Functional characterization of the nuclear localization signal of OREBP/TonEBP

The Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a transcription factor that mediates cellular adaptations to extracellular hypertonic stress. OREBP is regulated at multiple levels including nuclear import. Previous studies suggested that the consensus nuclear localization signal (NLS, amino acid residues 199–216) was crucial for the nuclear import of OREBP. To better delineate the role of NLS in OREBP nuclear import, we generated and studied the sub-cellular localization of different PEPCK fusion proteins that contains the minimal consensus NLS alone, or different fragments that cover NLS and its flanking sequences. Minimal NLS alone failed to direct PEPCK to the nucleus. Instead, the nuclear import function of NLS is reconstituted by including two flanking domains that were located in the N- and C-terminal to the NLS. Alanine-scanning mutagenesis of the corresponding region using FLAG-tagged OREBP further revealed two clusters of amino acids that are responsible for its nuclear translocation. Thus, although a general feature of monopartite and bipartite NLS is the present of multiple basic residues, and is thought to be sufficient for directing nuclear import of transcription factors, our data suggested that the NLS of OREBP is by itself essential but insufficient to direct nuclear import, and additional protein domains are required for its function.