Timing of protein-protein interactions involved in synaptic vesicle endocytosis
Carlos A. Villalba-Galea: 0000-0002-6489-4651
Conference Title/Conference Publication
Society for Neuroscience Annual Meeting
Society for Neuroscience
San Diego, CA
October 23-27, 2004
Date of Presentation
We have used Fluorescence Resonance Energy Transfer (FRET) to define the time course of interaction of clathrin with proteins involved in synaptic vesicle recycling. Pairs of interacting proteins were purified, fluorescently labeled, and microinjected into the squid giant presynaptic terminal. Trains of presynaptic action potentials evoked an increase in FRET between clathrin-Alexa350 and auxilin-Citrine, indicating that presynaptic activity promotes the interaction of clathrin with auxilin. Association between these proteins occurred without a measurable delay and could be described by an exponential function with a time constant of 32 ± 1.9 sec (n=3), consistent with the proposed role for auxilin in uncoating clathrin-coated vesicles. Dissociation of auxilin from clathrin was much slower, with a time constant of 330 sec. Resting terminals also exhibited FRET between clathrin and AP180, another endocytic protein. Presynaptic stimulation caused a remarkable decrease in FRET between this pair of interacting partners, after a delay of 10 sec, with a time constant of 16 ± 8.4 sec and a Hill coefficient of 2.1 ± 1.1 (n = 5). After stimulation ended, the FRET ratio recovered with a time constant of 25 ± 12 sec (n = 5). In summary, presynaptic activity promotes the interaction between clathrin and auxilin, while clathrin is already bound to AP180. The dissociation of AP180 from clathrin proceeds much more rapidly than the dissociation of auxilin. We conclude that FRET is a powerful and general technique for examining the timing of protein-protein interactions involved in synaptic vesicle trafficking. This work was supported by grants from the NIH and MDA and a Grass Fellowship. Supported by NIH and MDA and a Grass Fellowship.
Villalba-Galea, Carlos A.; Jin, S.; Prasad, K.; Augustine, G. J.; and Lafer, E. M., "Timing of protein-protein interactions involved in synaptic vesicle endocytosis" (2004). School of Pharmacy Faculty Presentations. 413.