Synaptic vesicles are formed primarily by endocytic budding from the plasma membrane of axon termini. Endocytosis usually involves clathrin-coated pits and is quite specific, in that several membrane proteins unique to the synaptic vesicles (e.g., neurotransmitter transporters) are specifically incorporated into the endocytosed vesicles and resident plasma membrane proteins (e.g., the voltage-sensitive Ca2+channel) remain. In this way, synaptic-vesicle membrane proteins can be reused and the recycled vesicles refilled with neurotransmitter (see Figure 22-26, step 6).
As in the formation of other clathrin/AP-coated vesicles, pinching off of endocytosed synaptic vesicles requires the GTP-binding protein dynamin (see Figure 14-19). Indeed, analysis of a temperature-sensitive Drosophila mutant called shibire (shi), which encodes the fly dynamin protein, provided early evidence for the role of dynamin in endocytosis. At the permissive temperature of 20 °C, the mutant flies are normal, but at the nonpermissive temperature of 30 °C, they are paralyzed (shibire, “paralyzed,” in Japanese) because pinching off of clathrin-coated pits in neurons and other cells is blocked. When viewed in the electron microscope, the shi neurons at 30 °C show abundant clathrin-coated pits with long necks but few clathrin-coated vesicles. The appearance of nerve termini in shi mutants at the nonpermissive temperature is similar to that of termini from normal neurons incubated in the presence of a nonhydrolyzable analog of GTP (see Figure 14-20). Because of their inability to pinch off new synaptic vesicles, the neurons in shi mutants eventually become depleted of synaptic vesicles when flies are shifted to the nonpermissive temperature, leading to a cessation of synaptic signaling and to paralysis.