MCDB428Lect12 - MCDB 428 Lecture 12: Monday February 4,...

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Lecture 12: Monday February 4, 2008 Today’s Outline Biochemical Analysis Revisited VSV-G GlcNAc transferase fusion assay Protein Components Cytosol requirements for fusion Membrane proteins required for fusion Overview of Vesicle Budding GTP-Binding proteins recruit coat proteins Coat proteins induce membrane curvature Coat proteins interact with cargo and v-SNAREs Overview of Uncoated Vesicle fusion Uncoated vesicles are fusion competent SNARE pairing between v- and t-SNAREs SNARE proteins Formation of the 4-helix coiled coil domain SNARE protein characteristics Importance for fusion: neurotoxin functions Rab GTPases Evidence for essential role in vesicle fusion The Rab GTPase cycle Interaction with Rab effector proteins Synaptic Vesicle Fusion Roles of Rab GTPase, SNAREs and NSF in fusion How SNAREs catalyze membrane fusion MCDB 428
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Setting Up a Cell-free Fusion Assay: I Isolating “Donor” and “Acceptor” Vesicle Fractions
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Setting Up a Cell-free Fusion Assay: II GlcNAc transferase Donor Fraction derived from VSV-G infected cells lacking GlcNAc transferase Acceptor Fraction derived from uninfected wild-type cells UDP- 3 H-GlcNAc as substrate for the GlcNAc transferase ATP Cytosol Donor Vesicles Acceptor Vesicles Manipulate cytosol to see what proteins are required to support fusion
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VSV-G Cytosolic Requirements for Golgi Vesicle Fusion + cytosol + UDP- 3 H-GlcNAc + ATP Donor Vesicles Acceptor Vesicles + GTP γ S coated vesicles + GTP γ S + NEM coated vesicles + NEM (N-ethylmaleimide) uncoated vesicles
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NSF: N -ethlymaleimide
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MCDB428Lect12 - MCDB 428 Lecture 12: Monday February 4,...

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