Portal de Periódicos da CAPES

The exomer cargo adaptor structure reveals a novel GTPase-binding domain

2012; Springer Nature; Volume: 31; Issue: 21 Linguagem: Inglês

10.1038/emboj.2012.268

1460-2075

Jon E. Paczkowski, Brian C. Richardson, Amanda M Strassner, J. Christopher Fromme,

Endoplasmic Reticulum Stress and Disease

Article21 September 2012free access Source Data The exomer cargo adaptor structure reveals a novel GTPase-binding domain Jon E Paczkowski Jon E Paczkowski Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Brian C Richardson Brian C Richardson Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Amanda M Strassner Amanda M Strassner Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author J Christopher Fromme Corresponding Author J Christopher Fromme Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Jon E Paczkowski Jon E Paczkowski Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Brian C Richardson Brian C Richardson Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Amanda M Strassner Amanda M Strassner Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author J Christopher Fromme Corresponding Author J Christopher Fromme Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Author Information Jon E Paczkowski1,‡, Brian C Richardson1,‡, Amanda M Strassner1 and J Christopher Fromme 1 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, 457 Weill Hall, Ithaca, NY 14853, USA. Tel.:+1 607 255 1016; Fax:+1 607 255 5961; E-mail: [email protected] The EMBO Journal (2012)31:4191-4203https://doi.org/10.1038/emboj.2012.268 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans-Golgi network (TGN)-derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (FN3–BRCT of exomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes. Introduction Eukaryotic cells control the subcellular localization of integral membrane proteins primarily through protein complexes that bind to the cytosolic domains of transmembrane cargo proteins to mediate trafficking of the cargo towards the target compartment (Schekman and Orci, 1996; Bonifacino and Glick, 2004). Many of these cargo adaptor complexes are recruited from the cytoplasm to the membrane surface of the donor compartment by activated GTPases of the Arf superfamily (Donaldson and Jackson, 2011). Trafficking adaptors usually function as integral components of vesicle coats, sorting cargo into nascent membrane vesicles and tubules and contributing to the morphogenesis of these transport carriers (Schekman and Orci, 1996; Bonifacino and Lippincott-Schwartz, 2003). Trafficking of cargo from the trans-Golgi network (TGN) is particularly complex, requiring several different cargo adaptors to sort cargo towards a host of different destinations, including the plasma membrane (PM) and various compartments of the endolysosomal system (De Matteis and Luini, 2008; Glick and Nakano, 2009). Much progress has been made in understanding the structural basis for the function of clathrin-dependent cargo adaptors and the COPI and COPII vesicle coats (Owen et al, 1999; Bi et al, 2002; Collins et al, 2002; Mossessova et al, 2003; Heldwein et al, 2004; Edeling et al, 2006; Bi et al, 2007; Jackson et al, 2010; Lee and Goldberg, 2010; Yu et al, 2012). However, outside of these well-described systems, comparatively little is known regarding the structural basis for the function of other cargo adaptors. Exomer is an Arf1 GTPase-dependent protein complex required for trafficking of specific cargo proteins from the TGN to the PM (Sanchatjate and Schekman, 2006; Trautwein et al, 2006; Wang et al, 2006). Exomer physically interacts with transmembrane cargo proteins that require it for trafficking (Sanchatjate and Schekman, 2006; Barfield et al, 2009). The exomer complex decorates membranes but is not sufficient to deform membranes (Wang et al, 2006), and is therefore most appropriately categorized as a cargo adaptor, if not a bona fide vesicle coat. Traffic of exomer-dependent cargo is highly regulated: the model cargo Chs3 is trafficked from the TGN to the PM by exomer in a cell-cycle-dependent manner, despite being expressed continuously throughout the cell-cycle (Chuang and Schekman, 1996; Santos and Snyder, 1997; Zanolari et al, 2011), and the exomer cargo Fus1 is targeted to the PM in response to pheromone signalling (Santos et al, 1997; Santos and Snyder, 2003; Barfield et al, 2009). It remains unknown how exomer-dependent trafficking of these cargos is controlled by these signalling pathways. The exomer complex consists of the Chs5 protein and four mutually paralogous proteins termed the ChAPs (for Chs5 and Arf1 binding Proteins): Chs6, Bud7, Bch1, and Bch2 (Trautwein et al, 2006). These five proteins interact in vivo (Trautwein et al, 2006) and co-purify as a complex (Sanchatjate and Schekman, 2006). The CHS5 and CHS6 genes are named based on their role in cell wall chitin synthesis (Roncero et al, 1988; Bulawa, 1992; Choi et al, 1994), and are required for trafficking the Chs3 chitin