TWIN AUTONOMOUS BIPARTITE NUCLEAR-LOCALIZATION SIGNALS DIRECT NUCLEARIMPORT OF GT-2

GT-2 is a DNA-binding protein with high target-sequence specificity to ward functionally defined, positively acting cis elements in the rice phytochrome A gene promoter. Using immunocytochemical procedures, it i s shown here that GT-2 is localized to the nucleus, consistent with a function in transcriptional regulation. Immunoblot and immunocytochemi cal analyses show that rice shoots contain higher levels of GT-2 prote in than roots, and that no photo-induced changes in GT-2 abundance or spatial distribution are detectable in these tissues, a result consist ent with the proposed constitutive activity of GT-2. In both shoots an d roots, GT-2 protein is undetectable in meristematic tissue but becom es expressed at later stages of cellular development, consistent with a role in contributing to the pattern of phytochrome A gene expression . By transfecting protoplasts with a series of constructs containing d eletion derivatives of GT-2 fused to beta-glucuronidase (GUS), followe d by in situ localization of GUS activity, two independent, functional ly active nuclear localization sequences (NLSs) have been identified i n GT-2. One NLS resides within each of a pair of previously identified , spatially separate, trihelix motifs in the protein. Sequence inversi on and alanine-scanning mutagenesis has identified residues within the se NLSs necessary for nuclear localization. Each NLS contains two basi c domains separated by 10 amino acids, conforming to the bipartite cla ss of NLS involved in the targeting of numerous other nuclear localize d proteins.