Activated kinesin-1 assembles into a dimer-of-dimers

Kinesin-1 mediates intracellular cargo transport along microtubules. The canonical kinesin-1 complex is composed of two KIF5 motor subunits (also known as kinesin heavy chain; KHCs) and two cargo-binding kinesin light chains (KLCs), and KIF5 is generally thought to function as a homodimer. Whether kinesin-1 forms higher-order assemblies beyond the dimer has remained unclear. Here, we show that the neuronal isotype KIF5C assembles into tetramers through reversible association of two dimers. Gel filtration and mass photometry demonstrate that KIF5C partitions between dimeric and tetrameric forms in vitro. Deletion of the autoinhibitory elbow region strongly shifts this distribution toward tetramers, indicating that conformational activation promotes intermolecular assembly. Tetramer formation requires the C-terminal tail but remains compatible with KLC binding. Single-molecule motility assays reveal that tetramer-enriched KIF5C exhibits increased landing rates and longer run lengths compared with the dimeric form. These findings identify reversible tetramerization as a previously unrecognized property of kinesin-1 and suggest that conformational activation not only relieves intramolecular inhibition but also promotes higher-order assembly, revealing an additional layer of regulation that may tune motor activity in neurons.