NUCLEOCYTOPLASMIC SHUTTLING FACTORS INCLUDING RAN AND CRM1 MEDIATE NUCLEAR EXPORT OF NFAT IN-VITRO

We have developed a permeabilized cell assay to study the nuclear expo rt of the shuttling transcription factor NFAT, which contains a leucin e-rich export signal. The assay uses HeLa cells that are stably transf ected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP-NFAT in digitonin-permeabilized cells occurs in a temper ature-and ATP-dependent manner and can be quantified by flow cytometry , In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30 degrees C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequen t export step. Using this assay, we have purified a second major expor t activity from cytosol. We found that it corresponds to CRM1, a prote in recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-depen dent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multipl e nucleocytoplasmic shuttling factors, including Ran and CRM1.