# 2007 The Authors Journal compilation # 2007 Blackwell Publishing Ltd Traffic 2007; 8: 582–593 Blackwell Munksgaard doi: 10.1111/j.1600-0854.2007.00554.x Multiple and Stepwise Interactions Between Coatomer and ADP-Ribosylation Factor-1 (Arf1)-GTP Zhe Sun1,†, Frank Anderl1,†, Kathrin Fro¨ hlich1, suggesting that these complexes are comprised of Liyun Zhao1, Stefan Hanke2, Britta Bru¨ gger1, conserved structural elements (13). The structure of the Felix Wieland1,*, Julien Be´ thune1,* core complexes of adaptor protein (AP) complex 1 and 2 (AP-1 and AP-2) were solved by X-ray crystallography 1Biochemie-Zentrum der Universita¨t Heidelberg (BZH), (14,15). Two 100-kDa subunits are built of a trunk domain Im Neuenheimer Feld 328, D-69120 Heidelberg, connected via a flexible linker to a smaller appendage Germany domain. The trunk domains together with the smaller sub- 2 Max-Planck-Institute for Biochemistry, Am Klopferspitz units m and s form a densely packed core that contains the 18, D-82152 Martinsried, Germany sites for specific interactions with a donor membrane. The †These authors contributed equally to this work coatomer subunits b and g together with d and z are likely to *Corresponding author: Julien Be´thune, [email protected] or Felix Wieland, represent a similar core of coatomer. Structural homology [email protected] was corroborated for the appendage domains of a2and b-adaptin and g-COP by X-ray crystallography (16,17). The small GTPase ADP-ribosylation factor-1 (Arf1) plays Formation of a vesicle includes binding to the donor mem- a key role in the formation of coat protein I (COP I)-coated brane of coat proteins and subsequent membrane deforma- vesicles. Upon recruitment to the donor Golgi membrane tion to sculpt out a bud. In the clathrin system, this is by interaction with dimeric p24 proteins, Arf1’s GDP is achieved by an Arf-GTP-dependent two-step process: bind- exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it ing of an adaptor complex to the GTPase followed by recruits coatomer, the heptameric coat protein complex recruitment of clathrin heavy and light chains. In contrast, of COP I vesicles, from the cytosol. In this process, Arf1 COP I vesicles are formed by an Arf-GTP-dependent one- was shown to specifically interact with the coatomer b step recruitment of coatomer, implying that both adaptor and and g-COP subunits through its switch I region, and with clathrin functions are unified in a single coatomer complex. e-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of b and g-COP. Site- We previously used an in vitro translation technique to directed photolabeling at position 167 in the C-terminal introduce photolabile amino acids at defined positions in helix of Arf1 revealed a novel interaction with coatomer Arf1. Through the use of these photolabile Arf1 derivatives, via a putative longin domain of d-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with we identified the coatomer subunits b-COP and g-COP as membrane-bound Arf1-GTP. These interactions are binding partners for the switch I region of Arf1-GTP located within the core, adaptor-like domain of coatomer, (18,19). In these functional binding assays with myristoy- indicating an organizational similarity between the COP I lated full-length Arf1 derivatives, Golgi enriched mem- coat and clathrin adaptor complexes. branes and coatomer, these GTP-dependent interactions were found after recruitment of coatomer to the mem- Key words: coat protein I, Golgi, membrane traffic, small branes, as well as in isolated COP I vesicles, indicating GTPase, vesicular transport their functional relevance. Another study described binding Received 19 September 2006, revised and accepted for of Arf1-GTP to b-COP and e-COP in a two-hybrid assay publication 8 February 2007, published online 26 March (20). In the clathrin system, GTP-dependent interaction of 2007 Arf1 through its switch I region with the adaptor com- plexes AP-1, 3 and 4, was attributed to the trunk domains of the larger adaptins. Coat protein I (COP I) vesicles mediate lipid and protein transport in the early secretory pathway of eukaryotic cells. In this study, we have extended our Arf1 photo-cross- Coatomer (1–3), the protein coat of these vesicles, is linking studies to map its interactions with coatomer on the recruited to Golgi membranes by the small, 20-kDa, Ras- Golgi membrane. We report that activated Arf1 binds the like GTPase Arf1 in its GTP-bound form (4,5). Inactivation trunk domains of b-COP and g-COP. In addition, we of Arf1 through GTP hydrolysis is sufficient to release describe two novel interactions between Arf1 and coat- coatomer from membranes and thus promotes vesicle omer: one involves a putative longin domain of d-COP and uncoating (6–8). Similarly, Arf1 plays a critical role in the the other the b0-COP subunit. These results underline