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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2000-5-22
pubmed:abstractText
We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/ADP-Ribosylation Factors, http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Coatomer Protein, http://linkedlifedata.com/resource/pubmed/chemical/G protein, vesicular stomatitis..., http://linkedlifedata.com/resource/pubmed/chemical/Guanosine 5'-O-(3-Thiotriphosphate), http://linkedlifedata.com/resource/pubmed/chemical/Guanylyl Imidodiphosphate, http://linkedlifedata.com/resource/pubmed/chemical/Membrane Glycoproteins, http://linkedlifedata.com/resource/pubmed/chemical/Membrane Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Phospholipid Transfer Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Sialoglycoproteins, http://linkedlifedata.com/resource/pubmed/chemical/Viral Envelope Proteins
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1046-2023
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:volume
20
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
437-54
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:10720465-ADP-Ribosylation Factors, pubmed-meshheading:10720465-Animals, pubmed-meshheading:10720465-Biological Transport, pubmed-meshheading:10720465-Carrier Proteins, pubmed-meshheading:10720465-Cell-Free System, pubmed-meshheading:10720465-Cells, Cultured, pubmed-meshheading:10720465-Coated Vesicles, pubmed-meshheading:10720465-Coatomer Protein, pubmed-meshheading:10720465-Cytosol, pubmed-meshheading:10720465-Dogs, pubmed-meshheading:10720465-Golgi Apparatus, pubmed-meshheading:10720465-Guanosine 5'-O-(3-Thiotriphosphate), pubmed-meshheading:10720465-Guanylyl Imidodiphosphate, pubmed-meshheading:10720465-Liver, pubmed-meshheading:10720465-Male, pubmed-meshheading:10720465-Membrane Glycoproteins, pubmed-meshheading:10720465-Membrane Proteins, pubmed-meshheading:10720465-Phospholipid Transfer Proteins, pubmed-meshheading:10720465-Rats, pubmed-meshheading:10720465-Rats, Sprague-Dawley, pubmed-meshheading:10720465-Sialoglycoproteins, pubmed-meshheading:10720465-Subcellular Fractions, pubmed-meshheading:10720465-Viral Envelope Proteins
pubmed:year
2000
pubmed:articleTitle
In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein.
pubmed:affiliation
Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.