Ahmad, B.; Lerma-Reyes, R.; Mukherjee, T.; Nguyen, H. V.; Weber, A. L.; Schulze, W. X.; Comer, J. R.; Schrick, K.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin (IMP) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMP-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMP isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMP triple mutants (imp-1,2,3) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMP isoforms in Arabidopsis. Taken together, our findings provide mechanistic evidence for IMP-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants.