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 protei n export. In this study we use the novel approach of simultaneous nucl ear/cytoplasmic microinjection of beta-galactosidase fusion proteins t o examine nuclear import and export conferred by the leucine-rich nucl ear export signals (NESs) of HIV-1 Rev and the cAMP-dependent protein kinase inhibitor PKI, comparing results to those for either a fusion p rotein containing a conventional nuclear localization sequence (NLS) o r 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 achiev ed within 40-45 min although not all NES-containing protein had been e xported from the nucleus at this time point. Interestingly, the Rev an d PKI NES fusion proteins, in stark contrast to beta-galactosidase its elf, exhibited nuclear entry in vivo and nuclear accumulation to level s about twofold those in the cytoplasm in vitro. We conclude that NESs , rather than exclusively conferring nuclear export, may be able to me diate shuttling between the nuclear and cytoplasmic compartments. (C) 1998 Academic Press.