A Novel System to Quantitate Nuclear-Cytoplasmic Fluxin 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 β-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 β-galactosidase itself. We also analyze nuclear transport of the proteinsin 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 β-galactosidase itself, exhibited nuclear entryin vivoand nuclear accumulation to levels about twofold those in the cytoplasmin vitro.We conclude that NESs, rather than exclusively conferring nuclear export, may be able to mediate shuttling between the nuclear and cytoplasmic compartments.