CAST controls presynaptic sequestration of Rab6 in neurons

CAST is a core active zone scaffold protein, yet its role in Rab6-dependent trafficking remains unclear. Here, we identify the coiled-coil domain of CAST (CC10) as a direct Rab6-binding module and show that CAST selectively interacts with the GTP-bound form of Rab6 in both heterologous cells and neurons. Biochemical mapping, isothermal titration calorimetry, and bimolecular fluorescence complementation demonstrate that CC10 is necessary and sufficient for Rab6 recognition. In cultured hippocampal neurons, CAST promotes the presynaptic accumulation of Rab6, whereas CC10-disrupting mutations abolish this effect without detectably altering Rab6 distribution within axonal regions under our imaging conditions. These results define a CAST-dependent mechanism that spatially restricts Rab6 at presynaptic boutons, extending ELKS-based models of Rab6 cargo capture and providing a structural basis for the organization of presynaptic trafficking.