A multivalent adaptor mechanism drives the nuclear import of proteasomes

Abstract

Nuclear protein homeostasis, including the turnover of transcription factors, critically depends on nuclear proteasomes. After each cell division, proteasomes need to be re-imported into the newly formed nucleus in a highly dynamic process that requires the largely unstructured protein AKIRIN2. However, how AKIRIN2 orchestrates this process and, more generally, how large protein complexes are translocated into the nucleus remains poorly understood. Here, we have used an integrated approach combining protein-wide saturation mutagenesis screens, cryoEM, and biochemical reconstitution to characterize AKIRIN2 as a scaffold protein that coordinates the stepwise assembly of an importin cluster around the proteasome. Through surveying every possible single amino acid substitution in AKIRIN2 using FACS- and microscopy-based genetic screens, we establish a comprehensive map of functionally relevant residues and binding interfaces in structured and disordered protein regions. Integrating these results with cryoEM analysis reveals a wing helix in a disordered region of AKIRIN2 that plays a crucial role in stabilizing proteasome interactions. Upon primary binding, AKIRIN2 homodimers recruit the importin IPO9, which in turn facilitates the binding of a second AKIRIN2 homodimer that recruits additional importins. Together, this multivalent molecular assembly amplifies the number of nuclear localisation signals and, thereby, triggers efficient proteasome translocation into the nucleus. Inside the nucleus, RanGTP rapidly dissociates importins, and AKIRIN2 is degraded by the proteasome in a ubiquitin-independent manner. Beyond mechanistically resolving the nuclear import of proteasomes, we propose that multivalent adaptor proteins like AKIRIN2 orchestrate the import of other macromolecular complexes and thereby dynamically control the composition of the nuclear proteome.