Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells.
Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.
de Curtis
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European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Howell
K E
KE
Simons
K
K
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000032:analysis / Q000648:ultrastructure*; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000032:analysis
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins