A COPI coat subunit interacts directly with an early-Golgi localized Arf exchange factor

Abstract

Arf (ADP-ribosylation factor) family small G proteins are crucial regulators of intracellular transport. The active GTP-bound form of Arf interacts with a set of proteins--effectors--which mediate the downstream signalling events of Arf activation. A well-studied class of Arf1 effectors comprises the coat complexes, such as the cis-Golgi-localized COPI (coat protein complex I) coat, and trans-Golgi network-endosomal clathrin coats. At least five different coats require Arf1-GTP to localize to organelle membranes. How a single Arf protein recruits different coat complexes to distinct membrane sites raises the question of how specificity is achieved. Here, we propose a molecular mechanism of this specificity for the COPI coat by showing a direct and specific interaction between a COPI subunit and a cis-Golgi localized subfamily of Arf guanine nucleotide exchange factors (GEFs) that takes place independently of Arf1 activation. In this way, a specific output on Arf1 activation can be programmed before the exchange reaction by the GEF itself.

Figure 1

Functional and physical interaction between GBF/Gea Arf guanine nucleotide exchange factors and the COPI coat. (A,B) Specificity of recruitment of COPI by GBF1 in vivo. HeLa cells were transfected with short interfering RNAs targeting (A) BIG1+BIG2, or (B) GBF1 and prepared for immunofluorescence analysis using antibodies against GBF1 and β-COP. (C) Yeast two-hybrid interaction between the yeast GBF1 homologue, Gea1p, and yeast γ-COP, Sec21p. Bait plasmid carrying full-length Sec21p was co-transformed into yeast with prey plasmid alone or carrying the indicated regions of Gea1p. Cells were grown on control (+LEU) and selective medium (−LEU) to monitor the expression of the reporter. (D) Lysates from yeast strain CJY104 SEC21∷3xGFP carrying a low-copy plasmid expressing HA-Gea1p, HA-Gea1p(5–754) or HA-Gea1p(5–611), were prepared and incubated with GFP antibodies (+) or IgG alone (−). Bound material was analysed by Western blot using HA antibodies. (E) Same as (D) except CJY104 carried plasmids expressing HA-Gea1p(5–310) or HA-Gea1p(5–223). (F) Summary of co-IPs using indicated truncations of Gea1p. Arf, ADP-ribosylation factor; BIG, brefeldin A-inhibited guanine nucleotide exchange protein; co-IP, co-immunoprecipitation; COPI, coat protein complex I; DCB, dimerization and cycophilin-binding domain; GBF1, Golgi-associated brefeldin A-resistant guanine nucleotide exchange factor 1; Gea, guanine nucleotide exchange on Arf; GFP, green fluorescent protein; HA, haemagglutinin; HUS, homology upstream from Sec7 domain; Sec7, catalytic domain; WT, wild type.

Figure 2

Gea1p interacts with the carboxy-terminal appendage domain of Sec21p. (A) Cell lysates were prepared from strain CJY110 GEA1∷13xMyc carrying plasmids expressing the indicated portions of Sec21p-HA (Sec21p-Nterm: aa 1–665; Sec21p-Cterm: aa 666–953), and immunoprecipitations (IP) were carried out with HA antibody (+) or IgG alone (−). Western blot (WB) analysis was performed using Myc antibody to detect endogenous Myc-tagged Gea1p. (B) Schematic diagram showing regions of Gea1p, Gea2p and Sec7p expressed as GST fusions in Escherichia coli. (C) Western blot analysis of GST fusion constructs expressed in E. coli. (D) The indicated GST fusion constructs were expressed in E. coli, purified on a glutathione-Sepharose column, and Sec21p(666–935)-His6 purified from E. coli was passed over the columns. Eluates were subject to western blot analysis with His6 antibodies. The first lane shows 10% of the input of Sec21p(666–935)-His6. DCB, dimerization and cycophilin-binding domain; Gea, guanine exchange factor on Arf; GST, glutathione S-transferase; HA, haemagglutinin; HUS, homology upstream from Sec7 domain.

