COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI.
ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.
Rowe
T
T
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Aridor
M
M
McCaffery
J M
JM
Plutner
H
H
Nuoffer
C
C
Balch
W E
WE
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure; D018699:Coated Vesicles / Q000737:chemistry* / Q000378:metabolism; D020756:Coatomer Protein; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D018189:Immunomagnetic Separation; D007668:Kidney / Q000166:cytology; D008322:Mammals; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D008854:Microscopy, Electron; D008861:Microsomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D020559:Monomeric GTP-Binding Proteins; D009154:Mutation / Q000502:physiology; D011817:Rabbits; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000097682:Lamin B Receptor
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D002352:Carrier Proteins; D020756:Coatomer Protein; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate; D008565:Membrane Proteins; D020559:Monomeric GTP-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000097682:Lamin B Receptor; C077395:SAR1 protein, S cerevisiae