Biochemical dissection of AP-1 recruitment onto Golgi membranes.
Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.
Traub
L M
LM
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Ostrom
J A
JA
Kornfeld
S
S
D000818:Animals; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014357:Trypsin
D006150:Guanine Nucleotides; D016755:Proto-Oncogene Proteins c-jun; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014357:Trypsin