A novel system to quantitate nuclear-cytoplasmic flux in vivo: kinetics of signal-dependent nuclear protein export.
Compared to signal-mediated nuclear protein import, there is a paucity of kinetic information with respect to signal-mediated nuclear protein export. In this study we use the novel approach of simultaneous nuclear/cytoplasmic microinjection of beta-galactosidase fusion proteins to examine nuclear import and export conferred by the leucine-rich nuclear export signals (NESs) of HIV-1 Rev and the cAMP-dependent protein kinase inhibitor PKI, comparing results to those for either a fusion protein containing a conventional nuclear localization sequence (NLS) or beta-galactosidase itself. We also analyze nuclear transport of the proteins in vitro. Both the Rev and PKI NESs confer nuclear export, in contrast to the NLS or mutated inactive NESs; steady state was achieved within 40-45min although not all NES-containing protein hadbeen exported from the nucleus at this time point. Interestingly, the Rev and PKI NES fusion proteins, in stark contrast to beta-galactosidase itself, exhibited nuclear entry in vivo and nuclear accumulation to levels about twofold those in the cytoplasm in vitro. We conclude that NESs, rather than exclusively conferring nuclear export, may be able to mediate shuttling between the nuclear and cytoplasmic compartments.
Efthymiadis
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Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia.
Dottorini
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Jans
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D000818:Animals; D001692:Biological Transport / Q000235:genetics; D006528:Carcinoma, Hepatocellular; D002451:Cell Compartmentation / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D003593:Cytoplasm / Q000378:metabolism*; D015694:Gene Products, rev / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D015694:Gene Products, rev; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D017868:Cyclic AMP-Dependent Protein Kinases