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© The Rockefeller University Press,
0021-9525/1997//313 $5.00
The Journal of Cell Biology, Volume 139, Number 2,
, 1997 313-325
Article |
Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor 
Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The
subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β 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.
Abbreviations used in this paper: aa, amino acid(s); IBB domain, importin-β–binding domain; MBP, maltose binding protein; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; RCC1. regulator of chromosome condensation 1; Rch1, Rag cohort 1.
A DAAD fellowship to Irene Boche under the Hochschulsonderprogramm II program, the support of the Deutsche Forschungsgemeinschaft (Fa 138/4-3), Vanderbilt University, and the National Science Foundation Shared Instrumentation grant BIR-9419667 are gratefully acknowledged.
Address all correspondence to Ellen Fanning, Department of Molecular Biology, Vanderbilt University, Box 1820, Station B, Nashville, TN 37235. Tel.: (615) 343-5677. Fax: (615) 343-6707. E-mail: Fannine{at}ctrvax.vanderbilt.edu
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