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. 1999 Feb 16;96(4):1224-8.
doi: 10.1073/pnas.96.4.1224.

Receptor-induced polymerization of coatomer

C Reinhard  1 , C Harter, M Bremser, B Brügger, K Sohn, J B Helms, F Wieland
Affiliations

Receptor-induced polymerization of coatomer

C Reinhard et al. Proc Natl Acad Sci U S A. .

Abstract

Coatomer, the coat protein complex of COPI vesicles, is involved in the budding of these vesicles, but the underlying mechanism is unknown. Toward a better understanding of this process, the interaction between coatomer and the cytoplasmic domain of a major transmembrane protein of COPI vesicles, p23, was studied. Interaction of coatomer with this peptide domain results in a conformational change and polymerization of the complex in vitro. This changed conformation also is observed in vivo, i.e., on the surface of authentic, isolated COPI vesicles. An average of four peptides was found associated with one coatomer complex after polymerization. Based on these results, we propose a mechanism by which the induced conformational change of coatomer results in its polymerization, and thus drives formation of the bud on the Golgi membrane during biogenesis of a COPI vesicle.

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Figures

Figure 1
Figure 1
Precipitation of coatomer with synthetic peptides corresponding to the COOH termini of p23wt, p23AS and Wbp1p. (A) Peptides used in this study. p23wt represents the cytoplasmic domain of p23, which binds coatomer depending on its dilysine and diphenylalanine motifs, whereas p23AS lacks these residues and therefore does not bind coatomer. Wbp1p represents the cytoplasmic domain of a subunit of the yeast N-oligosaccharyl transferase complex bearing a characteristic KKXX ER-retrieval motif known to bind coatomer. (B and C) Precipitation of coatomer (0.09 μM) with monomeric (B) and dimeric (C) peptides. Only p23wt (■) precipitates the complex, whereas mutated p23 peptide (p23AS, ●) and Wbp1p (▴) have no effect. The error bars indicate standard errors (n = 3).
Figure 2
Figure 2
Limited proteolysis of coatomer. Proteolysis of coatomer (0.09 μM) with thermolysin (0.008 μM) in the presence of dimerized peptides (50 μM) p23wt-d (A), p23AS-d (B), and Wbp1p-d (C). Western blot analysis of proteolytic fragments of the γ-COP subunit reveals a better susceptibility of this subunit to the protease in the presence of p23wt-d (A, lanes 2 and 3) as compared with p23AS-d (B, lanes 2 and 3 h) and Wbp1p-d (C, lanes 2 and 3 h).
Figure 3
Figure 3
Limited proteolysis of COPI vesicles. (A) Suspension of purified COPI vesicles containing ≈0.009 μM coatomer treated with thermolysin (0.009 μM) in the presence of p23AS-d (50 μM) as a substrate for the protease. Western blot analysis of proteolytic fragments of γ-COP reveals a prominent fragment of about 59 kDa (lane 1 h) that is highly susceptible to the protease (lane 2 h). (B) Coatomer (0.009 μM) incubated with p23wt-d (50 μM) and treated with thermolysin under the same conditions as used for the COPI vesicles yields a result comparable to that of COPI vesicles (lanes 1 and 2 h). In contrast, after partial digestion in the presence of p23AS-d (50 μM) of coatomer, the 59-kDa fragment is more stable (C, lanes 1 and 2 h).
Figure 4
Figure 4
Stoichiometry between coatomer and p23wt-d. 125I-labeled p23wt-d (■) or p23AS-d (●) were incubated with increasing concentrations of coatomer. After centrifugation, the amounts of peptide in the precipitates were determined by counting the 125I radioactivity. The error bars indicate standard errors (n = 4).
Figure 5
Figure 5
Size exclusion chromatography of p23-tail peptides. (A) Monomeric p23wt (p23wt-m: 1 mg/ml; sample corresponds to 20 μl). (B) Dimeric p23wt (p23wt-d: 1 mg/ml; sample corresponds to 20 μl). Peak 1 represents the dimerized form of p23wt-d, and peak 2 represents the monomeric form of p23wt-d. (C) Rechromatography of peak 1 of B (40 μl). The appearance of both peaks 1 and 2 demonstrates an equilibrium between two states of p23wt-d. (D) Dimeric p23wt (p23wt-d: 0.1 mg/ml; sample corresponds to 40 μl). The concentration dependence of the equilibrium of peak 1 and 2 indicates a bimolecular event.
Figure 6
Figure 6
Hypothetical model for bud formation. According to this model, interaction of coatomer with a tetramer of p23 induces a conformational change of the complex. This leads to its polymerization on the surface of a membrane, resulting in the formation of a coated bud. (Note that for simplicity, a role of ARF 1 is not included in this diagram.)
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