Functional Consequences of Necdin Nucleocytoplasmic Localization

Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown.We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin β1. Finally, exclusion of necdin from the nucleus triggered extensive cell death.These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization