ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes

Abstract

The assembly of clathrin-coated vesicles on Golgi membranes is initiated by the GTP-binding protein ADP ribosylation factor (ARF), which generates high-affinity membrane-binding sites for the heterotetrameric AP-1 adaptor complex. Once bound, the AP-1 recruits clathrin triskelia, which polymerize to form the coat. We have found that ARF.GTP also recruits AP-1 and clathrin onto protein-free liposomes. The efficiency of this process is modulated by the composition of the liposomes, with phosphatidylserine being the most stimulatory phospholipid. There is also a requirement for cytosolic factor(s) other than ARF. Thin-section electron microscopy shows the presence of clathrin-coated buds and vesicles that resemble those formed in vivo. These results indicate that AP-1-containing clathrin-coated vesicles can form in the absence of integral membrane proteins. Thus, ARF.GTP, appropriate lipids, and cytosolic factor(s) are the minimal components necessary for AP-1 clathrin-coat assembly.

Figure 1

Nucleotide- and temperature-dependent coat recruitment onto liposomes and Golgi-enriched membranes. Liposomes prepared from soybean 20% PC material (200 μg/ml) and Golgi-enriched membranes (50 μg/ml) were incubated with 5 mg/ml gel-filtered bovine adrenal cytosol in the presence or absence of various nucleotides at 1 mM, except for GTPγS, which was at 0.1 mM. Myristoylated ARF1 (mARF1; 4 μM) was added as indicated. Coat proteins were detected by probing with mAb TD.1 for the clathrin heavy chain (CHC; ref. 17), mAb 100/3 for the γ-subunit of AP-1 (γ; ref. 18), mAb 100/2 for the α-subunit of AP-2 (α; ref. 18), mAb M3A5 for β-COP (19), and mAb 1D9 for ARF1 (20). The high ARF background signals in samples with supplemented ARF are caused by nonspecific binding of ARF to the tubes.

Figure 2

AP-1 recruitment onto liposomes requires ARF and is BFA sensitive. (A) Liposomes prepared from soybean 20% PC material (200 μg/ml) and ARF-depleted Golgi membranes (50 μg/ml) were incubated with an AP-1-enriched pool of rat liver cytosol devoid of ARF in the absence or presence of 4 μM ARF and 100 μM GTPγS. The recruitment of the μ1 subunit of AP-1 and ARF was detected by immunoblotting with RY/1 (21) and 1D9, respectively. (B) Liposomes and Golgi-enriched membranes were incubated as in Fig. 1 in the absence or presence of 100 μg/ml BFA and 100 μM GTPγS as indicated. AP-1 binding was detected with mAb 100/3.

Figure 3

Lipid-dependent AP-1 recruitment. (A) Liposomes made from soybean lipid fractions containing 20% and 40% PC were incubated with 5 mg/ml gel-filtered rat liver cytosol in the presence or absence of 100 μM GTPγS. The binding of AP-1 and ARF was detected as in Fig. 2A. (B) Liposomes were prepared from 40% PC soybean material supplemented with 5–20% of PA, PS, or PI and incubated as in A. The binding of AP-1 and ARF was determined by immunoblotting and densitometry and was plotted as percentage of binding relative to the 20% PC liposomes.

Figure 4

(A–C) Coat binding to chemically defined liposomes. Liposomes were made from the combinations of phospholipids described below. Control liposomes (CL) consisted of DOPC/DOPE/cholesterol (50:40:10, wt/wt). The liposomes designated as PA, PS and PI contained 10% PA, PS or PI, respectively, added to CL. The liposomes designated as PI4P and PIP2 contained 5% of each lipid added to CL. The liposomes designated as PIP3 contained 2.5% PIP3 added to CL. The amount of DOPE in these liposomes was adjusted accordingly. SL designates liposome made from soybean 20% PC. The various liposomes were incubated as in Fig. 3. The binding of μ1, β-COP, and ARF was quantitated by immunoblotting and densitometry. Nonspecific binding in the absence of GTPγS was subtracted from the corresponding sample incubated with GTPγS. (D) The CL were supplemented with 5–20% PA and PS, and the binding of AP-1 was determined as above. The values are plotted as the percentage of binding of each protein relative to the 20% PC soybean liposomes.

Figure 5

AP-1 binding to liposomes requires cytosolic factor(s) recruited by ARF⋅GTPγS. (A) ARF-depleted Golgi membranes or liposomes prepared from soybean 20% PC material were incubated with 15 μg/ml soluble rat liver CCV coat fraction with or without ARF1 and GTPγS as noted. AP-1 binding was detected with the RY/1 antibody to μ1. The low level of AP-1 binding to the Golgi membranes in the absence of ARF1 may reflect incomplete depletion of ARF during the preincubation (see Materials and Methods). (A) Liposomes were primed with ARF⋅GTPγS in a first-stage incubation with 4 μM ARF1 and 100 μM GTPγS for 30 min at 37°C as noted. After recovery of the liposomes, either 5 mg/ml rat liver cytosol or 15 μg/ml soluble CCV coat fraction was mixed with the primed liposomes on ice for 15 min as indicated. The binding of AP-1 and ARF was determined by immunoblotting. (C) Liposomes were incubated with 4 μM ARF1 and 100 μM GTPγS, with or without 5 mg/ml AP-1-depleted bovine adrenal cytosol for 15 min at 37°C as noted. The liposomes were recovered and mixed with 15 μg/ml soluble CCV coat fraction with or without AP-1-depleted bovine adrenal cytosol on ice for 15 min. The binding of AP-1 and ARF was determined as above.

Figure 6

Electron-microscopic analysis of clathrin-coat assembly on Golgi membranes and liposomes. (A) Golgi membranes and soybean 20% PC liposomes were incubated with 5 mg/ml bovine adrenal cytosol supplemented with 15 μg/ml soluble CCV coat fraction, 4 μM mARF1, and 100 μM GTPγS as indicated. The recruitment of clathrin (CHC) and AP-1 (μ1) was determined by immunoblotting. (B) Membrane pellets recovered from Golgi membranes and liposomes that had been incubated as above were fixed and processed for electron microscopy as described in Materials and Methods. Golgi membranes incubated with cytosol and coat fraction in the absence (a) and presence (b) of GTPγS. Liposomes incubated with cytosol and coat fraction in the absence (d) or presence (e and f) of GTPγS. (c) Purified CCVs from rat liver. Clathrin-coat assembly occurred only in the presence of GTPγS (b, e, and f). (Bar = 100 nm.)