| Coordinate regulation of fusion and budding maintains membrane homeostasis and ensures the generation of functional vesicles that are capable of fusing with their target organelles. To learn more about the mechanisms that link these two processes, we studied coat recruitment in vesicles containing different fusion machineries and internalization of synaptotagmin VII, a protein known to be involved in calcium-regulated exocytosis. The coat recruitment studies involved performing reverse coating assays in which the AP-3 coat assembled on a vesicular substrate. When the coat assembled on vesicles containing VAMP-2, the v-SNARE associated with synaptic vesicles, the reaction was highly regulated, requiring GTPγS and ATP regenerating system. However, the assembly of coat on vesicles containing synaptotagmin I, the putative calcium sensor in synaptic vesicle exocytosis, was largely unregulated. These findings suggest that there are at least two subpopulations of synaptic vesicles, identifiable by their characteristic fusion machinery. These vesicles may arise from different donor membranes and may exocytose in response to different stimuli. The relationship between fusion machinery and vesicle budding was further investigated in the studies identifying the endocytic determinants of synaptotagmin VII. The synaptotagmin family of membrane proteins have been implicated in both exocytosis and endocytosis. Synaptotagmin VII, a ubiquitous family member, plays a role in multiple forms of regulated exocytosis. Here we show that the cytoplasmic C2 domains in synaptotagmin VII contain unique internalization signals and regulators of these signals. These two signals are a tryotophan-based signal in the extreme C-terminus and a second novel signal in the C2A. The carboxy-terminus is internalized in a dynamin- and eps15-dependent fashion whereas the C2A is internalized independently of both of these factors. Surprisingly, although synaptotagmin VII contains two functional internalization signals, it is not endocytosed in the cell types tested. This appears to be due, at least in part, to the presence of an inhibitory motif in the C2B domain of synaptotagmin VII. We hypothesize that the internalization of synaptotagmin VII is regulated in this way to allow it to couple the processes of regulated exocytosis and compensatory endocytosis. |
