Subcellular localization of interferon-inducible Myc/Stat-interacting protein Nmi is regulated by a novel IFP 35 homologous domain

Nmi was initially identified through a yeast two-hybrid interaction with N- Myc but it also interacts with c-Myc, Max, Fos, and several other transcrip tion factors, including signal transducer and activator of transcription (S tat) proteins. Nmi is an interferon (IFN)-inducible protein with 25% amino acid identity to the IFN-inducible protein IFP 35. We have found that this homology consists of a novel domain of approximately 90-92 amino acids (aa) that is repeated in tandem in each protein. This region, termed Nmi/IFP 35 domain (NID), is important for subcellular localization of Nmi. Full-lengt h Nmi protein or deletion constructs containing a single NID are localized to the cytoplasm, but amino-terminal Nmi fragments of up to 92 aa containin g neither NID are nuclear. Fusion of the amino-terminal end of Nmi to pyruv ate kinase, an exclusively cytoplasmic protein, results in a cytoplasmic fu sion protein, suggesting that the amino-terminal end of Nmi does not contai n a classic nuclear localization signal (NLS). Fusion of the amino-terminal end of Nmi to green fluorescent protein (GFP), which is normally found in both nuclear and cytoplasmic compartments, does not alter GFP distribution, whereas fusion of a single NID to GFP targets the fusion to the cytoplasm. Fusion of a nuclear localization signal (NLS) to full-length Nmi or NID re peats targets the hybrid to the nucleus, suggesting that a strong NLS is do minant to the cytoplasmic localization function of NID. NID may mediate cyt oplasmic localization of the full-length Nmi protein through NID-NID protei n interactions as demonstrated by yeast two-hybrid assay, immunoprecipitati on, and the presence of Nmi in a high molecular weight protein complex. The se results suggest that Nmi is composed of a modular structure with an amin o-terminal domain that when separated from the rest of the protein is nucle ar. The carboxy-terminal two thirds of the protein is composed of two NID t hat mediate cytoplasmic localization of the full-length protein.