Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis.
The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1.GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.
Meyer
Daniel M
DM
Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Crottet
Pascal
P
Maco
Bohumil
B
Degtyar
Elena
E
Cassel
Dan
D
Spiess
Martin
M
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D002966:Clathrin; D020690:GTPase-Activating Proteins; D008081:Liposomes; D021382:Protein Sorting Signals; D018808:Transcription Factor AP-1; D006160:Guanosine Triphosphate