Biochemical dissection of AP-1 recruitment onto Golgi membranes

L. M. Traub, J. A. Ostrom, S. Kornfeld

Research output: Contribution to journalArticle

245 Scopus citations

Abstract

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 GTPγS, 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°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 GTPγS-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.

Original languageEnglish
Pages (from-to)561-573
Number of pages13
JournalJournal of Cell Biology
Volume123
Issue number3
DOIs
StatePublished - Nov 9 1993

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