title: Analysis of Golgi complex functions: in vitro reconstitution systems.
abstract: In vitro reconstitution is prerequisite to investigate complex cellular functions at the molecular level. Reconstitution systems range from combining complete cellular cytosol with organelle-enriched membrane fractions to liposomal systems where all components are chemically defined and can be chosen at will. Here, we describe the in vitro reconstitution of COPI-coated vesicles from semi-intact cells. Efficient vesicle formation is achieved by simple incubation of permeabilized cells with the minimal set of coat proteins Arf1 and coatomer, and guanosine trinucleotides. GTP hydrolysis or any mechanical manipulations are not required for efficient COPI vesicle release.
authors.affiliation: Heidelberg University Biochemistry Center (BZH), University of Heidelberg, Im Neuenheimer Feld 328, Heidelberg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Adolf
authors.forename: Felix T
authors.initials: FT
authors.lastname: Wieland
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000502:physiology; D000818:Animals; D001692:Biological Transport; D022181:COP-Coated Vesicles / Q000502:physiology* / Q000648:ultrastructure; D020756:Coatomer Protein / Q000502:physiology; D006056:Golgi Apparatus / Q000502:physiology* / Q000648:ultrastructure; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D011817:Rabbits; D061987:Sf9 Cells
chemical_list: D020756:Coatomer Protein; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1
keywords: COPI; COPII; Semi-intact cells; Vesicle reconstitution; Vesicular transport