Loss of RCC1 leads to suppression of nuclear protein import in living cells
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Published on: August 12, 2015
T Tachibana1, N Imamoto, H Seino
1Department of Anatomy and Cell Biology, Osaka University Medical School, Japan.
Abstract:
The role of RCC1-Ran/TC4 in nuclear protein import was examined in living cells using a temperature-sensitive RCC1 mutant cell line, tsBN2, and tsBN2 transformed with a RCC1 cDNA lacking the nuclear localization sequence domain, delta 8-29. Substrate, containing a small number of SV40 T antigen nuclear localization sequence peptides, injected into the cytoplasm of tsBN2 cells cultured at the non-permissive temperature of 39.5 degrees C did not accumulate efficiently in the nucleus. When the same substrate was injected into the cytoplasm of heterokaryons of tsBN2 and wild type BHK21 cells, import efficiency into the tsBN2 nuclei was not restored. Import into the BHK21 nuclei gradually decreased after fusion. In contrast, import efficiency into tsBN2 nuclei gradually recovered after fusion with tsBN2 cells transformed with delta 8-29 in which functional RCC1 was diffusely distributed in both the nuclei and cytoplasm. Substrate did not accumulate in the nuclei of digitonin-permeabilized tsBN2 cells cultured at 39.5 degrees C even in the presence of normal cytosol. These results suggest that loss of RCC1 function leads to the decline of import competence of the nucleus and accumulation of a factor in the cytoplasm that suppresses nuclear import. These results indicate that the RCC1-Ran/TC4 system may regulate nuclear import.
Insights
Loss of RCC1 function impairs nuclear import. This suggests the RCC1-Ran/TC4 system regulates nuclear protein transport, potentially by preventing cytoplasmic factors from inhibiting import.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Nuclear protein import is crucial for cellular function.
- RCC1 (Regulator of Chromosome Condensation 1) and Ran/TC4 are key components of the nuclear transport machinery.
- Understanding their precise roles in nuclear import is essential.
Purpose of the Study:
- To investigate the role of the RCC1-Ran/TC4 system in nuclear protein import.
- To determine how RCC1 dysfunction affects the nucleus's ability to import proteins.
Main Methods:
- Utilized a temperature-sensitive RCC1 mutant cell line (tsBN2).
- Employed microinjection of substrate containing SV40 T antigen nuclear localization sequence peptides.
- Performed cell fusion experiments with wild-type BHK21 cells and tsBN2 cells expressing modified RCC1.
- Used digitonin-permeabilized cells to assess import in vitro.
Main Results:
- Nuclear import of substrate was inefficient in tsBN2 cells at the non-permissive temperature.
- Import was not restored in tsBN2 nuclei after fusion with wild-type cells.
- Import efficiency recovered in tsBN2 nuclei upon fusion with cells expressing a modified RCC1 (delta 8-29).
- Substrate failed to accumulate in permeabilized tsBN2 cells, even with normal cytosol.
Conclusions:
- Loss of RCC1 function compromises nuclear import competence.
- A cytoplasmic factor may accumulate, suppressing nuclear import upon RCC1 dysfunction.
- The RCC1-Ran/TC4 system likely plays a regulatory role in nuclear protein import.
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