ja5b08306_si_001.pdf (6.81 MB)
Reconstituting Intracellular Vesicle Fusion Reactions: The Essential Role of Macromolecular Crowding
journal contribution
posted on 2015-10-14, 00:00 authored by Haijia Yu, Shailendra
S. Rathore, Chong Shen, Yinghui Liu, Yan Ouyang, Michael H. Stowell, Jingshi ShenIntracellular vesicle fusion is mediated
by SNAREs and Sec1/Munc18
(SM) proteins. Despite intensive efforts, the SNARE-SM mediated vesicle
fusion reaction has not been faithfully reconstituted in biochemical
assays. Here, we present an unexpected discovery that macromolecular
crowding is required for reconstituting the vesicle fusion reaction
in vitro. Macromolecular crowding is known to profoundly influence
the kinetic and thermodynamic behaviors of macromolecules, but its
role in membrane transport processes such as vesicle fusion remains
unexplored. We introduced macromolecular crowding agents into reconstituted
fusion reactions to mimic the crowded cellular environment. In this
crowded assay, SNAREs and SM proteins acted in concert to drive efficient
membrane fusion. In uncrowded assays, by contrast, SM proteins failed
to associate with the SNAREs and the fusion rate decreased more than
30-fold, close to undetectable levels. The activities of SM proteins
were strictly specific to their cognate SNARE isoforms and sensitive
to biologically relevant mutations, further supporting that the crowded
fusion assay accurately recapitulates the vesicle fusion reaction.
Using this crowded fusion assay, we also showed that the SNARE-SM
mediated fusion reaction can be modulated by two additional factors:
NSF and α-SNAP. These findings suggest that the vesicle fusion
machinery likely has been evolutionarily selected to function optimally
in the crowded milieu of the cell. Accordingly, macromolecular crowding
should constitute an integral element of any reconstituted fusion
assay.