Functional reconstitution of COPI coat assembly and disassembly using chemically defined components - PubMed (original) (raw)

Functional reconstitution of COPI coat assembly and disassembly using chemically defined components

Constanze Reinhard et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Coat protein I (COPI)-coated transport vesicles mediate protein and lipid transport in the early secretory pathway. The basic machinery required for the formation of these transport intermediates has been elucidated based on the reconstitution of COPI-coated vesicle formation from chemically defined liposomes. In this experimental system, the coat components coatomer and GTP-bound ADP-ribosylation factor (ARF), as well as p23 as a membrane-bound receptor for COPI coat proteins, were shown to be both necessary and sufficient to promote COPI-coated vesicle formation. Based on biochemical and ultrastructural analyses, we now demonstrate that the catalytic domain of ARF-GTPase-activating protein (GAP) alone is sufficient to initiate uncoating of liposome-derived COPI-coated vesicles. By contrast, ARF-GAP activity is not required for COPI coat assembly and, therefore, does not seem to represent an essential coat component of COPI vesicles as suggested recently [Yang, J. S., Lee, S. Y., Gao, M., Bourgoin, S., Randazzo, P. A., et al. (2002) J. Cell Biol. 159, 69-78]. Thus, a complete round of COPI coat assembly and disassembly has been reconstituted with purified components defining the core machinery of COPI vesicle biogenesis.

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Figures

Fig. 1.

Fig. 1.

COPI budding from liposomes in the presence or absence of the catalytic domain of ARF-GAP. COPI budding assays using Golgi-like liposomes containing p23-lipopeptide, recombinant ARF1, and coatomer were performed in the presence (B and D) or absence (A and C) of the catalytic domain of ARF-GAP. The experiments shown in A and_B_ were performed in the presence of GTP; those shown in _C_and D are in the presence of GMPPNP. After incubation for 30 min at 37°C, samples were separated by flotation in sucrose density gradients. Fractions 3–13 (see Materials and Methods for details) were separated on SDS gels, followed by immunodetection of β′-COP and ARF1 based on Western blotting. Liposome-derived COPI-coated vesicles migrate in fractions 7–9 corresponding to a sucrose density of ≈40% (wt/wt).

Fig. 2.

Fig. 2.

Postincubation of COPI budding samples with ARF-GAP activity. Liposome-derived COPI-coated vesicles were generated in the presence of GTP as described in the legend of Fig. 1. Thereafter, the sample was split, with one half being mock-treated (A) and the other half incubated with the catalytic domain of ARF-GAP (B). Samples were analyzed by flotation in sucrose density gradients and SDS/PAGE–Western blotting as described in the legend to Fig. 1. For details, see Materials and Methods.

Fig. 3.

Fig. 3.

Uncoating of liposome-derived COPI vesicles depends on ARF1-GTP hydrolysis triggered by the catalytic domain of ARF-GAP. Liposome-derived COPI-coated vesicles were generated as described in the legend of Fig. 1. As indicated, a number of parameters such as the use of GTP versus GMPPNP, ARF1 wild-type versus ARF-Q71L, and temperature (37°C versus 4°C) were varied. Samples were separated by flotation in sucrose density gradients as described in the legend to Fig. 1. Fractions were collected as pool I (donor liposomes), pool II (coated vesicles), and pool III (load) as indicated in A. For each experimental condition shown in_B_, 2.5% of pool I and II as well as 0.5% of pool III were analyzed by SDS/PAGE and Western blotting using anti-β′-COP and anti-ARF1 antibodies as described in the legend to Fig. 1.

Fig. 4.

Fig. 4.

Ultrastructural analysis of ARF-GAP-induced uncoating of COPI-coated vesicles. Liposome-derived COPI-coated vesicles were generated in the presence of GTP as described in the legend of Fig. 1. (A) After isolation by flotation in sucrose density gradients the coated vesicle fraction (pool II, see legend of Fig. 3) was split, with one half being mock-treated and the other half incubated with the catalytic domain of ARF-GAP. Thereafter, coated vesicles were reisolated based on a second flotation gradient that was again fractionated into three pools as described in the legend to Fig. 3. For each experimental condition shown in A, 25% of pool I and II as well as 5% of pool III were analyzed by SDS/PAGE and Western blotting using anti-β′-COP and anti-ARF1 antibodies as described in the legend to Fig. 1. (B) COPI vesicles were generated in the presence of GTP and isolated as described in the legend to Fig. 3. After incubation in the presence or absence of the catalytic domain of ARF-GAP, samples were processed for electron microscopy as described under_Materials and Methods_. The bars in the main panels correspond to 100 nm; the bars in the Insets correspond to 50 nm.

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