Weberpost - Weber et al 1997 SNAREpins Minimal machinery...

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Weber et al. 1997 SNAREpins: Minimal machinery for membrane fusion. Cell 92:759-772. A. Major question •Membrane fusion is important for vesicle trafFcking as well as for cell fusion during development of certain organs (placenta, bone, muscle). •However, the cellular machinery that drives two membranes to fuse is still not well understood. B. Hypothesis : •“SNARE hypothesis” states that SNARE complex formation is important for proper targeting of membranes that are going to fuse. •Hypothesis in this paper: SNARE complex formation may also be sufFcient to drive membrane fusion as well as targeting •Already know SNARE complex is necessary for vesicle fusion
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•Create synthetic lipid vesicles (liposome bilayers) incorporating only v-SNARE or t-SNARE proteins •Assay fusion of these vesicles C. Approach: Cell Biology and Biochemistry Experimental Strategy: v-SNARE t-SNARE
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D. Tools required for these experiments - SNAREs Fig. 1A •Recombinant SNAREs •vSNARE - synaptobrevin 2/ VAMP2 •Puri±ed using 6X His tag on C-terminus •tSNAREs - SNAP25 and syntaxin •tSNAREs co-expressed and puri±ed using GST tag on SNAP25 •GST tag cleaved using thrombin protease •Puri±ed proteins shown in Fig 1A (lanes 1 & 2 coomassie stain)
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D. Tools -proteoliposomes •Vesicles with defned protein composition •Mix •recombinant proteins solubilized in detergent called OG •phospholipids (PS and PC) solubilized in detergent •Dilute and then dialyze the detergent •Spontaneous Formation oF liposomes •Separate vesicles From unincorporated components on a density gradient •Examine protein composition oF vesicles -SDS PAGE/coomassie stain ±ig. 1A •Vesicle with vSNARE VAMP2 •Vesicle with tSNAREs syntaxin and SNAP25
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D. Tools -FRET NBD rhodamine
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Weberpost - Weber et al 1997 SNAREpins Minimal machinery...

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