Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes

Abstract

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.

Fig. 1.

Coupling of a lipid anchor to synthetic peptides. (A) Chemical reaction for coupling synthetic peptides to a lipid anchor incorporated into liposomes. (B) Amino acid sequences of cytoplasmic domains of transmembrane proteins used in this study. Note that the gpI tail referred here as wild type (wt) contains a truncation of the C terminus devoid of any trafficking signal (13).

Fig. 2.

AP-1A binding is cargo-specific and requires intact sorting signals. (A) Liposomes with or without gpI or LimpII wild-type tails were incubated with cytosol in the presence of GTP or GTP-γS at 37°C or at 4°C. After incubation, AP-1A, AP-2, AP-3, COP-I, clathrin, and ARF-1 bound to liposomes were detected after SDS/PAGE and Western blotting using specific antibodies. AP-1A (black bars), AP-3 (white bars), AP-2 (dark gray bars), and COP-I (light gray bars) were quantified. (B) Liposomes with wild-type or mutated (Y10A, Y23A, ΔAC, Y10,23A ΔAC) gpI cytoplasmic domains or with glycine (-cd) were incubated at 37°C with cytosol in the presence of GTP-γS. After incubation, AP-1A, AP-3, and ARF-1 bound to liposomes were detected after SDS/PAGE and Western blotting using specific antibodies. AP-1A (black bars), AP-3 (white bars), and ARF-1 (gray bars) were quantified. The charts represent the typical results obtained in four independent experiments.

Fig. 3.

AP-1A binding and phosphoinositides. (A) gpI-liposomes containing various PIPs were incubated with cytosol (3 mg/ml) and GTP-γS. (B) Phosphatidylinositol 3-phosphate, PI-4P, or no PIP was added to liposomes with either gpI or glycine (-cd) and then incubated with cytosol (3 mg/ml) in the presence of GTP-γS. AP-1A, AP-2, AP-3, COP-I, and ARF-1 bound to liposomes were quantified after SDS/PAGE and Western blotting.

Fig. 4.

Proteomics of AP-1A-coated liposomes. Proteins bound to liposomes were fractionated by SDS/PAGE and identified by MALDI-TOF and LC-MS/MS spectrometry. The surface areas on the diagram reflect an estimated stoichiometry between the different machineries (taking AP-1-γ and CYFIP2 as markers) and GTPases obtained after Coomassie blue staining.

Fig. 5.

AP-1A coat assembly stabilizes machineries required for actin nucleation and membrane fusion. Liposomes with or without gpI and with or without PI-4P were incubated with cytosol and GTP-γS. After incubation and purification by flotation gradients, they were analyzed by Western blotting using specific antibodies as indicated. The recruitment of these markers was quantified (white bar, gpI and PI-4P; dark gray bar, gpI no PI-4P; light gray bar, PI-4P and no gpI; black bar, no gpI and no PI-4P).