Multiple and Stepwise Interactions Between Coatomer and ADP-Ribosylation Factor-1 (Arf1)-GTP

# 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 complexes 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 interactions. 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, photolabile 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. activated, GTP-loaded Arf1 on the trunk domains of AP-1, AP-3 (31) and AP-4 (11). Here,

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Figure 2: Photolabile Arf1 derivatives behave like wild-type Arf1 in a Golgi-binding experiment. Arf1-WT or photolabile derivatives were incubated with Golgi membranes in the presence of GDPbS (lane 1) or GTPgS (lane 2) and the membranes were then isolated by centrifugation. Membrane- bound Arf1 was detected by Western blotting with an anti-Arf1 (C-terminus) antibody. The signals detected for Golgi-bound Arf1-GDPbS and Arf1- GTPgS were quantified with the soft- ware UN-SCAN-IT gel (Silk Scientific) from three independent experiments. For wild-type and photoactive derivatives, the ratio between the two Arf1 forms is indicated on the right. The error bars represent the standard error of the mean. we have used limited proteolysis combined with Western with activated Arf1 through its trunk domain, and not its blotting to analyze the domains of b-COP and g-COP that appendage domain (Figure 5). interact with Arf1-GTP. In Figure 4, analysis of b-COP is depicted. After irradiation to induce covalent cross-linking, Golgi membranes were incubated with the polyspecific Novel interaction of Arf1-GTP with d-COP protease thermolysin and analyzed by Western blotting A novel product was observed upon irradiation of Arf1- with antibodies against Arf1 (Figure 4, upper panel). At low Y167Bp-GTP (Figure 3C, partner z). We used an immuno- protease concentration, the cross-link products seemed logic approach combined with mass spectrometry to unaffected (lane 4), whereas time-dependent degradation characterize the proteins cross-linked with this species of was observed at a higher protease concentration (lanes 6, Arf1-GTP. To this end, we upscaled the reaction and, after 8 and 10). A stable degradation product was observed with irradiation, Arf1-Y167Bp and cross-linked products were an apparent molecular mass of 70 kDa, a size that would isolated by immunoprecipitation with an anti-Arf1 antibody. be expected for a covalent adduct of the b-COP trunk Proteins were separated by SDS–PAGE and detected by domain with Arf1. Decoration with an antibody against the Coomassie staining (not shown). The region corresponding appendage domain of b-COP revealed, at high protease to 80 kDa was analyzed by mass spectrometry in both concentration, a protein fragment of 40 kDa, indicating irradiated and non-irradiated samples. As shown in a non-cross-linked appendage domain of b-COP (Figure 4, Table 1, the only proteins unique to the irradiated sample middle panel, lanes 5–10). In contrast, immunodetection are Arf1 and d-COP. The predicted apparent molecular with an antibody directed against the b-COP trunk domain mass of this pair (79 kDa) is consistent with their migration (Figure 4, lower panel) revealed a non-cross-linked trunk behavior on the gel. This previously unreported binding of domain at 55 kDa and an additional trunk domain of d-COP to Arf1 was further analyzed using recombinant higher apparent molecular mass, consistent with a cross- GST-tagged d-COP and ND17-Arf1, a truncated recombi- link to Arf1 (lanes 6, 8 and 10). This band co-migrates with nant version of Arf1 lacking the N-terminal a-helix and thus the Arf1 band in the upper panel of Figure 4, lanes 6, 8 and 10. soluble in both GDP- and GTP-bound forms. The two well- characterized ND17-Arf1 mutants Q71L and T31N were The interaction of g-COP with Arf1-I49Bp-GTP was ana- used: ND17-Arf1-Q71L is always bound to GTP as it is lyzed in a similar manner. Like b-COP, g-COP interacts unable to hydrolyze GTP and ND17-Arf1-T31N is locked in

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Figure 3: GTP-dependent interactions between Arf1 and coatomer. Arf1 photolabile derivatives (Arf1-I46-Bp in A; Arf1-I49-Bp in B; Arf1-Y167-Bp in C) were incubated in the presence of Golgi membranes and coatomer, with GDPbS (lanes 1 and 2 in A–C) or GTPgS (lanes 3–6 in A; lanes 3–8 in B; lanes 3 and 4 in C). Samples were then centrifuged to isolate membranes which were then resuspended in reaction buffer and UV irradiated (lanes 2, 4 and 6 in A; lanes 2, 4, 6 and 8 in B; lanes 2 and 4 in C). Photo-cross-linked products were analyzed by Western blotting with anti-Arf1 antibodies (lanes 1–4 in A–C), anti-b-COP antibodies (lanes 5 and 6 in A), anti-g1-COP antibodies (lanes 5 and 6 in B), or the antibody g-r (which recognizes both g1-COP and g2-COP; lanes 7 and 8 in B). Previously characterized cross-linked products are indicated. Arf1-I46-Bp gives rise to two previously unknown adducts (labeled as Arf1-I46 þ x and Arf1-I46 þ y). Arf1-Y167-Bp can be cross-linked to an unknown partner labeled as Arf1-Y167 þ z. the GDP bound form. Immobilized GST-d-COP was able to which d-COP is proteolytically nicked to a stable fragment specifically pull down ND17-Arf1 but in contrast to the of 31 kDa. This d-COP fragment is still part of a coatomer cross-linking assay where d-COP is part of the coatomer complex, as it is present in a coatomer sample immuno- complex, the interaction of the isolated d-COP with Arf1 is precipitated with an antibody directed against another COP not nucleotide dependent (Figure 6). This indicates that subunit (Figure 7A, lanes 1 and 2). Therefore, rather than the binding site for Arf1 on d-COP is not restricted to Arf1- cytosol as a source of coatomer, samples of the purified GTP per se. coatomer complex were further investigated. Incubation of Arf1-Y167Bp with Golgi membranes and purified coat- Further mapping of the interaction between Arf1-Y167Bp omer, followed by irradiation, gave rise to two bands, and d-COP was facilitated by our observation that coat- one which corresponds to Arf1-GTP cross-linked to full- omer purifies in two forms: one that is intact and one in length d-COP (at 80 kDa), and another at 50 kDa

