TurboID proximity labeling combined with structure prediction identifies C3H61/AtTZF5 as a tail-binding protein of Arabidopsis class VIII myosin ATM1

Abstract

Higher plants possess two classes of myosin molecular motors, class XI and class VIII, both unique to the plant lineage. The diverse cellular functions of class XI myosins, including organelle transport and nuclear positioning, have been elucidated largely through systematic identification of cargo adaptor proteins that bind to their globular tail domains (GTDs). In contrast, no proteome-wide screen for class VIII myosin tail-binding proteins has been reported; the few known interacting proteins were each discovered through studies focused on the binding partner rather than on the myosin itself, leaving the full repertoire of class VIII myosin-associated proteins largely unknown. Here, we employed TurboID-based proximity labeling to systematically identify proteins associated with the GTD of the class VIII myosin ATM1 in Arabidopsis thaliana, as this approach covalently biotinylates neighboring proteins in vivo, enabling their identification even after proteolytic degradation during cell lysis. We identified 233 non-redundant candidate ATM1-proximal proteins. Candidates were prioritized by AlphaFold3-based protein complex structure prediction and evaluated experimentally. By integrating AlphaFold3 prediction, co-immunoprecipitation, subcellular co-localization in living cells under stress, and structure-guided interface mutagenesis, we identified C3H61/AtTZF5 as an ATM1 tail-binding protein. C3H61/AtTZF5, a tandem CCCH zinc finger protein whose family members have been implicated in mRNA turnover, co-localized with the ATM1 tail in eIF4A2-positive granules under heat stress, and targeted substitutions at the AlphaFold3-predicted interface completely abolished the interaction. These findings strongly support a direct association between ATM1 and C3H61/AtTZF5, suggesting a possible link between this class VIII myosin and post-transcriptional gene regulation during stress responses.