Microtiter binding assays and in silico kinetic mapping reveal binding of TDP-43 nuclear localization signal to importin α is modulated by phosphorylation

Abstract

Importins transport cargos bearing a nuclear localization signal (NLS) from the cytoplasm into the nucleus. The NLS acts as a short recognition sequence that binds with high affinity to a heterodimer of importin α/β. Here we have studied the NLS of the RNA-binding protein TDP-43, which is involved in ALS. ELISA-based microtiter binding experiments revealed that phosphorylation of the TDP-43 NLS significantly perturbs importin α binding affinity, thereby regulating nuclear import. In particular, phosphorylation of threonine at position 88 (88pThr) within the NLS shows the most drastic reduction in affinity among single phosphorylations.