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Figure 4: Arf1 directly interacts through its SWI region with the trunk domains of b-COP. Binding of Arf1-I46-Bp to coatomer subunits and photo-cross-linking was performed as described in Figure 3. Samples without UV irradiation were treated as negative controls (lanes 1, 3, 5, 7 and 9). Samples were incubated with thermolysin as indicated, and analyzed by Western blotting with the anti-Arf1 (upper panel), anti-b-COP appendage domain (middle panel; anti-b-COP appendage) or anti-b-COP trunk domain antibodies (lower panel; anti-b-COP trunk).

Figure 5: Arf1 directly interacts through its SWI region with the trunk domains of g-COP. Same experiment as in Fig. 4 with Arf1-I49-Bp. The interaction between Arf1-I49-Bp and the g-COPI trunk domain is demonstrated by Western blotting with the anti-Arf1, g-r (recognizes trunk domains of g-COPs) and anti-g-COP appendage antibodies. Asterisks indicate bands that non-specifically cross-react with the antibodies.

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Table 1: Proteins identified in the non-irradiated and the irradiated which showed a UV-dependent mass shift for b0-COP samples upon incubation of coatomer with Golgi membranes and Arf1-I46Bp-GTPgS (Figure 8). The small amount of cross- UV þUV linked material has thus far prevented us from mapping Ig gamma chain C-region Ig gamma chain C-region more precisely the interaction site on b0-COP. Ig gamma heavy chain Ig gamma heavy chain constant region – rabbit constant region – rabbit The second additional band observed by photo-cross- Ig kappa chain Ig kappa chain linking of Arf1-I46Bp corresponded to a product of 150 ADP-ribosylation factor kDa (Figure 3A, indicated as Arf1-I46 þ y). This material Coatomer delta subunit was electroeluted from the gel and resolved on a gel with a larger pore size for a further purification (see Materials and Methods section). Mass spectrometry analysis of this (Figure 7A, lanes 3–7). This band was recognized by both band yielded peptides corresponding to Arf1 and b-COP the anti-Arf1 antibody (Figure 7A, lane 4) and the antibody (Table 3). The apparent molecular mass of this covalent against d-COP (Figure 7A, lane 7). The d-COP fragment product suggests that b-COP is exclusively bound to two that gave rise to this band was analyzed by mass spec- Arf1-GTP molecules. This was corroborated by the fact trometry. As shown in Figure 7B, a comparison of full- that this species was also observed after denaturation of length d-COP with the sequence of its 31-kDa degradation cross-linked samples and immunoprecipitation with anti- product shows that Arf1 binds the N-terminus of d-COP. bodies against either b-COP or Arf1, but not with antibodies against other coatomer subunits. Furthermore, in Interactions of Arf1-GTP with b0-COP and b-COP the mapping experiment depicted in Figure 4, after limited Upon incubation with Golgi membranes and cytosol, and proteolysis, an additional band appeared at 90 kDa after subsequent UV irradiation, Arf1-I46Bp produced two addi- longer exposure time, in fair agreement with a trunk tional cross-linked products (running at 130 and 150 kDa) domain cross-linked to two Arf1-GTP molecules. Taken of apparent higher molecular mass than Arf1 cross-linked together, these observations indicate that more than one to b-COP (Figure 3A, indicated as Arf1-I46 þ x and Arf1-I46 molecule of Arf1-GTP can bind to coatomer. þ y). To identify the 130-kDa band (Arf1-I46 þ x), we followed the protocol described above for the Arf1/d–COP Contribution of I46, I49 and Y167 to the binding of cross-linked product, by upscaling the size of the reaction, Arf1 to coatomer immunoprecipitation with the antibody directed against To estimate the contribution to the binding to coatomer of Arf1, and detection by Coomassie staining after separation each of the three sites in Arf1, the amino acids at positions by SDS–PAGE. Analysis by mass spectrometry of the 46, 49 and 167 were exchanged for alanines, and the material running at 130 kDa region led to the identifica- resulting Arf1 variants analyzed in a coatomer recruitment tion of peptides from Arf1 and the b0-COP subunit of assay on Golgi membranes. As a result, single Arf1 mutants coatomer in the irradiated but not in the non-irradiated (Arf1-I46A, Arf1-I49A and Arf1-Y167A) showed a mildly sample (Table 2). This was subsequently confirmed by reduced ability to recruit coatomer to membrane (Figure 9), Western blotting analysis with an anti-b0-COP antibody, while an Arf1 variant with all three positions mutated to alanines showed a more pronounced reduction of coatomer binding. This suggests that all three interfaces occur syner- gistically during coatomer recruitment to Golgi membranes. As expected, none of the Arf1 variants could abolish coatomer binding completely, indicating that these individual positions represent only parts of binding interfaces. These data do not, however, completely rule out the possibility of a sequential binding to coatomer of the three sites.

