Cytokine-induced nuclear translocation of STAT1 via a non-transferable NLS

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin β, Ran-GTP, and the importin α5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin α5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin α5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-α5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin α5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.