A single binding site for dilysine retrieval motifs and p23 within the gamma subunit of coatomer

Abstract

Coatomer, the major component of the coat of COPI transport vesicles, binds both to the dilysine motif of resident membrane proteins of the endoplasmic reticulum and to the cytoplasmic domain of p23, a major type I membrane protein of COPI vesicles. Using a photocrosslinking approach, we find that under native conditions a peptide analogous to the cytoplasmic domain of p23 interacts with coatomer exclusively through its gamma subunit and shares its binding site with a KKXX retrieval motif. However, upon dissociation of coatomer, interaction with various subunits, including an alpha-, beta'-, epsilon-COP subcomplex, of the photoreactive peptide is observed. We suggest that, under physiological conditions, interaction of coatomer with both endoplasmic reticulum retrieval motifs and the cytoplasmic domain of p23 is mediated by gamma-COP.

Figure 1

Peptides used in this study. (A) Peptide sequences of p23wt, p23A, p23AS, and Wbp1p. p23wt represents the cytoplasmic domain of p23 known to bind coatomer. p23A binds coatomer less efficiently than the wild-type peptide, whereas the peptide p23AS has lost this capability. Wbp1p represents the cytoplasmic domain of a subunit of the yeast N-oligosaccharyltransferase complex and contains a characteristic KKXX ER-retrieval motif also known to interact with coatomer. (B) Immobilized, photoreactive p23wt peptide (¹²⁵I-F*-p23wt). The natural Phe at position −8 was replaced by the photoreactive analogue l-4-[3-(trifluoromethyl-3H-diazirin-3-yl)]phenylalanine (F*, Tmd-Phe). F*-p23wt peptide was immobilized by coupling to thiopropyl-Sepharose by a disulfide bond and was radioactively labeled with [¹²⁵I]iodine.

Figure 2

Photocrosslinking of coatomer with immobilized ¹²⁵I-F*-p23wt. Coatomer was incubated with ¹²⁵I-F*-p23wt Sepharose for 90 min on ice prior to irradiation for 2 min on ice. Lanes 1, total photocrosslinked products. Lanes 2–9, samples immunoprecipitated with antibodies against individual COP proteins or the cytoplasmic domain of p23. (A) Immunoblot. (B) Autoradiogram of A. The samples were analyzed by SDS/PAGE, immunoblotting, and autoradiography. COPs were detected with a mixture of antibodies against all seven COPs. Total protein was separated on SDS/7.5–16.5% polyacrylamide gradient gels (lanes 1), α-, β-, β′-, γ-, and δ-COP were separated on 7.5% gels (lanes 2–7); ɛ- and ζ-COP were separated on 15% gels (lanes 8 and 9).

Figure 3

Photocrosslinking of cell lysate with ¹²⁵I-F*-p23wt in solution. CHO cell lysate was incubated with ¹²⁵I-F*-p23wt in solution for 1 h on ice prior to photoactivation for 2 min on ice. (A) Lane 1, immunoblot of total cell lysate analyzed with a mixture of antibodies against all seven coatomer subunits. The asterisk indicates a cross-reactivity of the anti-ɛ-COP antiserum with a protein present in the lysate. Lane 2, immunoblot analysis of coatomer immunoprecipitated with an anti-γ-COP antibody without prior dissociation. Lanes 3–5, immunoblot analysis of β′-, β-, and γ-COPs individually immunoprecipitated (IP) upon dissociation of coatomer by SDS treatment. Samples were separated on 7.5–16.5% (lanes 1 and 2) or 7.5% polyacrylamide gels (lanes 3–5) in the presence of SDS. (B) Autoradiogram of A.

Figure 4

Peptide competition experiments. CHO lysate was incubated with ¹²⁵I-F*-p23wt in solution in the absence (lane 1) or presence (lanes 2, 3, 4, and 5) of a 10-fold excess of nonradioactive peptides, and irradiated. Coatomer was immunoprecipitated with an anti-γ-COP antibody. Samples were separated on SDS/7.5–16.5% polyacrylamide gradient gels. (A) Immunoblot with antibodies against α-ɛ-COPs. (B) Autoradiogram of A.

Figure 5

Photocrosslinking of frozen CHO lysate with ¹²⁵I-F*-p23wt in solution. Incubations and photocrosslinking were performed as described in the legend to Fig. 3 with the exception that lysates used for the experiments shown in lanes 1–3 were frozen in liquid nitrogen, stored at −80°C, and thawed shortly before use. (A) Immunoblot. Lane 1, immunoprecipitation of COPs with an antibody against α-COP; lane 2, against β-COP; lane 3, against γ-COP. Lanes 4–6, immunoprecipitations analogous to those shown in lanes 1–3 but from freshly prepared cell lysates. (B) Autoradiogram of A. Coatomer as a standard, with COPs visualized by immunoblotting.