VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis

The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed. •VAMP4 is the first identified ADBE cargo•VAMP4 is essential for ADBE•Most synaptic vesicle cargoes are not selectively recovered by ADBE Nicholson-Fish et al. show that VAMP4 is selectively retrieved by activity-dependent bulk endocytosis (ADBE) and is essential for ADBE to proceed. Thus, synaptic vesicles generated by ADBE will have a specific molecular composition that defines their physiological function.