A yeast model reveals the SMAD3 nuclear import mechanism

ABSTRACT TGF-β signaling is important for patterning tissues during embryonic development and relies on the nuclear import of SMADs. Basal SMAD nuclear import mechanisms remain to be fully understood and could benefit from advances in our understanding of nuclear transport machinery. To assess the molecular determinants of SMAD nuclear transport, we take advantage of the unicellular S. cerevisiae model that lacks the TGF-β signaling complexity of vertebrates but has conserved nuclear transport machinery. In this minimal system, we find that the steady-state distribution of SMAD2/3, SMAD4, and SMAD1/5 are Ran-dependent and either accumulate in, or are depleted from the nucleus. Conditionally inhibiting each of the karyopherin/importins demonstrates that SMAD3 is imported by orthologues of importin-β, importin-7 and importin-8. The previously defined nuclear localization signal is insufficient to confer nuclear import. Instead, our data suggest the entire MH1 domain may act as an importin binding surface. Deleting a portion of this domain impairs SMAD3 function in Xenopus and SMAD3 nuclear enrichment in HEK293T cells. Thus, the yeast platform provides an efficient strategy to illuminate the nuclear transport mechanisms of embryonic signaling effectors. Competing Interest Statement Mustafa K. Khokha is a co-founder of Victory Genomics Inc.