synthase to the PM (Santos and Snyder, 1997; Ziman et al, 1998). BUD7 was originally identified in a screen for genes affecting bud-site selection (Zahner et al, 1996); BCH1 and BCH2 are named for their homology (BUD7 and CHS6 homologues). The contrast of specific phenotypes of individual ChAP deletion mutants with the overlapping phenotypes of chs5Δ mutants suggests that ChAPs act as cargo-specific adaptors and Chs5 serves a more general, perhaps structural, role in exomer function (Trautwein et al, 2006). Subunits of the exomer complex have been identified in a diverse set of single-celled eukaryotes (Trautwein et al, 2006), but no clear homologues have been found in metazoans. Nevertheless, exomer serves as an important model system for understanding the regulation of cargo trafficking from the TGN in higher eukaryotes, based on the tightly regulated trafficking of its cargos and the functional interaction between exomer and other trafficking machinery that is conserved in metazoans, including the Arf1 GTPase, the TGN-localized Rab proteins Ypt31/32 (Rab11 homologues), and the Myosin V motor Myo2 (Santos and Snyder, 1997; Ortiz and Novick, 2006; Trautwein et al, 2006). To gain insight into exomer function we determined the structure of a functional exomer complex. This structure, with accompanying functional studies, establishes the molecular architecture of the complex and the domains involved in membrane recruitment. The structure reveals that the tandem FN3–BRCT domain of Chs5 projects from the exomer core complex. We demonstrate that this domain binds directly to Arf1, and is required for stable recruitment of exomer to membranes in vitro and in vivo. Results The structure of a functional exomer complex We first determined that residues 1–274 represent the minimal functional fragment of Chs5, based on proper in vivo trafficking of Chs3–GFP (Figure 1A and B; Supplementary Figure 1). This finding is consistent with a recent report, demonstrating that the C-terminus of Chs5 is dispensable for exomer function (Martin-Garcia et al, 2011). On the basis of this result, we determined the crystal structure of a functional exomer complex consisting of Chs6 and residues 1–299 of Chs5 (Chs5/6 exomer complex). The final model (Figure 1C; Supplementary Figures 2 and 3A; Supplementary Movie 1) was refined against a 2.75 Å native data set resulting in good statistics (Supplementary Table 1). The asymmetric unit contains one molecule of Chs6 in complex with one molecule of Chs5(1–299). Figure 1.Molecular architecture of the Chs5/6 exomer heterodimer. (A) Schematic of exomer subunit constructs used, with domains and motifs identified by searching the SMART and TPRpred databases. Exomer complexes form through association of Chs5 with one or more of the ChAPs (Chs6, Bud7, Bch1, and Bch2). (B) Chs3–GFP localization of yeast cells expressing residues 1–299 of Chs5 phenocopies that of cells expressing full-length Chs5 (residues 1–658). Plasmids expressing Chs5 constructs or empty vector were introduced into chs5Δ Chs3–GFP cells (yeast strain CFY264). Images show a single focal plane, with GFP and DIC channels merged. Arrows indicate proper localization of Chs3–GFP to incipient bud sites and the mother-bud neck of very small buds. Arrowhead indicates lack of proper localization in cells lacking functional Chs5. Scale bar, 2 μm. (C) The structure of the Chs5(1–299)/Chs6 heterodimer asymmetric unit, shown as a ribbon diagram. Chs5 is red and Chs6 is blue. (D) Ribbon diagram of the Chs5(1–299) structure, indicating the four different structural motifs. (E) Ribbon diagram of the Chs6 structure, from a top–down perspective relative to (C). Inset shows the same perspective as a surface diagram to visualize the solvent channel in Chs6. See also Supplementary Movie 1. Download figure Download PowerPoint Chs5 is an elongated protein with four small domains (Figure 1D; Supplementary Figure 3B). From N-terminus to C-terminus, it consists of an anti-parallel β-sheet motif, a long α-helix, a fibronectin 3 (FN3) domain, and a BRCA1 C-terminus (BRCT) domain. The linker between the α-helix and the FN3 domain may be flexible, as it consists of a glycine followed by three hydrophilic residues lacking regular secondary structure. As a result of this architecture, the FN3 and BRCT domains project away from the bulk of the complex. For simplicity, we refer to this fragment of Chs5 as the FBE domain (FN3–BRCT of exomer). Chs6 forms a large ring structure (Figure 1E) and comprises mainly α-helices, including several tetratricopeptide repeat (TPR) motifs, and a single five-stranded mixed β-sheet. The topology of Chs6 is complex, with the polypeptide chain meandering back and forth about the ring structure, forming a single structural domain of ∼700 residues (Supplementary Figure 2F). Although there is some resemblance to the helical solenoid structures found in other trafficking and membrane sculpting proteins (Brohawn et al, 2008), the fold of Chs6 is unique, as