Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes

Abstract

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large ( approximately 300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5'-[gamma-thio]triphosphate. Coated vesicles are 40-70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5'-[gamma-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.

Figure 1

Formation of COPI-coated vesicles from liposomes consisting of 49 mol % DOPC, 21 mol % DOPE, 8 mol % PI, 8 mol % DOPS, 5 mol % DOPA, 2.2 mol % PIP, 0.8 mol % PIP₂, 2 mol % cytidine 5′-diphosphate–diacylglycerol, 2 mol % NBD- derivative of DOPE, and 2 mol % 1-oleoyl-2-NBD-N-aminododecanoyl-sn-glycero-3-phosphocholine. Liposomes were incubated with combinations of Arf1p, coatomer, and nucleotides. After incubation, membrane morphology was analyzed by conventional electron microscopy. (A) Liposomes were incubated with Arf1Q71L p and GTP-γ-S for 1 h at 30°C, followed by the addition of coatomer and further incubation for 15 min at 4°C. A mixture of large, partially coated and small, vesicle-like coated liposome profiles are present. (Inset) A coated bud-like element on a large liposome. Free coated vesicular profiles also are seen. (B) Liposomes were incubated with GTP-γ-S for 1 h at 30°C and then with coatomer for 15 min at 4°C without Arf1p. No coats are present on the liposome profiles. (C) Liposomes were incubated with Arf1p and GDP-β-S for 1 h at 30°C. After addition of coatomer, the mixture was kept at 4°C for 15 min. The liposome population is uncoated. [Bars = 200 nm (A–C) and = 100 nm (Inset).]

Figure 2

Size distribution of liposomes after incubation with COPI-coat components and nucleotides. Liposomes were incubated either in the presence of Arf1Q71Lp and GTP-γ-S, Arf1p and GDP-β-S, or GTP-γ-S alone for 1 h at 30°C. Coatomer was added to all of the samples and was incubated for 15 min at 4°C. The percentage of coated or uncoated, <70-nm, vesicle-like profiles was evaluated on electron microscopy pictures of 120 liposomes in each condition. The mean diameter of the liposomes after incubation was 136 ± 5nm, 152 ± 5 nm, and 159 ± 6 nm (SEM), respectively.

Figure 3

Enrichment of COPI-coated vesicles. (A) Distribution of lipids on a sucrose-density gradient after a binding reaction followed by centrifugation to equilibrium. (B) COPI-coated vesicles present in fractions 14–16 of the sucrose-density gradient. The mean size of the coated vesicles was 50 ± 16 nm (SD). (Bar = 100 nm.) (Bottom) Gallery of coated vesicles in equatorial section. (Bars = 50 nm.)

Figure 4

Lipid requirement for binding of COPI and COPII components. (A) Liposomes composed of various combinations of phospholipids were tested for binding of COPI proteins in the presence of 0.1 mM GTP-γ-S or 0.1 mM GDP-β-S. Liposomes were floated through a sucrose cushion as described in Materials and Methods. Liposome-bound proteins were separated by SDS/PAGE, were stained with SYPRO-Red, and visualized with a storm860 image analyzer. COPII mix, liposomes used for COPII budding (ref. ; K.M., unpublished material); POPC/POPE, liposomes made of 55 mol % 1-palmitoyl-2-oleoyl-phosphatidylcholine, 44 mol % 1-palmitoyl-2-oleoyl-PE, and 1 mol % of NBD-PE; DOPE/DOPC, liposomes made of 55 mol % DOPC, 44 mol % DOPE, and 1 mol % NBD-PE; DOPC/DOPE/DOPS, liposomes made of 40 mol % DOPC, 44 mol % DOPE, 15 mol % DOPS, and 1 mol % NBD-PE; DOPC/DOPE/DOPA, liposomes made of 40 mol % DOPC, 44 mol % DOPE, 15 mol % DOPA, and 1 mol % NBD-PE; DOPC/DOPE/PI, liposomes made of 40 mol % DOPC, 45 mol % DOPE, and 15 mol % PI from soybean; DOPC/DOPE/PIP, liposomes made of 50 mol % DOPC, 45 mol % DOPE, and 5 mol % PIP from bovine brain; DOPC/DOPE/DOPS, liposomes made of 50 mol % DOPC, 45 mol % DOPE, and 5 mol % PIP2 from bovine brain. (B) Comparison of the binding of coatomer, Arf1p, and COPII proteins to liposomes made of various mixtures of phospholipids. Protein-liposome complexes were recovered from fractions floated in a sucrose density gradient. The yield of Arf1p and δ-COP from incubations conducted in the presence of GTP-γ-S and of Sar1p and Sec23p from incubations containing guanosine 5′-[β,γ- imidylyl]triphosphate are shown.

Figure 5

Arf1p is essential for the formation of COPI-coated vesicles. Liposomes consisting of 40 mol % DOPC, 44 mol % DOPE, 15 mol % DOPA, and 1 mol % NBD-PE were incubated for 1 h at 30°C with Arf1Q71Lp and GTP-γ-S (A) or GTP-γ-S (B). Coatomer was added to both samples, and binding was allowed to occur for 15 min at 30°C and 15 min at 4°C. Profiles of 120 liposomes of each condition were analyzed. In A, coated vesicle-like profiles are mixed with large liposomes showing coated segments, one in the form of a coated bud-like profile (arrow). In this incubation, 27% of the liposomes were <70 nm and were coated; only 1% of <70-nm profiles are uncoated. (B) Mostly large liposomes with loose coats were present. In this incubation, 50% of the >70-nm profiles had coated segments; only 5% of the liposomes were <70 nm, and 80% of those were uncoated. The mean diameter of the liposomes was 145 ± 7 nm in A and 296 ± 14 nm (SEM) in B. The bars in (A and B) represent 200 nm.