Abstract
Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle. Using a reconstituted system for COPI vesicle formation from purified components, we find that a dimerization-deficient Arf1 mutant, which does not display the ability to modulate membrane curvature in vitro or to drive formation of coated vesicles, is able to recruit coatomer to allow formation of COPI-coated buds but does not support scission. Chemical cross-linking of this Arf1 mutant restores vesicle release. These experiments show that initial curvature of the bud is defined primarily by coatomer, whereas the membrane curvature modulating activity of dimeric Arf1 is required for membrane scission.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP-Ribosylation Factor 1 / genetics
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ADP-Ribosylation Factor 1 / metabolism*
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Amino Acid Substitution / physiology
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Animals
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Autoantigens / metabolism
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COP-Coated Vesicles / physiology*
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COP-Coated Vesicles / ultrastructure
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Coatomer Protein / metabolism*
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Cross-Linking Reagents / metabolism
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Cross-Linking Reagents / radiation effects
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Cryoelectron Microscopy
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Cysteine / genetics
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Cysteine / metabolism
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Cytoskeletal Proteins
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism
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Golgi Apparatus / pathology
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Golgi Apparatus / physiology
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Guanosine Triphosphate / metabolism
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HeLa Cells
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Humans
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Intracellular Membranes / physiology
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Lipid Bilayers / metabolism
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Membrane Proteins / metabolism
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Microscopy, Electron, Transmission
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Microscopy, Fluorescence
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Mutation / physiology
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N-Acetyllactosamine Synthase / metabolism
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Nuclear Proteins / metabolism
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Photochemical Processes
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Protein Binding / physiology
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Protein Multimerization / physiology
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Rabbits
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Rats
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Rats, Inbred Strains
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Recombinant Fusion Proteins / metabolism
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Transfection
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Unilamellar Liposomes / metabolism
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alpha-Mannosidase / metabolism
Substances
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Autoantigens
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Coatomer Protein
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Cross-Linking Reagents
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Cytoskeletal Proteins
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GTPase-Activating Proteins
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Golgin subfamily A member 2
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Lipid Bilayers
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Membrane Proteins
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Nuclear Proteins
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Recombinant Fusion Proteins
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TRIP11 protein, human
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Unilamellar Liposomes
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cytohesin-2
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Guanosine Triphosphate
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N-Acetyllactosamine Synthase
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alpha-Mannosidase
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ADP-Ribosylation Factor 1
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Cysteine