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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) Effect of RCC1 depletion on Rch1 recycling. tsBN2  cells were injected with Rch133–529 into the cytoplasm followed by  incubation at 33.5°C (a and b) or 39.5°C (c and d). Fluorescence  micrographs show the localization of Rch1 in cells that were fixed  after 0 min (a and c) or 4 h (b and d) of incubation under the conditions indicated. (B) Time course of nuclear accumulation of cytoplasmically injected Rch133–529 in the absence and presence of  RCC1. tsBN2 and BHK21 cells were injected into the cytoplasm  with Rch133–529 and incubated at 39.5°C or, as a control, at 33.5°C  for various time periods, fixed and stained. Quantification was  done as in Fig. 1 B and the percentage of Rch1 in the nucleus was  expressed as a function of time after injection. Bar, 25 μm.
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Figure 7: (A) Effect of RCC1 depletion on Rch1 recycling. tsBN2 cells were injected with Rch133–529 into the cytoplasm followed by incubation at 33.5°C (a and b) or 39.5°C (c and d). Fluorescence micrographs show the localization of Rch1 in cells that were fixed after 0 min (a and c) or 4 h (b and d) of incubation under the conditions indicated. (B) Time course of nuclear accumulation of cytoplasmically injected Rch133–529 in the absence and presence of RCC1. tsBN2 and BHK21 cells were injected into the cytoplasm with Rch133–529 and incubated at 39.5°C or, as a control, at 33.5°C for various time periods, fixed and stained. Quantification was done as in Fig. 1 B and the percentage of Rch1 in the nucleus was expressed as a function of time after injection. Bar, 25 μm.

Mentions: Addition of RanGTP to the heterodimeric complex of karyopherin α and β leads to the dissociation of the heterodimer in vitro (Rexach and Blobel, 1995; Görlich et al., 1996b). The fact that the nuclear protein RCC1 is the only known Ran guanine exchange factor, and plays a role in nuclear protein import in vivo (Tachibana et al., 1994; Dickmanns et al., 1996), suggested that RCC1 could be involved in the release of Rch1 from the import complex by providing RanGTP. Since the disassembly of the import complex must be a prerequisite for the recycling of the import factors, RCC1 would then indirectly affect the shuttling of the NLS receptor α subunit. To investigate the possible effect of RCC1 on receptor recycling, we used tsBN2 cells, mutant derivatives of BHK21 cells that express thermolabile RCC1 (for review see Dasso, 1993). To compare shuttling of Rch1 in the presence and absence of RCC1, Rch133–529 was injected into the cytoplasm of tsBN2 cells. The cells were then fixed immediately or incubated for 4 h at either the restrictive or permissive temperature. The subcellular distribution of Rch133–529 was then determined by immunofluorescent staining. Fig. 7 A shows that in the presence of RCC1, cells displayed the predominantly cytoplasmic staining typical of injected Rch133–529 (Fig. 7 A, a and b). In contrast, Rch1 was predominantly nuclear in the absence of RCC1 (Fig. 6 A, compare b [33.5°C] and d [39.5°C]).


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) Effect of RCC1 depletion on Rch1 recycling. tsBN2  cells were injected with Rch133–529 into the cytoplasm followed by  incubation at 33.5°C (a and b) or 39.5°C (c and d). Fluorescence  micrographs show the localization of Rch1 in cells that were fixed  after 0 min (a and c) or 4 h (b and d) of incubation under the conditions indicated. (B) Time course of nuclear accumulation of cytoplasmically injected Rch133–529 in the absence and presence of  RCC1. tsBN2 and BHK21 cells were injected into the cytoplasm  with Rch133–529 and incubated at 39.5°C or, as a control, at 33.5°C  for various time periods, fixed and stained. Quantification was  done as in Fig. 1 B and the percentage of Rch1 in the nucleus was  expressed as a function of time after injection. Bar, 25 μm.
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Figure 7: (A) Effect of RCC1 depletion on Rch1 recycling. tsBN2 cells were injected with Rch133–529 into the cytoplasm followed by incubation at 33.5°C (a and b) or 39.5°C (c and d). Fluorescence micrographs show the localization of Rch1 in cells that were fixed after 0 min (a and c) or 4 h (b and d) of incubation under the conditions indicated. (B) Time course of nuclear accumulation of cytoplasmically injected Rch133–529 in the absence and presence of RCC1. tsBN2 and BHK21 cells were injected into the cytoplasm with Rch133–529 and incubated at 39.5°C or, as a control, at 33.5°C for various time periods, fixed and stained. Quantification was done as in Fig. 1 B and the percentage of Rch1 in the nucleus was expressed as a function of time after injection. Bar, 25 μm.
Mentions: Addition of RanGTP to the heterodimeric complex of karyopherin α and β leads to the dissociation of the heterodimer in vitro (Rexach and Blobel, 1995; Görlich et al., 1996b). The fact that the nuclear protein RCC1 is the only known Ran guanine exchange factor, and plays a role in nuclear protein import in vivo (Tachibana et al., 1994; Dickmanns et al., 1996), suggested that RCC1 could be involved in the release of Rch1 from the import complex by providing RanGTP. Since the disassembly of the import complex must be a prerequisite for the recycling of the import factors, RCC1 would then indirectly affect the shuttling of the NLS receptor α subunit. To investigate the possible effect of RCC1 on receptor recycling, we used tsBN2 cells, mutant derivatives of BHK21 cells that express thermolabile RCC1 (for review see Dasso, 1993). To compare shuttling of Rch1 in the presence and absence of RCC1, Rch133–529 was injected into the cytoplasm of tsBN2 cells. The cells were then fixed immediately or incubated for 4 h at either the restrictive or permissive temperature. The subcellular distribution of Rch133–529 was then determined by immunofluorescent staining. Fig. 7 A shows that in the presence of RCC1, cells displayed the predominantly cytoplasmic staining typical of injected Rch133–529 (Fig. 7 A, a and b). In contrast, Rch1 was predominantly nuclear in the absence of RCC1 (Fig. 6 A, compare b [33.5°C] and d [39.5°C]).

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