BIOC 212 Lecture Notes - Lecture 16: Clathrin, Cell Membrane, Dynamin
29 May 2018
School
Department
Course
Professor
5- Intracellular Trafficking
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Vesicle Fusion
• Next step in targeting vesicles to acceptor membrane
• Nobel Prize 2013
o James Rothman: SNARE mechanisms
o Randy Shekman: Sec genes, including for SNAREs
o Thomas Südhof: Synaptic vesicle fusion
• All found same mechanism at the same time
Targeting & Fusion
• SNARE proteins: family of related membrane proteins
• Rabs and tethers can recruit SNAREs to fusion site (where vesicles attached)
• V-SNAREs on vesicles recognize partner t-SNAREs on target membranes
o Function after tethering
o V-SNAREs and t-SNAREs form complex together, giving rise to fusion
o Final determinant of targeting specificity (second level)
§ Specific partners
• Formation of SNARE complex mediates fusion
o What physically drives membrane fusion itself
SNAREs
• V-SNAREs are monomers with single TM helical domain
• T-SNAREs are trimers: combinations of TM and peripheral subunits
• The correct set of v- and t-SNAREs form a stable tetramer
o 4 tightly wound α-helices in a bundle, coiled up against each other
o Tightly bound, giving physical tension that drives vesicle fusion
SNARE Folding
• V-SNARE monomer is not stably folded
o Does not have other helices around so does not form stable secondary
structure
o Flops around as flexible polypeptide
• T-SNARE trimer is a partially stable 3-helix bundle
o But not fully stable since missing fourth helix
• V- and t-SNARE fold into a very stable 4-helix bundle
• The folding process pulls membranes close together
o Folding process forming final alpha helix
§ 4-helix bundle lies sideways between target membrane & vesicle
o Generates physical strain like a spring
§ Ends of the helix where join to the membrane wants to form completely
folded stable structure that is straight
§ Wants to be straight in terms of its folding
§ Acts as a spring, pushing the two membranes together
o Not dependent on ATP or GTP
§ Use energy of protein folding
Fusion Model
• As the 4-helix bundle forms, the TM anchors are bent and strained
• This holds the lipid bilayers together
• Water is excluded from area, allowing hydrophobic contacts between lipids of
membranes
o As membrane come close together, pushing out water molecules
o Membrane is lipid bilayer with one outer and one inner layer
o Outer leaflet fuses first, and then proceeded with inner leaflets that fuse
together
• Outer leaflet fusion is unstable, because of the strong membrane curvature
o Membrane wants to be straight
o Strain gives fusion of inner leaflets
• Inner leaflet fusion relieves the strain in the SNARE complex and membrane
curvature
o At last, the membrane can straighten out and the SNAREs can also
straighten out on either side into the cytosol
SNARE Dissociation
• SNARE complex is stable, unstrained and inactive after fusion
o Membrane is flat and SNAREs are completely folded
o Need energy to reset the system (energy not used for fusion)
• NSF is a AAA-family ATPase that dissociates v- and t-SNAREs
o AAA like p97 and proteasome subunit --tend to pull things apart
o Essential for continuation of vesicle traffic
o Pulls the SNAREs apart using ATP such that can go through other cycles of
fusion
o If did not have NSF, all SNAREs would get caught up in 4 alpha helices
complex
• T-SNAREs become active again as 3 helix bundle
o On the acceptor membrane; stay there
• V-SNAREs are recycled back to their donor membrane by vesicles
Homotypic Fusion
• In some fusion events, donor and target membranes are the same
o Fusion of COP-II vesicles into vesicular-tubular cluster
Document Summary
Vesicle fusion: next step in targeting vesicles to acceptor membrane, nobel prize 2013, james rothman: snare mechanisms, randy shekman: sec genes, including for snares, thomas s dhof: synaptic vesicle fusion, all found same mechanism at the same time. Specific partners: formation of snare complex mediates fusion, what physically drives membrane fusion itself. 4-helix bundle lies sideways between target membrane & vesicle: generates physical strain like a spring. Ends of the helix where join to the membrane wants to form completely folded stable structure that is straight. Wants to be straight in terms of its folding. Acts as a spring, pushing the two membranes together: not dependent on atp or gtp. Inner leaflet fusion relieves the strain in the snare complex and membrane curvature: at last, the membrane can straighten out and the snares can also straighten out on either side into the cytosol.
