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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 accumulates in the nuclei of Vero cells in vivo. Vero cells were injected into the cytoplasm with either Rch133–529  (a and b) or Rch1244–529 (c and d) and fixed immediately (a and c) and 2.5 h (b and d) after injection. Immunofluorescence was performed using an antibody against the T7-tag of the extracellular Rch1 proteins and a Cy3-coupled secondary antibody. Micrographs  were taken with a digital camera mounted on a fluorescence microscope. (B) Time course of nuclear accumulation of Rch1244–529. Vero  cells were injected either with Rch133–529 or Rch1244–529 and fixed at various time points as indicated on the x-axis. Cells were immunostained, and the amount of Rch1 fluorescence was measured as described in Materials and Methods. The staining of nuclear Rch1 is expressed as the percentage of total cell fluorescence residing in the nucleus and plotted against time after microinjection. For each time  point, the average value obtained from 10–20 cells in two to three independent experiments is shown; the standard deviation of the  mean is indicated by error bars. Bar, 25 μm.
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Figure 1: (A) Rch1244–529 accumulates in the nuclei of Vero cells in vivo. Vero cells were injected into the cytoplasm with either Rch133–529 (a and b) or Rch1244–529 (c and d) and fixed immediately (a and c) and 2.5 h (b and d) after injection. Immunofluorescence was performed using an antibody against the T7-tag of the extracellular Rch1 proteins and a Cy3-coupled secondary antibody. Micrographs were taken with a digital camera mounted on a fluorescence microscope. (B) Time course of nuclear accumulation of Rch1244–529. Vero cells were injected either with Rch133–529 or Rch1244–529 and fixed at various time points as indicated on the x-axis. Cells were immunostained, and the amount of Rch1 fluorescence was measured as described in Materials and Methods. The staining of nuclear Rch1 is expressed as the percentage of total cell fluorescence residing in the nucleus and plotted against time after microinjection. For each time point, the average value obtained from 10–20 cells in two to three independent experiments is shown; the standard deviation of the mean is indicated by error bars. Bar, 25 μm.

Mentions: To determine the subcellular localization of Rch133–529 and Rch1244–529, each protein was microinjected into the cytosol of Vero cells. The cells were either fixed immediately after injection or after a 2.5-h incubation at 37°C. The exogenous Rch1 was visualized by staining its T7-tag with anti-T7 antibodies. As shown in Fig. 1 A, a and b, Rch133–529 was localized predominantly in the cytoplasm at both time points. Although cells injected with Rch1244–529 showed cytoplasmic staining immediately after injection (Fig. 1 A, c), strong nuclear staining was observed 2.5 h later (Fig. 1 A, d). The time course of nuclear uptake of the two proteins was followed by injecting cells with either Rch133–529 or Rch1244–529, fixing them at various time points as indicated (Fig. 1 B), and determining the amount of nuclear fluorescence as a percentage of the total cell fluorescence in the injected cells. The graph shows that the fraction of nuclear Rch133–529 remained at ∼20% over a period of 2.5 h, consistent with the predominantly cytoplasmic importin α observed in Xenopus cells (Görlich et al., 1996a). However, nuclear staining of Rch1244–529 increased slowly from 12 to 38% over the same period of time. Since nuclear protein import took place over the entire 2.5-h period, these results suggest that Rch133–529 was rapidly recycled to the cytoplasm, but Rch1244–529 was not.


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 accumulates in the nuclei of Vero cells in vivo. Vero cells were injected into the cytoplasm with either Rch133–529  (a and b) or Rch1244–529 (c and d) and fixed immediately (a and c) and 2.5 h (b and d) after injection. Immunofluorescence was performed using an antibody against the T7-tag of the extracellular Rch1 proteins and a Cy3-coupled secondary antibody. Micrographs  were taken with a digital camera mounted on a fluorescence microscope. (B) Time course of nuclear accumulation of Rch1244–529. Vero  cells were injected either with Rch133–529 or Rch1244–529 and fixed at various time points as indicated on the x-axis. Cells were immunostained, and the amount of Rch1 fluorescence was measured as described in Materials and Methods. The staining of nuclear Rch1 is expressed as the percentage of total cell fluorescence residing in the nucleus and plotted against time after microinjection. For each time  point, the average value obtained from 10–20 cells in two to three independent experiments is shown; the standard deviation of the  mean is indicated by error bars. Bar, 25 μm.
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Related In: Results  -  Collection

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Figure 1: (A) Rch1244–529 accumulates in the nuclei of Vero cells in vivo. Vero cells were injected into the cytoplasm with either Rch133–529 (a and b) or Rch1244–529 (c and d) and fixed immediately (a and c) and 2.5 h (b and d) after injection. Immunofluorescence was performed using an antibody against the T7-tag of the extracellular Rch1 proteins and a Cy3-coupled secondary antibody. Micrographs were taken with a digital camera mounted on a fluorescence microscope. (B) Time course of nuclear accumulation of Rch1244–529. Vero cells were injected either with Rch133–529 or Rch1244–529 and fixed at various time points as indicated on the x-axis. Cells were immunostained, and the amount of Rch1 fluorescence was measured as described in Materials and Methods. The staining of nuclear Rch1 is expressed as the percentage of total cell fluorescence residing in the nucleus and plotted against time after microinjection. For each time point, the average value obtained from 10–20 cells in two to three independent experiments is shown; the standard deviation of the mean is indicated by error bars. Bar, 25 μm.
Mentions: To determine the subcellular localization of Rch133–529 and Rch1244–529, each protein was microinjected into the cytosol of Vero cells. The cells were either fixed immediately after injection or after a 2.5-h incubation at 37°C. The exogenous Rch1 was visualized by staining its T7-tag with anti-T7 antibodies. As shown in Fig. 1 A, a and b, Rch133–529 was localized predominantly in the cytoplasm at both time points. Although cells injected with Rch1244–529 showed cytoplasmic staining immediately after injection (Fig. 1 A, c), strong nuclear staining was observed 2.5 h later (Fig. 1 A, d). The time course of nuclear uptake of the two proteins was followed by injecting cells with either Rch133–529 or Rch1244–529, fixing them at various time points as indicated (Fig. 1 B), and determining the amount of nuclear fluorescence as a percentage of the total cell fluorescence in the injected cells. The graph shows that the fraction of nuclear Rch133–529 remained at ∼20% over a period of 2.5 h, consistent with the predominantly cytoplasmic importin α observed in Xenopus cells (Görlich et al., 1996a). However, nuclear staining of Rch1244–529 increased slowly from 12 to 38% over the same period of time. Since nuclear protein import took place over the entire 2.5-h period, these results suggest that Rch133–529 was rapidly recycled to the cytoplasm, but Rch1244–529 was not.

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