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PMID: 8276887 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.

The Journal of cell biology ·Vol. 123 ·No. 6 Pt 2 ·1993-12-00 ·Pages 1649-59

Melchior F, Paschal B, Evans J, Gerace L

Abstract

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.

MeSH Terms
Amino Acid Sequence Cell Nucleus/drug effects,metabolism,ultrastructure Cytosol/metabolism Electrophoresis, Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay GTP Phosphohydrolases/metabolism GTP-Binding Proteins/analysis,metabolism Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Guanosine Triphosphate/analogs & derivatives,pharmacology HeLa Cells Humans Kinetics Models, Biological Molecular Sequence Data Molecular Weight Nuclear Proteins/metabolism Oligopeptides/chemical synthesis,immunology ran GTP-Binding Protein
Chemicals
Nuclear Proteins Oligopeptides Guanosine 5'-O-(3-Thiotriphosphate) Guanosine Triphosphate GTP Phosphohydrolases GTP-Binding Proteins ran GTP-Binding Protein
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Melchior F
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Paschal B
Evans J
Gerace L
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50 references, click to expand
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.123.6.1649
Published
1993-12-00
Pages
1649-59
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2290879
Subset
IM
Corrections
ErratumIn
-
External Links
PubMed source DOI: 10.1083/jcb.123.6.1649 PMC Full Text View journal details
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