Discussion

The basic mechanisms of membrane deformation that lead to the formation of transport vesicles are still poorly Figure 6: Recombinant d-COP binds to Arf1 regardless of its understood. The goal of our investigations is to understand nucleotide state. Recombinant GST-tagged d-COP was coupled how coat proteins are recruited to and polymerize on to glutathione sepharose beads and incubated with ND17-Arf1- membranes to form COP I vesicles. Arf1 is a key player Q71L (GTP-bound) or ND17-Arf1-T31N (GDP bound) as indicated; empty beads were used as a negative control. After extensive in the recruitment of the coat protein complex coatomer washing the samples were analyzed by Western blot with anti- to Golgi membranes, and thus a detailed characterization bodies directed against d-COP or Arf1. Beads coupled to GST of this interaction will provide significant insight into the showed no binding to Arf1 (not shown). mechanism of coat assembly. In this study, we used

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Figure 7: Arf1-Y167-Bp interacts directly with an N-terminal 31-kD fragment of d-COP. A) Rabbit liver coatomer was purified by immunoprecipitation under native conditions with antibodies directed against b0-COP. The immunoprecipitated product was separated by SDS-PAGE and visualized by Coomassie staining (lane 1). Part of the sample was run simultaneously on SDS–PAGE and analyzed by Western blotting. The band labeled as d-COP and the band marked by an arrow correspond to the two bands that were immunoreactive with antibodies against d-COP (lane 2). Lanes 3–7: Arf1-Y167-Bp was incubated with GTPgS in the presence of Golgi membranes and rabbit liver coatomer. Samples were then centrifuged and the isolated membranes were UV irradiated (lane 4 and 7). Membrane-bound photo- cross-linked products were analyzed by Western blotting with anti-Arf1 antibodies (lanes 3 and 4) and anti-d-COP antibodies (lanes 5–7). In lane 5, a sample containing only Golgi membranes was loaded. The bands in lanes 5–7 indicated with asterisks represent cross-reacting Golgi membrane proteins. B) The bands marked by arrows in A) were identified by mass spectrometry as d-COP and an N-terminal fragment thereof. Identified peptides are highlighted in bold. The box indicates the anti-d-COP antibody epitope.

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Table 2: Proteins identified in the non-irradiated and the irradiated Table 3: Proteins identified in the non-irradiated and the irradiated samples samples

UV þUV UV þUV

Golgi-associated Golgi-associated particle Carbamoylphosphate Carbamoylphosphate particle 102K chain 102K chain synthetase synthetase Ig gamma Ig gamma Integrin, alpha 2 Integrin, alpha 2 heavy chain constant heavy chain constant Leucyl/cystinyl Leucyl/cystinyl region-rabbit region-rabbit aminopeptidase 2 aminopeptidase 2 ADP-ribosylation factor IQ motif containing IQ motif containing Coatomer beta’ subunit GTPase-activating GTPase-activating protein 1 protein 1 CLTC protein CLTC protein site-specific cross-linking of photolabile Arf1-GTP derivatives Coatomer alpha subunit Coatomer alpha subunit to identify and map several direct interactions with co- Epidermal growth Epidermal growth atomer. Usage of photolabile protein species has three factor receptor factor receptor major advantages over chemical cross-linking: (i) formation Oncostatin M receptor Oncostatin M receptor of a covalent bond is completely independent of the chem- CD109 CD109 Phospholipase C Phospholipase C istry of the reaction partner, so no selectivity exists for ADP-ribosylation factor chemical groups, (ii) the possibility to freely select the posi- Coatomer beta subunit tion of the photoactivable group allows to map the interaction site within the protein, and (iii) the method is highly specific because it probes only proximity (up to 3 A˚ )andis Arf1-GTP interacts with the trunk domains of b-COP monovalent avoiding serial cross-links leading to aggregates. and g-COP We demonstrated that Arf1-GTP binds to b-COP and g- Here, we mapped the interactions of Arf1-GTP with b-COP COP through their trunk domains. Although the structure and g-COP to their trunk domains, and described a pre- of coatomer is not known, it is believed that b-, g-, d- and viously unknown interaction between Arf1-GTP and the N- z-COP share structural similarities with the g (or a, d or e), terminus of d-COP, and an interaction with b0-COP. Finally, b-, m-, and s-adaptins, respectively, of clathrin adaptor com- we provided evidence that multiple Arf1-GTP molecules plexes (13). It is, therefore, interesting that in a functional may bind to coatomer simultaneously. Importantly, this diversity of interactions exists in the context of full-length, myristoylated Arf1 and complete coatomer bound to Golgi membranes, and thus are likely to be functional.

Figure 9: Contribution of I46, I49 and Y167 to the binding of Arf1 to coatomer. Arf1 variants as labeled were incubated with Figure 8: Arf1 interacts through the I46 position directly with GTPgS in the presence of HeLa Golgi membranes and coatomer. b0-COP. Arf1-I46-Bp was incubated with GTPgS in the presence of After incubation, the samples were centrifuged and analyzed by HeLa Golgi membranes and coatomer. After incubation, the sam- Western blot with antibodies directed against b0-COP for coat- ples were centrifuged, the membranes were UV irradiated (lane 2; omer and against Arf1. The ratio between the signal observed for lane 1: no UV irradiation). The photo-cross-linked proteins were b0-COP and that of Arf1 were calculated and normalized to the then immunoprecipitated with anti-Arf1 antibodies under native value obtained when wild-type Arf1 was used. The resulting data conditions. Immunoprecipitated proteins were subjected to SDS– from four independent experiments are presented as an histo- PAGE, and analyzed by Western blot with an anti-b0-COP antibody. gram where the error bars are the standard error of the mean.

