubation at 37°C. Fig. 3 A shows that shortly after injection, Rch1185–529 was still in the nucleus of injected cells (Fig. 3 A, a), whereas 30 min later, translocation to the cytoplasm had occurred (Fig. 3 A, b). Comparison of the export kinetics of Rch1185–529 and Rch133–529 (Fig. 3 B) confirmed that the rate of export of Rch1185–529 was comparable to that of Rch133–529. These results support the idea that the sequence between aa 185 and 244 of Rch1 is necessary to direct 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) Rch1185–529 is efficiently exported from the nucleus.  Rch1185–529 was injected into the nuclei of Vero cells, which were  fixed and immunostained 0 min (a) or 30 min (b) later. (B) Cells  were injected into the nucleus with either Rch1185–529 or Rch133–529  to compare export kinetics of the two proteins. After injection,  cells were fixed and stained. Nuclear fluorescence was determined and plotted against time after microinjection as in Fig. 1 B.  Bar, 25 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139786&req=5

Figure 3: (A) Rch1185–529 is efficiently exported from the nucleus. Rch1185–529 was injected into the nuclei of Vero cells, which were fixed and immunostained 0 min (a) or 30 min (b) later. (B) Cells were injected into the nucleus with either Rch1185–529 or Rch133–529 to compare export kinetics of the two proteins. After injection, cells were fixed and stained. Nuclear fluorescence was determined and plotted against time after microinjection as in Fig. 1 B. Bar, 25 μm.
Mentions: Another Rch1 deletion mutant, Rch1185–529, was used to narrow down the region that might contain the information necessary for nuclear export of the NLS receptor subunit. Rch1185–529 was injected into the nuclei of Vero cells, and the cells were fixed and stained either immediately after injection or after a 30-min incubation at 37°C. Fig. 3 A shows that shortly after injection, Rch1185–529 was still in the nucleus of injected cells (Fig. 3 A, a), whereas 30 min later, translocation to the cytoplasm had occurred (Fig. 3 A, b). Comparison of the export kinetics of Rch1185–529 and Rch133–529 (Fig. 3 B) confirmed that the rate of export of Rch1185–529 was comparable to that of Rch133–529. These results support the idea that the sequence between aa 185 and 244 of Rch1 is necessary to direct 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