Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus.
Using a cytosol and nucleotide dependent assay that we previously developed, we have investigated the requirement for coat proteins in the in vitro production of trans-Golgi network (TGN)-derived vesicles from a Madin-Darby canine kidney (MDCK) cell Golgi fraction that contains the 35S-labeled, terminally glycosylated, envelope glycoprotein of vesicular stomatitis virus (VSV-G) accumulated in the TGN. We found that the TGN-derived vesicles, like those involved in intra-Golgi transport and in retrograde transport to the endoplasmic reticulum, contain a coatomer coat and that coatomer is required for their formation. Thus, after they are produced with GTPgammaS, the coated vesicles could be captured on beads containing anticoatomer antibody. Moreover, a cytosolic protein fraction depleted of coatomer could not support vesicle formation but it did so after purified coatomer was added. We also determined that P200/myosin II does not play an essential role in the in vitro generation of TGN-derived vesicles. Thus, removal of this protein from the cytosol, by differential salt precipitation or binding to phalloidin-induced actin filaments, had no effect on vesicle generation. Nevertheless, immunodepletion of cytosol using the anti-P200/myosin II AD7 antibody abolished vesicle generation and that antibody was capable of effectively immunocapturing coated vesicles, even when these were generated in the absence of P200/myosin II. These effects, however, are explained by the unexpected finding that the AD7 antibody interacts with undenatured coatomer.
Simon
J P
JP
Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
Shen
T H
TH
Ivanov
I E
IE
Gravotta
D
D
Morimoto
T
T
Adesnik
M
M
Sabatini
D D
DD
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002460:Cell Line; D018699:Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D020756:Coatomer Protein; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D009218:Myosins / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000906:Antibodies; D020756:Coatomer Protein; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D009218:Myosins