Traffic 2007; 8: 582–593 589 Sun et al. assay similar to ours, Arf1-GTP (but not Arf1-GDP) was found that Arf1 interacts with the putative longin domain of shown to interact through its switch I region with the trunk d-COP through its C-terminal helix rather than the switch domains of the g and b1 adaptins of AP-1, and with the region, so it appears that the interface between longin trunk domains of the d and b3 adaptins of AP-3 (31). domains and small GTPases is not conserved. Furthermore, a two-hybrid assay revealed that the switch I and II regions of Arf1 mediate its GTP-dependent interac- The apparent cross-linking efficiency between Arf1-I46Bp tion with the trunk domain of AP-4 e adaptin (structurally and b0-COP was notably weaker than for the other inter- related to b-COP) (11). Together, these data strongly actions we reported. Indeed, quantification of immunoblot suggest that Arf1-GTP interacts with coatomer and clathrin signals obtained for b-COP before and after cross-linking adaptor complexes in a similar manner. This underlines revealed that 60% of the protein was cross-linked to that, although critical to coat recruitment to and release Arf1-GTP (Figure 4, middle panel, lanes 1 and 2). Similarly, from membranes, Arf1 does not confer compartmental 50% of g-COP (Figure 5, middle panel, lanes 1 and 2) and specificity to the budding process. This is consistent with 100% of d-COP (Figure 7A, middle panel, lanes 6 and 7) several studies that have shown that other players, in were cross-linked to Arf1. In contrast, only 5% of b0-COP addition to Arf1, restrict recruitment of a given coat to was cross-linked to Arf1-I46Bp (Figure 6). This indicates a defined location in the endomembrane system. These that either this interaction is weak and therefore not as include transmembrane proteins (32), phosphatidylinositi- significant as those involving other coatomer subunits, or des (33), and additional cytosolic factors (34). that the position 46 is not within an optimal range to support efficient cross-linking with b0-COP. Binding of 0 Arf1-GTP interacts with the putative longin domain of Arf1-GTP to b -COP represents a difference between the 0 d-COP and with b0-COP COP I and clathrin coats: a and b -COP are predicted to be Using an Arf1 derivative with a photolabile amino acid at functionally similar to clathrin heavy chains (13), but there position 167, we found a novel GTP-dependent interaction is currently no evidence of clathrin binding to Arf1. This between the C-terminal a-helix of Arf1 and the d-COP N- might reflect the fact that, in contrast to adaptor com- terminus. d-COP is predicted to have a fold similar to the plexes and clathrin, which are recruited to membranes as medium-size m subunits of clathrin adaptor complexes two separate complexes, coatomer is a stable complex (13). Corresponding interactions of Arf1 with the m1 and probably comprising both adaptor and coat functions 0 m3 adaptins of AP-1 and AP-3 have not been reported. (37,38). However, as the cross-link to b -COP was weak Although photolabile derivatives of Arf1 have been used to as compared with the other COP subunits, more sensitive investigate its binding to AP-1 and AP-3, the photolabile methods would be needed to investigate a potential amino acids were restricted to the switch regions of Arf1 interaction of Arf1 with clathrin. (9,31). Therefore, it will be interesting to determine whether m1orm3 interacts with the C-terminal helix of Sequence of the binding events between Arf1 and Arf1. The m4 adaptin of AP-4 was reported to interact with coatomer Arf1 in a yeast two-hybrid assay (11). Mutations in the Interestingly, as an individual recombinant potein, d-COP switch regions of Arf1 did not affect the interaction, binds to Arf1 regardless of its nucleotide state. In contrast, indicating that another part of Arf1 binds m4 adaptin. The in the context of coatomer, the binding of d-COP to Arf1 was authors reported that the C-terminal cargo binding domain strictly GTP dependent. Moreover, it was shown that coat- of m4 contains the binding site for Arf1. However, the omer does not bind to Arf1-GDP. Thus, it is likely that within fragment that retained binding to Arf1 in the two-hybrid coatomer the binding site for Arf1 on d-COP is not directly assay comprised not only the C-terminal domain but also accessible. This strongly indicates that prior binding of Arf1- part of the N-terminus, leaving open the possibility that GTP to b-andg-COP opens the binding site for Arf1 on d- Arf1 interacts with m adaptins and d-COP analogously. COP, i.e. d-COP per se is not restricted to bind to Arf1-GTP, but is only exposed to Arf1-GTP. This is in good agreement Primary structure analysis suggests that the N-terminal with the location of its binding site on Arf1 (amino acid 167 region of m-COP adopts the fold of a longin domain (13,35). in the C-terminal region and not in the switch regions). In Longin domains are characterized by a structurally con- addition, this seems analogous to the binding of Arf1 to the served profilin-like fold and are found at the N-terminus of m4 subunit of the AP-4 clathrin adaptor complex, which several proteins important for membrane transport: certain was described as not to be nucleotide dependent (11). SNARE proteins, m- and s-adaptins, and SRa, a subunit of the signal recognition particle receptor (36). Recently, longin Multiple and stepwise interactions between domains were suggested to represent generic motifs that coatomer and Arf1 interact with small membrane-associated GTPases (35). We have shown that Golgi-bound Arf1-GTP directly inter- Notably, based on the structure of the longin domain of acts with three, if not four, different coatomer subunits. As SRa complexed with the small GTPase SR54, the interface we have used GTPgS in our functional assay, once coat- between longin domains and small GTPases was pro- omer is bound through Arf1-GTPgS to Golgi membranes it posed to contain the GTPase switch I and II regions. We should not be released anymore. As a consequence, in