recruitment of the clathrin adaptor complexes AP-1 (9), AP- structural and functional similarities between clathrin 3 (10) and AP-4 (11). Subunits of coatomer and clathrin adaptor complexes and coatomer and predict a potential adaptor complexes share weak sequence homology (12), interaction of adaptor m-chains with Arf1-GTP. In addition, 582 www.traffic.dk Arf1 and Coatomer Interactions they show differences that might relate to the mechanistic differences in vesicle formation that exist between the clathrin and the COP I systems. Results Expression of Arf1 proteins with site-directed photolabile amino acids Photolabile amino acid derivatives within polypeptides facilitate the identification of specific protein–protein inter- actions. Upon UV irradiation, highly reactive species are generated that covalently cross-link to binding partners within 3-A˚ distance (21). Until recently, generation of site- directed photolabile proteins was dependent on chemical synthesis or in vitro translation systems. Hence, photo- labile proteins were available in very limited quantities, precluding proteomic analysis and restricting identification of cross-link partners to Western blotting for known candidates. Novel binding partners could not be identified using these methods. Recently, an in vivo expression system was described that allows the production of large amounts of photolabile proteins in Escherichia coli using Figure 1: Expression of site-directed photolabile human Arf1 a benzophenone (Bp) amino acid derivative (22). We have proteins in E. coli. A) Sites within Arf1 where photolabile amino adapted this system to produce 16 site-directed photo- acid derivatives were introduced. SW1: switch I region, ISW: labile derivatives of full-length, myristoylated Arf1. Three of interswitch region, SW2: switch II region. The three labeled them could be cross-linked to putative binding partners and positions indicate the derivatives that yielded cross-linked inter- were characterized. In these derivatives, Bp residues action partners and are characterized in the present study. B) replaced amino acid positions I46 and I49 in the switch I Expression of wild-type Arf1 (WT) and photolabile derivatives region, and Y167 in the C-terminus (Figure 1A); each was (lane 2, 3) or was not (lane 1) induced at 278C by arabinose (Ara.) in the presence (lane 3) or absence (lanes 1 and 2) of derivative was obtained in up to microgram quantities p-benzoyl-L-phenylalanine (Bp). Expression products were ana- (Figure 1B). lyzed by Western blotting with an anti-Arf1 (C-terminus) antibody. Note that when produced in E. coli, Arf1 has a mixture of The photoactivable derivatives behave like myristoylated (myr.) and non-myristoylated (nonmyr.) forms (47). wild-type Arf1 For COP I-vesicles formation, Arf1 in its GDP bound form is transiently recruited by dimeric p23 protein to the Golgi Golgi membranes, photolabile Arf1-I46Bp, I49Bp and membrane (23–25). Thereafter, exchange of GDP for GTP Y167Bp were incubated with Golgi membranes and is catalyzed by a guanine nucleotide exchange factor coatomer in the presence of GDPbS or GTPgS, a non- (26,27), and the activated GTP-bound Arf1 is stably asso- hydrolyzable GTP analog. Membranes were then separated ciated with the membrane. As a result, about threefold from unbound material by centrifugation and irradiated at more Arf1-GTP is bound to Golgi membranes than Arf1- 366 nm to drive photo-cross-linking of Bp residues. Analysis GDP (28). by SDS–PAGE and Western blotting with antibodies against Arf1 and coatomer subunits revealed the known interac- To test their functionality, we assessed nucleotide- tions of Arf1 with b-COP and g-COP (Figure 3A and B, lane dependent binding of the Arf1 photolabile proteins to Golgi 4, as indicated). The lower band (Figure 3B, lane 4) repre- membranes. For all Arf1 species, replacement of GDP with sents Arf1 covalently linked to g2-COP, an isotype of g-COP GTP caused a threefold enhancement of Golgi membrane (29,30). Three additional Arf1 complexes were observed binding (Figure 2). This ratio is in very good agreement (Figure 3A and C, indicated as Arf1-I46 þ x, Arf1-I46 þ y, with the previous quantification of nucleotide-dependent Arf1-Y167 þ z) with apparent molecular masses of about binding of Arf1 to Golgi membranes using radiolabeled 150 kDa (Arf1 þ partner y), 130 kDa (Arf1 þ partner x) and Arf1 (28). Thus these photolabile Arf1 species were then 80 kDa (Arf1 þ partner z). used to map the binding sites on b-COP and g-COP and to search for additional binding partners. Arf1-GTP interacts with the trunk domains of b-COP and g-COP GTP-dependent interactions of Arf1 with coatomer Cross-linking and two-hybrid experiments have revealed To probe the interfaces of Arf1 with coatomer under that Arf1-GTP binds clathrin adaptor complexes through functional conditions, i.e.