Figure 3

The amino-terminal region of human GBF1 interacts with mammalian γ1-COP. Yeast strain BY4742 carrying GBF1(1–662)-2 × HA (A,B) or YEp352-GBF1 (B) and pEG202-LexA-γ1-COP was subjected to immunoprecipitation (IP) analysis as described in Fig 1 with HA or GBF1 antibodies, respectively. Western blot (WB) analysis was carried out using antibodies against LexA. (C) Schematic diagram showing the series of carboxy-terminal truncations of human GBF1 tested for interaction with bovine HA-γ1-COP. (D) Lysates from COS7 cells expressing YFP alone or the indicated human Venus-GBF1 construct and bovine HA-γ1-COP were incubated with GFP antibodies. Immunoprecipitates were subjected to Western blot analysis using HA antibodies. (E) Co-IP experiments as in (D) were carried out with COS7 cells expressing YFP alone or human Venus-GBF1. Immunoprecipitates were subjected to Western blot analysis using β′-COP antibodies to detect endogenous COPI (upper panel). Lysate from cells expressing Venus-GBF1 was analysed by Western blot using GBF1 antibodies to detect YFP-GBF1 and endogenous (End.) GBF1 (lower panel). A representative blot of three independent experiments is shown. (F) Co-IP experiments as in (E) were carried out with COS7 cells expressing YFP alone or human Venus-GBF1, and the immunoprecipitated proteins were detected using GFP antibodies. A representative blot of three independent experiments is shown. (G) Co-IPs were carried out with cells expressing no YFP-tagged protein (untransfected), YFP alone, human Venus-GBF1 or Venus-GBF1-D544A, and endogenous β′-COP was detected in co-IPs using β′-COP antibodies (upper panel). Cells were co-transfected with human HA-γ1-COP and YFP alone, human Venus-GBF1 or Venus-GBF1-D544A, and Western blots were carried out with HA antibodies (lower panel). A representative blot of three independent experiments is shown. (H) The indicated human GBF1 or BIG1 constructs were co-expressed with human HA-γ1-COP in COS7 cells and co-IPs were carried out as in (D). BIG, brefeldin A-inhibited guanine nucleotide exchange protein; co-IP, co-immunoprecipitation; COPI, coat protein complex I; DCB, dimerization and cycophilin-binding domain; GBF1, Golgi-associated brefeldin A-resistant guanine nucleotide exchange factor 1; GFP, green fluorescent protein; HA, haemagglutinin; HUS, homology upstream from Sec7 domain; WT, wild type; YFP, yellow fluorescent protein.

Figure 4

Interaction between Gea1p/GBF1 and γ-COP occurs independently of Arf activation. (A) CJY104 erg6Δ SEC21∷3xGFP cells expressing the indicated Gea1p construct were subjected to immunoprecipitations (IP) as described in Fig 1. Cells were treated with (+) or without (−) 100 μg/ml BFA for 10 min before the preparation of cell lysates, and 100 μg/ml BFA was maintained throughout the rest of the experiment. (B) CJY110 erg6Δ GEA1∷13xMyc expressing YEp352-SEC21-3 × HA cells in the absence or presence of 100 μg/ml BFA were subjected to immunoprecipitation analysis using HA antibodies, and the Western blot was probed with Myc antibodies to detect Gea1p. (C) COS7 cells expressing YFP alone or the Venus-GBF1-D544A and human HA-γ1-COP were treated with or without 25 μg/ml BFA for 5 min before the preparation of cell lysates, and 25 μg/ml BFA was maintained throughout the remainder of the experiment. Immunoprecipitation was performed with GFP antibodies, and Western blot analysis with HA antibodies. (D) Model for the interaction of the COPI coat with GBF/Gea Arf GEFs before Arf activation. Arf1, Arf GEFs and COPI all cycle rapidly between membranes and the cytosol. Receptors (R) such as p23/p24 have been identified for both Arf1-GDP and COPI (Bethune et al, 2006). Once associated with the membrane, Arf1-GDP, GBF/Gea and COPI form a complex. After nucleotide exchange, the Arf GEF is released and active Arf1-GTP stabilizes the association of COPI with the membrane. Arf, ADP-ribosylation factor; BFA, brefeldin A; COPI, coat protein complex I; GBF1, Golgi-associated BFA-resistant GEF1; Gea, guanine nucleotide exchange on Arf; GEF, guanine nucleotide exchange factor; HA, haemagglutinin; WB, Western blot; YFP, yellow fluorescent protein.