title: Synaptic vesicle recycling at CNS snapses without AP-2.
abstract: Synaptic vesicles (SVs) are composed of approximately 10 types of transmembrane proteins that must be recycled after exocytosis of neurotransmitter. The mechanisms for resorting these proteins into synaptic vesicles once incorporated into the plasma membrane after exocytosis are poorly understood. The adaptor complex AP-2 is the major clathrin-associated adaptor for cargo recognition at the plasma membrane. Here, we have investigated its role in synaptic vesicle endocytosis. shRNA-mediated knockdown of the AP-2 complex results in an approximately 96% reduction of this protein complex in primary neurons. We used simultaneous expression of shRNA and pHluorin-tagged vesicle components to show that the absence of AP-2 significantly slows but does not prevent the endocytosis of four of the major synaptic vesicle transmembrane proteins. We show that in the absence of AP-2, the AP-1 adaptor complex appears to functionally substitute for AP-2 but results in complex internalization kinetics that are now sensitive to the guanine-nucleotide exchange factor for ADP-ribosylation factor GTPase (ARF-GEF) inhibitor brefeldin-A (BFA). Simultaneous removal of both AP-2 and AP-1 prevents this compensatory substitution and results in slowed but functional endocytosis. These results demonstrate that in the absence of AP-2, SV proteins still become endocytosed, and synaptic vesicle recycling remains operational.
authors.affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Ryan