it only shares a resemblance to known structures at the level of the TPR motifs that are scattered throughout the domain (Supplementary Table 2). Surprisingly, a solvent channel lies at the centre of the Chs6 ring (Figure 1E, inset). Within this solvent channel there is a cleft containing electron density unattributable to any protein atoms. We could reasonably model a molecule of HEPES, a component of the buffers used for purification and crystallization, into the electron density at this site. The Chs5–Chs6 interaction interface Chs5 binds to Chs6 through two different motifs (Figure 2A). The long α-helix of Chs5 (residues 54–76) binds to the surface of Chs6 primarily by interacting with a single long α-helix in Chs6 (residues 300–321) otherwise orphaned in the middle of several TPR repeats. This helix in Chs6 runs anti-parallel to the long helix of Chs5, and extensive hydrophobic and electrostatic interactions occur between the two helices (Figure 2B), similar to the helix–helix interactions of TPR motifs. Thus, these two helices together form an intermolecular TPR-like motif. Additionally, the small β-sheet at the N-terminus of Chs5 packs against a concave hydrophobic surface of Chs6, adjacent to where the Chs5 α-helix binds. The two surfaces of Chs6 contacting Chs5 represent the most highly conserved regions of ChAP protein surface, as determined from sequence alignments generated using all four ChAP proteins (Figure 2A). Figure 2.The Chs5–Chs6 interaction interface. (A) Residues 1–80 of Chs5(1–299) are shown as a red ribbon. Chs6 is coloured by conservation based on a sequence alignment of the four S. cerevisiae ChAP proteins (Chs6, Bud7, Bch1, Bch2). Blue indicates residues that are most conserved, yellow indicates residues that are least conserved, according to a normalized conservation score calculated by ConSurf (Ashkenazy et al, 2010). (B) Close-up view of the interaction of the Chs5 α-helix (residues 50–74) with Chs6. Chs5 is red and Chs6 is blue. The outlined residue numbers denote the positions that disrupt the interaction when mutated, as shown in parts (C, D). (C) Purification from E. coli of recombinant exomer complexes with mutations in the Chs5 subunit. Purified samples were loaded after normalizing for Chs6 levels. The immunoblot indicates the relative expression levels of the mutant Chs5 subunits in the E. coli lysates, as detected by anti-Chs5. (D) Purification of exomer complexes with mutations in the Chs6 subunit, performed as in (C). The asterisk denotes a resin-binding E. coli contaminant that is more prominent in the I305R mutant. (E) Plasmids expressing Myc-tagged Chs5 constructs were introduced into chs5Δ cells (CFY857). Serial dilutions were spotted onto the indicated media and imaged after 2 days of growth at 30°C. Calcofluor white was used at 50 μg/ml. Cells with disrupted Chs3 trafficking (i.e., exomer mutants) are able to grow in the presence of this toxin. (F) Imaging of chromosomal Chs6–GFP in chs5Δ cells (CFY857) expressing plasmid borne Myc-tagged Chs5 constructs. Single confocal sections are shown. Scale bar, 2 μm. Figure source data can be found with the Supplementary data. Source Data for Figure 2 [embj2012268-sup-0004.pdf] Download figure Download PowerPoint We used structure-based mutagenesis to test the importance of these regions for the Chs5–Chs6 interaction. We first found that a Chs5 construct lacking the N-terminal 50 amino acids comprising the β-sheet domain formed a robust interaction with Chs6 and with another ChAP, Bch1, when co-expressed in Escherichia coli (Supplementary Figure 4A and B), indicating that this region of Chs5 is dispensable for the Chs5–ChAP interaction. In contrast, introducing a two-residue mutation in the long α-helix of Chs5, F63E/Q67E, disrupted the Chs5–Chs6 interaction (Figure 2C). We note that neither the F63E nor the Q67E single mutants disrupted the Chs5–Chs6 interaction, suggesting that this interface is stable enough to tolerate moderate perturbation. We found that introduction of a mutation in the Chs6 α-helix that contacts the Chs5 α-helix, I305R, also destabilized the Chs5–Chs6 interaction (Figure 2D). We found that disrupting the Chs5–Chs6 interaction compromised exomer function in vivo, as monitored by cellular sensitivity to the toxin calcofluor white conferred by exomer-mediated trafficking of Chs3 to the PM (Valdivieso et al, 1991; Santos and Snyder, 1997). Consistent with the in vitro interaction results, cells expressing either the F63E or Q67E Chs5 single mutants were sensitive to calcofluor, whereas cells expressing the F63E/Q67E Chs5 double mutant were as resistant to calcofluor as chs5Δ cells (Figure 2E), despite this mutant being expressed at a level similar to that of wild-type Chs5 (Supplementary Figure 4C). We then examined the localization of Chs6–GFP in cells expressing these Chs5 mutants. Correlating with the results of the calcofluor sensitivity and in vitro interaction assays, we found that Chs6–GFP was properly localized