Biochemical characterization of the coating mechanism of the endosomal donor compartment of synaptic vesicles.
The heterotetrameric adaptor protein complex, AP-3, sorts proteins to both the endosome/lysosome and the synaptic vesicles. We have characterized the recruitment of pure AP-3 complex and ADP-ribosylation factor (ARF) onto the endosomal donor compartments that give rise to synaptic vesicles. We demonstrated that endosomes become heavier in a sucrose gradient after incubation with rat brain cytosol and a nonhydrolyzable GTP analog, GTPgammaS. This process requires a small GTPase, ARF-1. Furthermore, the endosomal coating is specific for AP-3 but not the AP-2 complex. This process requires only two soluble proteins AP-3 and ARF, with the recruitment of AP-3 being saturable at about 30 nM. These results establish that the synaptic vesicle's donor membrane is coated with AP-3 before vesiculation, in a coat-protein-specific and dose-dependent fashion.
Horng
Jim-Tong
JT
Department of Biochemistry, Chang Gung University, Kweishan, Taiwan. jimtong@mail.cgu.edu.tw
Tan
Chung-Yueh
CY
D033963:Adaptor Protein Complex 3 / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002458:Cell Fractionation; D011992:Endosomes / Q000648:ultrastructure*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D013572:Synaptic Vesicles / Q000648:ultrastructure*
D033963:Adaptor Protein Complex 3; D016244:Guanosine 5'-O-(3-Thiotriphosphate)