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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 MBP–Rch1 fusion constructs. BHK21  cells were injected into the nucleus with purified proteins MBP (a  and b), MBP fused to Rchl residues 218–244 (c and d), or MBP  fused to Rchl residues 197–217 (e and f), fixed 0 min (a, c, and e)  or 10 min (b, d, and f) later, and examined by immunofluorescence. (B) Export kinetics of MBP–Rch1 fusion constructs. After  nuclear injection of MBP, MBP218–244, and MBP197–217, the cells  were fixed and immunostained using an anti-MBP polyclonal serum. Nuclear fluorescence at different times after injection was  determined and plotted against time as in Fig. 1 B. Bar, 25 μm.
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Figure 4: (A) Export of MBP–Rch1 fusion constructs. BHK21 cells were injected into the nucleus with purified proteins MBP (a and b), MBP fused to Rchl residues 218–244 (c and d), or MBP fused to Rchl residues 197–217 (e and f), fixed 0 min (a, c, and e) or 10 min (b, d, and f) later, and examined by immunofluorescence. (B) Export kinetics of MBP–Rch1 fusion constructs. After nuclear injection of MBP, MBP218–244, and MBP197–217, the cells were fixed and immunostained using an anti-MBP polyclonal serum. Nuclear fluorescence at different times after injection was determined and plotted against time as in Fig. 1 B. Bar, 25 μm.

Mentions: The fluorescent micrographs (Fig. 4 A) show that both MBP and MBP218–244 were nuclear at the 0-min (Fig. 4 A, a and c) and the 10-min (Fig. 4 A, b and d) time points. In contrast, cells injected with MBP197–217 showed nuclear staining only at the 0-min time point (Fig. 4 A, e) and cytoplasmic staining after a 10-min incubation (Fig. 4 A, f). The export kinetics shown in Fig. 4 B confirm that neither MBP nor MBP218–244 were exported over the time period monitored. However, MBP197–217 was very rapidly exported from the nucleus to the cytoplasm. We therefore conclude that the amino acids 197–217, encompassing the putative NES, were sufficient to direct nuclear export of MBP.


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 MBP–Rch1 fusion constructs. BHK21  cells were injected into the nucleus with purified proteins MBP (a  and b), MBP fused to Rchl residues 218–244 (c and d), or MBP  fused to Rchl residues 197–217 (e and f), fixed 0 min (a, c, and e)  or 10 min (b, d, and f) later, and examined by immunofluorescence. (B) Export kinetics of MBP–Rch1 fusion constructs. After  nuclear injection of MBP, MBP218–244, and MBP197–217, the cells  were fixed and immunostained using an anti-MBP polyclonal serum. Nuclear fluorescence at different times after injection was  determined and plotted against time as in Fig. 1 B. Bar, 25 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139786&req=5

Figure 4: (A) Export of MBP–Rch1 fusion constructs. BHK21 cells were injected into the nucleus with purified proteins MBP (a and b), MBP fused to Rchl residues 218–244 (c and d), or MBP fused to Rchl residues 197–217 (e and f), fixed 0 min (a, c, and e) or 10 min (b, d, and f) later, and examined by immunofluorescence. (B) Export kinetics of MBP–Rch1 fusion constructs. After nuclear injection of MBP, MBP218–244, and MBP197–217, the cells were fixed and immunostained using an anti-MBP polyclonal serum. Nuclear fluorescence at different times after injection was determined and plotted against time as in Fig. 1 B. Bar, 25 μm.
Mentions: The fluorescent micrographs (Fig. 4 A) show that both MBP and MBP218–244 were nuclear at the 0-min (Fig. 4 A, a and c) and the 10-min (Fig. 4 A, b and d) time points. In contrast, cells injected with MBP197–217 showed nuclear staining only at the 0-min time point (Fig. 4 A, e) and cytoplasmic staining after a 10-min incubation (Fig. 4 A, f). The export kinetics shown in Fig. 4 B confirm that neither MBP nor MBP218–244 were exported over the time period monitored. However, MBP197–217 was very rapidly exported from the nucleus to the cytoplasm. We therefore conclude that the amino acids 197–217, encompassing the putative NES, were sufficient to direct nuclear export of MBP.

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