ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes.
At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation.
Crottet
Pascal
P
Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Meyer
Daniel M
DM
Rohrer
Jack
J
Spiess
Martin
M
D020823:ADP-Ribosylation Factor 1 / Q000302:isolation & purification / Q000378:metabolism*; D033961:Adaptor Protein Complex 1 / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D008055:Lipids; D008081:Liposomes; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D014443:Tyrosine; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1