Proteomics of tau repeat domain interactome via split TurboID proximity labeling

In Alzheimer's disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized this split TurboID TauRD displaying the requirement for both ligase fragment co-expression for robust enzyme activity and nuclear and cytoplasmic localization of the recombinant proteins. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins (RBPs), and nuclear speckles. These in vitro results were further supported by integrating the TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from human AD and related tauopathies. This revealed an overlap with the in vitro TauRD interactome and several modules enriched with RBPs and increased in AD and PSP. These findings emphasize the importance of nuclear pathways in tau pathology, such as RNA splicing and nuclear-cytoplasmic transport and establishes the sTurbo TauRD system as a valuable tool for exploring the tau interactome.