Coat assembly directs v-SNARE concentration into synthetic COPII vesicles.
COPII proteins are required to create transport vesicles and to select cargo molecules for transit from the ER. A reconstituted liposome budding reaction was used to detect the capture and concentration of membrane-associated v-SNARE molecules into synthetic COPII vesicles. A novel glutathione-phosphatidyl-ethanolamine conjugate (Glut-PE) was synthesized and incorporated into chemically defined liposomes to provide binding sites for GST hybrid proteins. Large liposomes containing bound cytoplasmic domains of the v-SNAREs, Sec22p or Bos1p, or of the ER resident proteins, Sec12p and Ufe1p, were exposed to COPII proteins and GMP-PNP. v-SNAREs but not resident proteins were concentrated in synthetic COPII vesicles generated from donor liposomes. We conclude that COPII proteins are necessary and sufficient for cargo selection and vesicle morphogenesis.
Matsuoka
K
K
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720, USA.
Morimitsu
Y
Y
Uchida
K
K
Schekman
R
R
D001692:Biological Transport; D002352:Carrier Proteins / Q000502:physiology*; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D018699:Coated Vesicles / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003593:Cytoplasm; D005656:Fungal Proteins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes / Q000138:chemical synthesis / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D016339:Spectrometry, Mass, Fast Atom Bombardment; D033921:Vesicular Transport Proteins
C068037:BOS1 protein, S cerevisiae; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D010750:Phosphoproteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C071239:SEC12 protein, S cerevisiae; C106113:SEC31 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C106909:Sec22 protein, S cerevisiae; C103868:UFE1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase; D005978:Glutathione