title: Liposome-based assays to study membrane-associated protein networks.
abstract: Transport carriers regulate the bidirectional flow of membrane between the compartments of the secretory and endocytic pathways. Their biogenesis relies on the recruitment of a number of cytosolic proteins and protein complexes on specific membrane microdomains with defined protein and lipid compositions. The timely assembly of these cellular machines onto membranes involves multiple protein-protein and protein-lipid interactions and is necessary to select membrane proteins and lipids into nascent carriers, to bend the flat membrane of the donor compartment, to change the shape of this nascent carrier into a tubular-vesicular structure, and to operate its scission from the donor compartment. A challenge in this field of membrane cell biology has been to identify these machineries and to understand their precise function, in particular by studying their spatial and temporal dynamics during carrier biogenesis. During the past years, liposome-based synthetic biology fully recapitulating the fidelity of carrier biogenesis as seen in vivo has proved to be instrumental to identify these key cytosolic components using mass spectrometry and their dynamics using fluorescence microscopy. We describe here the methods to isolate on synthetic membranes the protein networks needed for carrier biogenesis, to identify them using label-free quantitative proteomics, and to visualize their dynamics on giant unilamellar vesicles.
authors.affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Niehage
authors.affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Stange
authors.affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Anitei
authors.affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany. Electronic address: bernard.hoflack@biotec.tu-dresden.de.
authors.forename: Bernard
authors.initials: B
authors.lastname: Hoflack
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism
chemical_list: D002966:Clathrin; D008081:Liposomes; D058619:Maltose-Binding Proteins; D010455:Peptides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1
keywords: Giant unilamellar vesicles; Imaging; Label-free mass spectrometry; Liposomes; Proteomics; Transport vesicles