Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor
We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysi...
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| Main Authors: | , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
December 15, 1993
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| In: |
The journal of cell biology
Year: 1993, Volume: 123, Issue: 6 pt 2, Pages: 1649-1659 |
| ISSN: | 1540-8140 |
| DOI: | 10.1083/jcb.123.6.1649 |
| Online Access: | Resolving-System, lizenzpflichtig, Volltext: https://doi.org/10.1083/jcb.123.6.1649 Resolving-System, kostenfrei, Volltext: https://rupress.org/jcb/article/123/6/1649/29381/Inhibition-of-nuclear-protein-import-by 
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| Author Notes: | Frauke Melchior, Bryce Paschal, Janice Evans, and Larry Gerace |
| Summary: | We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities. |
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| Item Description: | Gesehen am 07.09.2023 |
| Physical Description: | Online Resource |
| ISSN: | 1540-8140 |
| DOI: | 10.1083/jcb.123.6.1649 |