Synaptobrevin Trans-Membrane Domain forms a Complex that Enhances the Rate of “Stalk” and Pore Formation in PEG-Mediated Vesicle Fusion

Abstract

Synaptobrevin (SB), a membrane anchored protein in the neuronal cell membrane, complexes with syntaxin (SX) and SNAP-25 to facilitate membrane fusion in neurotransmitter release. SB and SX promote Poly(ethylene glycol) (PEG)-mediated fusion whether or not they are assembled into a SNARE complex (Dennison et al., BJ, 2006, 1661). Viral fusion protein trans-membrane domains (TMDs) enhance the fusion. Thus, we hypothesized that the SB-TMD may also affect fusion kinetics. The kinetics of PEG-mediated fusion of DOPC/DOPE/sphingomyelin/cholesterol/DOPS (32/25/15/20/8) 25 nm vesicles (SUVs) was examined in the presence and absence of SB TMD. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to a 3-state sequential model (Weinreb & Lentz, BJ, 2007, 4012), from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state. SB TMD enhanced the rates of “stalk” and fusion pore (FP) formation in a cooperative fashion (maximum effect at 3 peptide/vesicle). TMD ordered the bilayer interior in a similarly cooperative fashion. The effects of hexadecane and TMD on fusion kinetics were quite distinct and not mutual. SB TMD increased the probability of both LM and CM in the initial intermediate, suggesting that it promoted formation of transient pores before the final pore state. Transition state thermodynamic changes indicate that the effects on “stalk” and FP formation involved different mechanism, although both showed that in presence of TMD the change in entropy is always greater than enthalpy. Supported by NIH grant GM32707 to BRL.