590 Traffic 2007; 8: 582–593 Arf1 and Coatomer Interactions these conditions binding equilibria are not reversible, but with the amber stop codon (TAG) using the QuickChange site-directed shifted to completion. Therefore, we assume that within mutagenesis kit (Stratagene, La Jolla, CA, USA). Mutant ARF1 (hARF- the time scale of the assay (a 15-min incubation time mutant-TAG) and yeast N-myristoyltransfase (NMT) genes were inserted as a bicistron into the E. coli expression plasmid pBAD-tRNA using standard followed by 45 min of UV irradiation), the interactions molecular biology techniques. The plasmids pBAD-tRNA and pBK-pAFRS between coatomer and Arf1 have reached completeness. (22) were generously donated by Dr Peter G. Schultz. The pBAD-tRNA- Moreover, we have shown, using single- and multiple- hARF-mutant-TAG constructs were confirmed by DNA sequencing. For the point mutants of Arf1, that all three interfaces cooperate expression of GST-tagged d-COP, a cDNA encoding bovine d-COP was during coatomer binding. It is then likely that stepwise cloned into the pPRO-GST-TEV (41) downstream the GST gene using standard techniques, this plasmid was called pPRO-GST-d-COP. A pET-15b binding events as described above lead to a binding of plasmid carrying a cDNA encoding bovine ND17-Arf1 downstream a His-tag coatomer to Arf1 through b and g-COP simultaneously, (23) was modified using the QuickChange site-directed mutagenesis kit to opening an additional nucleotide independent site on d- introduce the Q71L or T31N in Arf1. COP. Thus, the cross-linked products we observe probably represent simultaneous interactions of multiple subunits Expression of site-specific photolabile ARF-mutant-Bp of coatomer with Arf1 rather than sequential binding in E. coli events corresponding to different stages of COP I vesicle Both pBAD-tRNA-hARF-mutant-TAG and pBK-pAFRS plasmids were co- formation, although the latter cannot fully be excluded. transformed into E. coli strain DH10B. Cells were grown in 2 YT medium containing kanamycin at 30 mg/L and tetracycline at 25 mg/L before being washed in PBS and used to inoculate 4 L glycerol minimal medium þ Taking our data together we propose a model to describe leucine medium with appropriate antibiotics, 1 mM myristic acid and 1 mM the interaction of Arf1-GTP with coatomer (Figure 10). Bp. Cultures were grown to an OD600 of 0.6, and recombinant protein After activation on the Golgi membrane, Arf1-GTP in a first production was induced by the addition of arabinose to 0.02%, followed by step interacts with the trunk domains of b and g-COP. This 22 h growth at 278C. Cells were harvested by centrifugation and lysed in first binding then induces a conformational change within 25 mM Tris–HCl (pH 7.2), 50 mM KCl and 1 mM DTT. After centrifugation at 100 000 g for 60 min, the supernatant was used directly in photo- coatomer, exposing an additional binding site located cross-linking experiments or frozen at 808C. within the longin domain of d-COP; this binding site is not specific for activated Arf1, but can only be presented to Expression and expression of GST-d-COP and Arf1-GTP. After this second step, Arf1-GTP is bound to ND17-Arf1-Q71L/T31N in E. coli three different COP subunits. In addition, our observation The plasmids pPRO-GST-d-COP and pET-15b-ND17-Arf1-Q71L/T31N were that more than one Arf1 can bind to coatomer indicates that transformed into E. coli strain BL21star (Invitrogen, Carlsbad, NM, USA).

Arf1-GTP might oligomerize on the membrane and thus Cells were grown at 378C in LB medium until OD600 reached 0.6. IPTG was provide multiple binding sites for one coatomer complex. then added to 0.1 mM (for GST-d-COP) or 1 mM (for ND17-Arf1) and This is in agreement with our previous data indicating the the incubations resumed at 168C overnight (for GST-d-COP) or at 378Cfor existence of dimers of Arf1-GTP on membranes (19) and 3 h (for ND17-Arf1). After induction, cells were harvested by centrifugation and the resulting pellets were lyzed using an Emulsiflex C5 (Avestin, with previous quantification of Arf1 and coatomer on COP I Mannhein, Germany). After two centrifugations (30 000 g for 10 min vesicles (39,40) indicating a stoichiometry of at least four and 100 000 g for 1 h), GST-d-COP was purified using glutathione Arf1 molecules for one coatomer complex. sepharose fast flow material (Amersham biosciences, Freiburg, Germany) and ND17-Arf1 with Ni-sepharose fast flow (Amersham biosciences) according to manufacturer’s instructions. Materials and Methods Pulldown experiments Construction of E. coli expression plasmids Glutathione sepharose beads (10 mL) coupled with GST-d-COP were For the introduction of the photolabile amino acid p-benzoyl-L-phenylalanine incubated with 1 mgofND17-Arf1-Q71L or ND17-Arf1-T31N dissolved in

(Bp), the codons for I46, I49 or Y167 in human Arf1 cDNA were replaced 200 mL of 50 mM Hepes, pH 7.4, 150 mM KCL, 2 mM MgCl2 for 1 h at room

Figure 10: Model for the interaction of Arf1-GTP with coatomer. After activation on the Golgi membrane (1), Arf1 now bound to GTP can interact with coatomer. In a first step (2), coatomer binds Arf1-GTP via two binding sites located on the trunk domains of b and g-COP. This first interaction induces a conformational change within coatomer that leads to the exposition of an additional binding site for Arf1 located within the longin domain of d-COP (2).

