title: Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling.
abstract: Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.
authors.affiliation: Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany.
authors.forename: Jun-Yong
authors.initials: JY
authors.lastname: Jia
authors.forename: Stephanie
authors.initials: S
authors.lastname: Lamer
authors.forename: Michael
authors.initials: M
authors.lastname: Schümann
authors.forename: Michael R
authors.initials: MR
authors.lastname: Schmidt
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D001923:Brain Chemistry; D002772:Cholera Toxin / Q000032:analysis; D002784:Cholesterol / Q000737:chemistry*; D002853:Chromatography, Liquid; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000648:ultrastructure; D017830:Octoxynol / Q000737:chemistry / Q000494:pharmacology; D040901:Proteomics; D051381:Rats; D013570:Synaptic Membranes / Q000737:chemistry* / Q000187:drug effects; D013572:Synaptic Vesicles / Q000737:chemistry / Q000648:ultrastructure*; D053719:Tandem Mass Spectrometry; D033921:Vesicular Transport Proteins / Q000032:analysis*
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D017830:Octoxynol; D002772:Cholera Toxin; D002784:Cholesterol