ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes - PubMed (original) (raw)

ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes

Pascal Crottet et al. Mol Biol Cell. 2002 Oct.

Abstract

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.

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Figures

Figure 1

Figure 1

Peptidoliposomes to assay AP-1 recruitment in vitro. The maleimide derivative of PE MMCC-DHPE was used to couple synthetic peptides via an N-terminal cysteine to a lipid (A). The peptides used correspond to the cytoplasmic domain of Lamp1 (B, Lamp1Y) or the segment of TGN38 that has previously been shown to contain the functional tyrosine motif (Boll et al., 1996). Lamp1A and TGN38A are the control peptides with the critical tyrosine mutated to alanine. After incubation of peptidoliposomes with AP-1 and with or without ARF1, they were floated from the bottom of a sucrose step gradient (C). Four fractions were collected as indicated, with fraction I containing the floated liposomes with bound proteins and fraction IV including the loading zone with unbound proteins.

Figure 2

Figure 2

AP-1 recruitment to peptidoliposomes is signal-, ARF1- and lipid-dependent. (A) Peptidoliposomes made of 100% PC or 50% PC/50% soybean lipids and presenting Lamp1Y or Lamp1A peptides were incubated with a mixed adaptor preparation and with or without myristoylated ARF1 and GMP-PNP. After flotation on a sucrose step gradient, four fractions (I–IV, as shown in Figure 1C) were collected from the top and analyzed by immunoblotting for γ-adaptin or ARF1. (B) The same experiments were performed using peptidoliposomes made of 50% PC/50% soybean lipids and presenting TGN38Y or TGN38A peptides.

Figure 3

Figure 3

Lipid requirement for AP-1 recruitment to peptidoliposomes. (A) Three percent of the indicated lipid was incorporated into PC peptidoliposomes exposing Lamp1Y. After incubation with a mixed adaptor preparation and with myristoylated ARF1·GMP-PNP, fractions I and IV of a flotation gradient were analyzed by immunoblotting. PIPs indicates a commercial mixture of phosphoinositides. (B) Two percent of PI-monophosphates and 1% of PI-bis- and trisphosphates were incorporated into PC peptidoliposomes exposing Lamp1Y and analyzed as in A. (C) The recruitment of AP-1 and ARF1 to liposomes containing different phosphoinositides (2% of PI-monophosphates and 1% of PI-bis- and trisphosphates) were densitometrically quantified. The amount recovered in fraction I is expressed in percent of the total in fractions I plus IV. The average and SDs of at least three experiments, including those shown in B, are presented.

Figure 4

Figure 4

Nucleotide dependence of AP-1 recruitment to peptidoliposomes. The indicated nucleotide was incubated with myristoylated or nonmyristoylated ARF1 and peptidoliposomes containing 3% of mixed inositides and exposing Lamp1Y. The analysis was performed as in Figure 3.

Figure 5

Figure 5

Nucleotide exchange on ARF1. Myristoylated ARF1 was incubated at 37°C with [35S]GTPγS and either buffer only (▴), PC liposomes (●), or PC with 10% mixed phosphoinositides (□). At the indicated times, samples were quickly filtered through a nitrocellulose filter. After washing, the radioactivity on the filter, corresponding to GTPγS bound to ARF1, was counted.

Figure 6

Figure 6

Recruitment of pure AP-1 to peptidoliposomes. (A) Aliquots of the mixed adaptor preparation (lane 1) and of hydroxyapatite-purified AP-1 (lane 2) containing the same amount of AP-1 (as judged by immunoblot analysis) were separated by SDS-gel electrophoresis and stained with silver. AP-1 subunits β1, γ, and μ1 are indicated by filled arrowheads, whereas AP180 and AP-2 subunits αa, αc, β2, and μ2 are indicated by open arrowheads. (B) AP-1 recruitment assays were performed using liposomes made of PC with or without 1% PI(4,5)P2 and exposing Lamp1Y (LY) or Lamp1A (LA) peptides in the presence of myristoylated ARF1 loaded with GMP-PNP or GDP. The analysis was performed as in Figure 3.

Figure 7

Figure 7

Recruitment of AP-1 from cytosol. AP-1 recruitment assays were performed using brain cytosol (A) or adrenal gland cytosol (B), and peptidoliposomes made of soybean lipids (lanes 1–4), PC with 1% PI(4,5)P2 (lanes 5–8), or pure PC (lanes 9–12), exposing Lamp1Y (LY) or Lamp1A (LA) peptides. Cytosol supplemented with purified ARF1 and GMP-PNP was incubated with the peptidoliposomes for 30 min at 37°C before separation by gradient centrifugation. (C) To determine the kinetics, AP-1 recruitment assays were performed using brain cytosol and liposomes exposing Lamp1Y peptides prepared of either PC alone (white bars) or PC containing 1% PI(4,5)P2 (dark bars) at different incubation times (average and SD of 3 determinations).

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