Traffic 2007; 8: 582–593 591 Sun et al. temperature. The beads were then washed extensively and resuspended Mass spectrometry in SDS–PAGE loading buffer. Proteins after electrophoresis were subjected to in-gel trypsin digestion. The resulting peptide mixtures were separated by online reversed-phase nanoscale capillary liquid chromatography and analyzed by electrospray Golgi membranes and cytosol from HeLa cells and tandem mass spectrometry (ESI-MS/MS) using an LTQ-FT mass spec- purified rabbit liver coatomer trometer (Thermo Electron, Bremen, Germany), or by a combination of Golgi membranes and cytosol were isolated from spinner cultures of HeLa standard ESI and matrix-assisted laser desorption ionization techniques. cells as described in (42); rabbit liver coatomer was purified as described in (3).

Acknowledgments Binding of Arf1 and coatomer to Golgi membranes and photo-cross-linking We would like to thank Dr Tracy LaGrassa for critically reading the Golgi membranes, HeLa cytosol or rabbit liver coatomer, and Arf1 photo- manuscript, Dr Thomas Ruppert (ZMBH, Heidelberg, Germany) and Dr labile derivatives were incubated in 25 mM HEPES–KOH (pH 7.2), 2.5 mM Johannes Lechner (BZH, Heidelberg, Germany) for mass pectrometry Mg(OAc)2, 20 mM KCl, 1 mg/mL ovalbumin, 1 mM DTT, 0.2 M sucrose and 50 mM nucleotide (GDPbS or GTPgS) at 378C for 20 min. Membranes analyses. The reagents for expression of site-directed photolabile proteins were pelleted by centrifugation for 30 min at 20 800 g,48C. Membranes in E. coli were generously provided by Dr Peter G. Schultz (The Scripps were resuspended in 25 mM Hepes-KOH (pH 7.2), 2.5 mM Mg(OAc)2, Research Institute, La Jolla, CA). This work was supported by a grant of the 20 mM KCl and 0.2 M sucrose and then irradiated at 366 nm light for Deutsche Forschungsgemeinschaft (SFB 638). 45 min. Photo-cross-linked membranes were re-isolated centrifugation for 30 min, 20 800 g,48C.

