The assembly of AP-3 adaptor complex-containing clathrin-coated vesicles on synthetic liposomes.
The heterotetrameric adaptor protein complex AP-3 has been shown to function in the sorting of proteins to the endosomal/lysosomal system. However, the mechanism of AP-3 recruitment onto membranes is poorly understood, and it is still uncertain whether AP-3 nucleates clathrin-coated vesicles. Using purified components, we show that AP-3 and clathrin are recruited onto protein-free liposomes and Golgi-enriched membranes by a process that requires ADP-ribosylation factor (ARF) and GTP but no other proteins or nucleotides. The efficiency of recruitment onto the two sources of membranes is comparable and independent of the composition of the liposomes. Clathrin binding occurred in a cooperative manner as a function of the membrane concentration of AP-3. Thin-section electron microscopy of liposomes and Golgi-enriched membranes that had been incubated with AP-3, clathrin, and ARF.GTP showed the presence of clathrin-coated buds and vesicles. These results establish that AP-3-containing clathrin-coated vesicles form in vitro and are consistent with AP-3-dependent protein transport being mediated by clathrin-coated vesicles.
Drake
M T
MT
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Zhu
Y
Y
Kornfeld
S
S
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000138:chemical synthesis* / Q000378:metabolism*; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008055:Lipids; D008081:Liposomes; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D009711:Nucleotides; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C116691:ARF5 protein, human