VSV Trans-Membrane Domain Promotes Content Mixing to occur Early in the Fusion Process

Abstract

Polyethylene glycol (PEG)-mediated fusion of 25 nm vesicles was studied at different temperatures in the presence of the trans-membrane domain (TMD) of the G protein (fusion protein) of vesicular stomatitis virus (VSV). Vesicles were composed of dioleyol-phosphatidylcholine (DOPC), dioleyol-phosphatidylethanolamine (DOPE), bovine brain sphingomyelin (SM), and cholesterol (CH) in a molar ratio of 35:30:15:20. Kinetic parameters of the fusion process were determined by fitting lipid mixing (LM), content mixing (CM), and content leakage (L) time courses globally to a three state sequential model that allowed for leakage from the final fusion pore state. This yielded the rate constants for conversion between different states as well as the probabilities of the occurrence of LM and CM in each state. The TMD enhanced the initial rate of lipid mixing and content mixing in agreement with our previous report (Biochemistry, 2002, 14925). This resulted partly from enhanced extent of CM, although the extent of LM remained unaltered. In addition, the rate of intermediate formation was increased slightly, although, the principal effect of TMD was to increase the probability of CM in the initial intermediate at the expense of the final step of the process. This suggests that the presence of TMD enhanced flickering pore formation, leading to an increase in CM early in the process. Supported by NIGMS grant 32707 to BRL.