The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.
We have established systems that reconstitute the biogenesis of coated transport vesicles with liposomes made of pure lipids and purified coat proteins. Optimization of the lipid composition in the liposomes allowed the efficient binding of both coat protein I and coat protein II (COPII) coat subunits. Coated vesicles of approximately the size generated from biomembranes were detected and characterized by centrifugation analysis and electron microscopy. A variation of this budding reaction allowed us to measure the sorting of v-SNARE proteins into synthetic COPII vesicles. We developed a novel system to tether glutathione S-transferase (GST)-hybrid proteins to the surface of liposomes formulated with a glutathione-derivatized phospholipid. This system allowed us to detect the positive role of cytoplasmic domains of two v-SNARE proteins that are packaged into COPII vesicles. Therefore, both generation of coated vesicles and protein sorting into the vesicles can be reproduced with liposomes and purified proteins.
Matsuoka
K
K
Department of Molecular and Cell Biology, Howerd Hughes Medical Institute, University of California, Berkeley, CA 94706, USA.
Schekman
R
R
D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D020755:Coat Protein Complex I / Q000378:metabolism*; D018699:Coated Vesicles / Q000378:metabolism*; D004337:Drug Carriers; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D020755:Coat Protein Complex I; D004337:Drug Carriers; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C106113:SEC31 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase; D005978:Glutathione