Antibodies and immunoprecipitation References For immunoprecipitations, samples were incubated with the indicated antibodies in 200 mL of 20 mM Tris–HCl (pH 5.2), 2 mM EDTA, 0.15 M 1. Duden R, Griffiths G, Frank R, Argos P, Kreis TE. Beta-COP, a 110 kd NaCl, and 0.5% Triton X-100 (immunoprecipitation buffer) for 2 h at 48Cby protein associated with non-clathrin-coated vesicles and the Golgi head-over-head rotation. In experiments in which proteins were denatured complex, shows homology to beta-adaptin. Cell 1991;64:649–665. before immunoprecipitation, SDS was added to a final concentration of 1% 2. Serafini T, Stenbeck G, Brecht A, Lottspeich F, Orci L, Rothman JE, and samples were incubated for 3 min at 958C.The amount of Triton X-100 Wieland FT. A coat subunit of Golgi-derived non-clathrin-coated was increased to 0.9% by dilution to have a 10-fold excess of Triton X-100 vesicles with homology to the clathrin-coated vesicle coat protein over SDS. Samples were incubated with protein A Sepharose (Amersham beta-adaptin. Nature 1991;349:215–220. Biosciences) for 2 h at 48C by head-over-head rotation. Beads were washed five times in immunoprecipitation buffer and once in PBS. Bound proteins 3. Waters MG, Serafini T, Rothman JE. ‘Coatomer’: a cytosolic protein were eluted in SDS–PAGE sample buffer and analyzed by Western blotting complex containing subunits of non-clathrin-coated Golgi transport and mass spectrometry. For immunoprecipitation of the native coatomer vesicles. Nature 1991;349:248–251. complex, an anti-b0-COP antibody was used (43). For immunoprecipitation 4. Palmer DJ, Helms JB, Beckers CJ, Orci L, Rothman JE. Binding of of dissociated coatomer, anti-b-COP appendage [M3A5, described in (44)], coatomer to Golgi membranes requires ADP-ribosylation factor. J Biol anti-b-COP trunk (B1.2, rabbit antisera against the peptide EAGELK- Chem 1993;268:12 083–12 089. PEEEITVGPVQK), antibody against the g1-COP and g2-COP trunk domains 5. Donaldson JG, Cassel D, Kahn RA, Klausner RD. ADP-ribosylation [g-r, described in (45,46)] and anti-Arf1 C-terminus (23) antibodies were factor, a small GTP-binding protein, is required for binding of the used. Antiserum against the appendage domain of g-COP (residues 555– coatomer protein beta-COP to Golgi membranes. Proc Natl Acad Sci 874 of the mouse 1-COP sequence) was raised in rabbits. The same g U S A 1992;89:6408–6412. antibodies were also used for Western blot analysis. 6. Tanigawa G, Orci L, Amherdt M, Ravazzola M, Helms JB, Rothman JE. Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi- Limited proteolysis derived COP-coated vesicles. J Cell Biol 1993;123:1365–1371. 7. Reinhard C, Schweikert M, Wieland FT, Nickel W. Functional reconsti- Purified rabbit liver coatomer or pelleted Golgi membranes were resuspended in 50 mM Tris–HCl (pH 7.4) and the thermolysin was added at tution of COPI coat assembly and disassembly using chemically the concentrations indicated. Digestion was performed at 378C for the defined components. Proc Natl Acad Sci U S A 2003;100:8253–8257. indicated times. The reaction was stopped by the addition of EDTA (final 8. Bigay J, Gounon P, Robineau S, Antonny B. Lipid packing sensed by concentration of 50 mM) and analyzed by Western blotting. ArfGAP1 couples COPI coat disassembly to membrane bilayer curva- ture. Nature 2003;426:563–566. 9. Austin C, Hinners I, Tooze SA. Direct and GTP-dependent interaction of Electroelution ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on imma- The cross-linking experiment was performed with 1 mg of purified Golgi ture secretory granules. J Biol Chem 2000;275:21 862–21 869. membranes from Hela cells. Afterwards the samples were separated 10. Ooi CE, Dell’Angelica EC, Bonifacino JS. ADP-Ribosylation factor 1 through a 10% SDS–PAGE gel and stained with colloidal Coomassie blue. (ARF1) regulates recruitment of the AP-3 adaptor complex to mem- The gel slices running at the appropriate size were cut out and collected in branes. J Cell Biol 1998;142:391–402. a glass tube. For the subsequent electroelution an electroelution chamber 11. Boehm M, Aguilar RC, Bonifacino JS. Functional and physical inter- (Electro-Eluter, Model 422, BioRad, Hercules, CA, USA) was used. To actions of the adaptor protein complex AP-4 with ADP-ribosylation reduce the buffer volume, the samples were centrifuged for 1 h at 378Cin a SpeedVac vacuum centrifuge. The proteins were then precipitated twice factors (ARFs). Embo J 2001;20:6265–6276. with methanol/chloroform (4:1) to remove salts. Finally the samples were 12. Schledzewski K, Brinkmann H, Mendel RR. Phylogenetic analysis of resuspended in sample buffer, separated through a 6% SDS–PAGE gel and components of the eukaryotic vesicle transport system reveals a com- stained with colloidal Coomassie blue. The gel slices running at the mon origin of adaptor protein complexes 1, 2, and 3 and the F appropriate size were cut out and analyzed by mass spectrometry. subcomplex of the coatomer COPI. J Mol Evol 1999;48:770–778.

