Abstract
While predominantly studied in cytoplasmic contexts, actin plays critical roles in the nucleus, regulating chromatin accessibility and remodeling, transcription, and DNA damage repair. Cell-based studies have contributed to a widely accepted model in which the import factor Importin 9 (IPO9) acts in concert with the actin filament-severing protein cofilin to transport actin into the nucleus. The classical nuclear localization signal on cofilin is thought to anchor IPO9 to cofilin-bound actin monomers, driving the formation of an import-competent tripartite actin•cofilin•IPO9 complex. In striking contradiction to this established model of actin import, we demonstrate that IPO9 directly binds to monomeric actin with mid-nanomolar affinity and, rather than promoting IPO9•actin complex formation, cofilin competitively inhibits binding of IPO9 to actin. We further report competitive binding for monomeric actin between IPO9 and the canonical actin monomer-sequestering protein, profilin. As cofilin and profilin are both capable of binding actin monomers at the barbed face, our results are consistent with a model in which IPO9 binds an overlapping portion of this interface. In further support, we demonstrate that IPO9 modestly occludes the barbed face of actin monomers, decreasing the rate of filament formation, and exhibits minimal filamentous actin binding. Finally, we identify unexpected affinity between the nuclear import release factor RanGTP and monomeric actin; however, a tripartite IPO9•actin•RanGTP complex does not form. The competitive interactions observed between IPO9 and cytoplasmic actin-binding proteins suggest dynamically coupled equilibria mediate the nuclear transport of actin monomers.