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Subunit In Vivo Is Dependent on a
Nuclear Export Signal, Energy, and RCC1
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
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 
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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