DIFFERENTIAL IMPORTIN-ALPHA RECOGNITION AND NUCLEAR TRANSPORT BY NUCLEAR-LOCALIZATION SIGNALS WITHIN THE HIGH-MOBILITY-GROUP DNA-BINDING DOMAINS OF LYMPHOID ENHANCER FACTOR-1 AND T-CELL FACTOR-1

The transcription factor lymphoid enhancer factor 1 (LEF-1) is directe d to the nucleus by a nine-amino-acid nuclear localization signal (NLS ; KKKKRKREK) located in the high-mobility-group DNA binding domain. Th is NLS is recognized by two armadillo repeat proteins (pendulin/Rch1/a lpha-P1/hSrp1 alpha and Srp1/karyopherin-alpha/alpha-S1/NPI-1) which f unction in nuclear transport as the importin-alpha subunit of NLS rece ptors, T-cell factor 1 (TCF-1), a related transcription factor, contai ns a similar sequence (KKKRRSREK) in the identical position within its HMG DNA binding domain. We show that this sequence functions as an NL S in vivo but is not recognized by these two importin-alpha subtypes i n a yeast two-hybrid assay and only weakly recognized in an in vitro b inding assay. Transfer of the LEF-1 NLS to TCF-1 can confer pendulin/R ch1 binding, demonstrating that the NLS is the primary determinant for recognition. We have constructed a set of deletion mutations in pendu lin/Rch1 to examine the differential NLS recognition more closely. We find that the entire armadillo repeat array of pendulin/Rch1 is necess ary to maintain high affinity and specificity for the LEF-1 NLS versus the TCF-1 NLS, Importin-beta, the second subunit of the NLS receptor complex, does not influence in vitro NLS binding affinity or specifici ty. To test whether this differential recognition is indicative of dis tinct mechanisms of nuclear transport, the subcellular localization of LEF-1 and TCF-1 fused to green fluorescent protein (GFP) was examined in an in vitro nuclear transport assay. GFP-LEF-1 readily localizes t o the nucleus, whereas GFP-TCF-1 remains in the cytoplasm. Thus, LEF-1 and TCF-1 differ in several aspects of nuclear localization.