NUCLEOCYTOPLASMIC RECYCLING OF THE NUCLEAR-LOCALIZATION SIGNAL RECEPTOR-ALPHA SUBUNIT IN-VIVO IS DEPENDENT ON A NUCLEAR EXPORT SIGNAL, ENERGY, AND RCC1

Nuclear protein import requires a nuclear localization signal (NLS) re ceptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probabl y 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 fa ctors 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 cel ls was found primarily in the cytoplasm. Rch1 injected into the nucleu s was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nu clei of Vero cells after cytoplasmic injection. After nuclear injectio n, the truncated Rch1 was retained in the nucleus, but either Rch1 res idues 207-217 or a heterologous nuclear export signal, but not a mutan t form of residues 207-217, restored nuclear export. Loss of the nucle ar transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsB N2 caused nuclear accumulation of wild-type Rch1 injected into the cyt oplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that R CC1 acts at an earlier step in Rch1 recycling, possibly the disassembl y of an import complex that contains Rch1 and the import substrate. Co nsistent 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.