592 Traffic 2007; 8: 582–593 Arf1 and Coatomer Interactions

13. McMahon HT, Mills IG. COP and clathrin-coated vesicle budding: 32. Bremser M, Nickel W, Schweikert M, Ravazzola M, Amherdt M, different pathways, common approaches. Curr Opin Cell Biol 2004;16: Hughes CA, Sollner TH, Rothman JE, Wieland FT. Coupling of coat 379–391. assembly and vesicle budding to packaging of putative cargo receptors. 14. Collins BM, McCoy AJ, Kent HM, Evans PR, Owen DJ. Molecular Cell 1999;96:495–506. architecture and functional model of the endocytic AP2 complex. Cell 33. Wang YJ, Wang J, Sun HQ, Martinez M, Sun YX, Macia E, Kirchhausen 2002;109:523–535. T, Albanesi JP, Roth MG, Yin HL. Phosphatidylinositol 4 phosphate 15. Heldwein EE, Macia E, Wang J, Yin HL, Kirchhausen T, Harrison SC. regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Crystal structure of the clathrin adaptor protein 1 core. Proc Natl Acad Cell 2003;114:299–310. Sci U S A 2004;101:14108–14113. 34. Zhu Y, Drake MT, Kornfeld S. ADP-ribosylation factor 1 dependent 16. Hoffman GR, Rahl PB, Collins RN, Cerione RA. Conserved structural clathrin-coat assembly on synthetic liposomes. Proceedings Of The motifs in intracellular trafficking pathways: structure of the gamma National Academy Of Sciences Of The United States Of America COP appendage domain. Mol Cell 2003;12:615–625. 1999;96:5013–5018. 17. Watson PJ, Frigerio G, Collins BM, Duden R, Owen DJ. Gamma-COP 35. Schlenker O, Hendricks A, Sinning I, Wild K. The structure of the appendage domain – structure and function. Traffic 2004;5:79–88. mammalian signal recognition particle (SRP) receptor as prototype for 18. Zhao L, Helms JB, Brugger B, Harter C, Martoglio B, Graf R, Brunner J, the interaction of small GTPases with Longin domains. J Biol Chem Wieland FT. Direct and GTP-dependent interaction of ADP ribosylation 2006;281:8898–8906. factor 1 with coatomer subunit beta. Proc Natl Acad Sci U S A 1997; 36. Rossi V, Banfield DK, Vacca M, Dietrich LE, Ungermann C, D’Esposito 94:4418–4423. M, Galli T, Filippini F. Longins and their longin domains: regulated 19. Zhao L, Helms JB, Brunner J, Wieland FT. GTP-dependent binding of SNAREs and multifunctional SNARE regulators. Trends Biochem Sci ADP-ribosylation factor to coatomer in close proximity to the binding 2004;29:682–688. site for dilysine retrieval motifs and p23. J Biol Chem 1999;274: 37. Hara-Kuge S, Kuge O, Orci L, Amherdt M, Ravazzola M, Wieland FT, 14198–14203. Rothman JE. En bloc incorporation of coatomer subunits during the 20. Eugster A, Frigerio G, Dale M, Duden R. COP I domains required for assembly of COP-coated vesicles. J Cell Biol 1994;124:883–892. coatomer integrity, and novel interactions with ARF and ARF-GAP. 38. Lowe M, Kreis TE. In vivo assembly of coatomer, the COP-I coat Embo J 2000;19:3905–3917. precursor. J Biol Chem 1996;271:30725–30730. 21. Brunner J. New photolabeling and crosslinking methods. Annu Rev 39. Serafini T, Orci L, Amherdt M, Brunner M, Kahn RA, Rothman JE. Biochem 1993;62:483–514. ADP-ribosylation factor is a subunit of the coat of Golgi-derived 22. Wang L, Brock A, Herberich B, Schultz PG. Expanding the genetic code COP-coated vesicles: a novel role for a GTP-binding protein. Cell of Escherichia coli. Science 2001;292:498–500. 1991;67:239–253. 23. Gommel DU, Memon AR, Heiss A, Lottspeich F, Pfannstiel J, Lechner 40. Sohn K, Orci L, Ravazzola M, Amherdt M, Bremser M, Lottspeich F, J, Reinhard C, Helms JB, Nickel W, Wieland FT. Recruitment to Golgi Fiedler K, Helms JB, Wieland FT. A major transmembrane protein of membranes of ADP-ribosylation factor 1 is mediated by the cytoplas- Golgi-derived COPI-coated vesicles involved in coatomer binding. mic domain of p23. Embo J 2001;20:6751–6760. J Cell Biol 1996;135:1239–1248. 24. Majoul I, Sohn K, Wieland FT, Pepperkok R, Pizza M, Hillemann J, 41. Bethune J, Kol M, Hoffmann J, Reckmann I, Brugger B, Wieland F. Soling HD. KDEL receptor (Erd2p)-mediated retrograde transport of the Coatomer, the coat protein of COPI transport vesicles, discriminates cholera toxin A subunit from the Golgi involves COPI, p23, and the endoplasmic reticulum residents from p24 proteins. Mol Cell Biol 2006; COOH terminus of Erd2p. J Cell Biol 1998;143:601–612. 26:8011–8021. 25. Contreras I, Ortiz-Zapater E, Aniento F. Sorting signals in the cytosolic 42. Clary DO, Rothman JE. Purification of three related peripheral mem- tail of membrane proteins involved in the interaction with plant ARF1 brane proteins needed for vesicular transport. J Biol Chem 1990; and coatomer. Plant J 2004;38:685–698. 265:10109–10117. 26. Helms JB, Rothman JE. Inhibition by brefeldin A of a Golgi membrane 43. Harter C, Draken E, Lottspeich F, Wieland FT. Yeast coatomer contains enzyme that catalyses exchange of guanine nucleotide bound to ARF. a subunit homologous to mammalian beta’-COP. FEBS Lett 1993; Nature 1992;360:352–354. 332:71–73. 27. Donaldson JG, Finazzi D, Klausner RD. Brefeldin A inhibits Golgi 44. Allan V, Kreis T. A microtubule-binding protein associated with mem- membrane-catalysed exchange of guanine nucleotide onto ARF pro- branes of the Golgi apparatus 10.1083/jcb.103.6.2229. J Cell Biol tein. Nature 1992;360:350–352. 1986;103:2229–2239. 28. Helms JB, Palmer DJ, Rothman JE. Two distinct populations of ARF 45. Harter C, Pavel J, Coccia F, Draken E, Wegehingel S, Tschochner H, bound to Golgi membranes. J Cell Biol 1993;121:751–760. Wieland F. Nonclathrin coat protein gamma, a subunit of coatomer, 29. Futatsumori M, Kasai K, Takatsu H, Shin HW, Nakayama K. Identifica- binds to the cytoplasmic dilysine motif of membrane proteins of the tion and characterization of novel isoforms of COP I subunits. early secretory pathway. Proc Natl Acad Sci U S A 1996;93:1902–1906. J Biochem (Tokyo) 2000;128:793–801. 46. Reinhard C, Harter C, Bremser M, Brugger B, Sohn K, Helms JB, 30. Wegmann D, Hess P, Baier C, Wieland FT, Reinhard C. Novel isotypic Wieland F. Receptor-induced polymerization of coatomer. Proc Natl gamma/zeta subunits reveal three coatomer complexes in mammals. Acad Sci U S A 1999;96:1224–1228. Mol Cell Biol 2004;24:1070–1080. 47. Franco M, Chardin P, Chabre M, Paris S. Myristoylation-facilitated 31. Austin C, Boehm M, Tooze SA. Site-specific cross-linking reveals a dif- binding of the G protein ARF1GDP to membrane phospholipids is ferential direct interaction of class 1, 2, and 3 ADP-ribosylation factors with required for its activation by a soluble nucleotide exchange factor. adaptor protein complexes 1 and 3. Biochemistry 2002;41:4669–4677. J Biol Chem 1996;271:1573–1578.

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