Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes.
Coat components localize to specific membrane domains, where they sort selected transmembrane proteins. To study how clathrin coats are stabilized on such domains and to identify the protein networks involved, we combined proteomic screens and in vitro liposome-based assays that recapitulate the fidelity of protein sorting in vivo. Our study identifying approximately 40 proteins on AP-1A-coated liposomes revealed that AP-1A coat assembly triggers the concomitant recruitment of Rac1, its effectors, and the Wave/Scar complex as well as that of Rab11 and Rab14. The coordinated recruitment of these different machineries requires a mosaic of membrane components comprising the GTPase ADP-ribosylation factor 1, sorting signals in selected transmembrane proteins, and phosphatidylinositol 4-phosphate. These results demonstrate that the combinatorial use of low-affinity binding sites present on the same membrane domain accounts not only for a selective coat assembly but also for the coordinated assembly of selected machineries required for actin polymerization and subsequent membrane fusion.
Baust
Thorsten
T
Biotechnological Center, Dresden University of Technology, Germany.
Czupalla
Cornelia
C
Krause
Eberhard
E
Bourel-Bonnet
Line
L
Hoflack
Bernard
B
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology; D011485:Protein Binding; D040901:Proteomics; D013552:Swine; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D008081:Liposomes; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D020691:rab GTP-Binding Proteins