to the TGN in cells expressing either the F63E or Q67E single mutant versions of Chs5, but was severely mislocalized in the F63E/Q67E Chs5 mutant, similar to its localization in chs5Δ cells (Figure 2F). This result confirms a previous finding that Chs6 requires Chs5 in order to localize to the TGN (Trautwein et al, 2006). Taken together, these results demonstrate that the α-helical Chs5–Chs6 interface observed in the crystal structure, which buries 817 Å2 of solvent accessible surface, is functionally relevant in vivo. Exomer is a heterotetramer Previous analysis of the exomer complex in detergent-solubilized cell extracts found that immunoprecipitation of Chs5 or of any single ChAP resulted in co-immunoprecipitation of all four ChAP proteins (Sanchatjate and Schekman, 2006; Trautwein et al, 2006). Size analysis of solubilized extracts (Sanchatjate and Schekman, 2006) and of recombinant exomer subunits expressed in insect cells (Wang et al, 2006) also suggested that exomer likely exists as a multimeric complex. We performed sedimentation velocity and Stokes radius analysis (Figure 3A and B) to determine that the recombinant Chs5/6 exomer complex is a heterotetramer, based on a native molecular weight of 205 kDa (two copies of Chs6 and two copies of Chs5(1–299) have an expected molecular weight of 240 kDa). Complementary analysis by a different technique, multiangle light scattering (MALS) (De et al, 2010), yielded a similar value of 195±10 kDa (Supplementary Figure 5A), confirming this result (we note that the experimentally determined molecular weight may be slightly lower than expected due to partial proteolysis or complex dissociation during production and purification of the recombinant proteins). A heterotetramer is also consistent with an experiment, demonstrating that exomer complexes isolated from cells using epitope-tagged Chs6 contain the other ChAPs in significantly lower abundance (Sanchatjate and Schekman, 2006). Figure 3.The Chs5 N-terminus mediates formation of the exomer heterotetramer. (A) Sedimentation velocity analysis, using sedimentation on a sucrose gradient relative to standards to determine the S-value of the Chs5(1–299)/Chs6 exomer complex. (B) Stokes radius analysis, using retention volume on a Superdex 200 gel fitration column. (C) View of a crystal-packing interaction that relates two asymmetric units via homodimeric interaction of the Chs5 N-terminus. The symmetry mate is coloured dark blue (Chs6) and green (Chs5). In both (C, D), the red arrow denotes the Chs5 N-terminus belonging to the heterodimer on the left. (D) Domain swap model of the heterotetramer, with colouring otherwise as in (C). Note the red arrow now points to the Chs5 N-terminus in the symmetry-related position. (E) Close-up view of the crystal-packing interface of the domain swap model, demonstrating the lack of continuity in the model between the N-terminus (residues 1–50) and the long α-helix of Chs5 (starting after residue 50). The loop expected to contain His25 and His26 is coloured orange (both of these residues are mutated to Asp in Chs5(HH/DD)/Chs6). The modelled position of Met47 (site of the M47P mutation) in the polypeptide is coloured blue. See also Supplementary Movie 2. Download figure Download PowerPoint We found that deletion of the N-terminal β-sheet of Chs5 reduced the Stokes radius of the Chs5–Chs6 complex by 21% (Figure 3B), corresponding to a 50% reduction in the hydrodynamic volume of the complex. In addition, this construct exhibited a native molecular weight of 117 kDa as determined by MALS (Supplementary Figure 5B), closely matching the 115 kDa calculated molecular weight of a single Chs5(51–299)/Chs6 heterodimer. Deletion of residues 1–50 of Chs5 did not disrupt the Chs5–Chs6 interaction (Supplementary Figure 4A). Therefore, this motif mediates formation of an exomer heterotetramer via dimerization of two Chs5/6 heterodimers. Importantly, loss of this 50-residue N-terminal homodimerization motif abrogated exomer function in vivo (Supplementary Figure 1C). Chs5-mediated heterotetramerization is consistent with the finding that ChAP–ChAP interactions were lost in chs5Δ mutant cells, but not in cells lacking individual ChAP genes (Sanchatjate and Schekman, 2006). The observation that exomer is a tetramer is at odds with the presence of only a single Chs5/6 pair in the crystal asymmetric unit. Examining crystallographic symmetry revealed that exomer packs in a tight hexamer of heterodimers mediated by Chs6–Chs6 crystal contacts (Supplementary Figure 5G). Within this hexamer, two-fold crystallographic axes relate pairs of Chs5/6 heterodimers contacting each other by their Chs5 N-termini (Figure 3C), as would be expected from the biochemical data, indicating that this domain mediates formation of the tetramer. The electron density of the Chs5 N-terminal β-sheets is quite weak, consistent with partial disruption of the dimeric interaction by the extensive crystal contacts, resulting from the hexameric packing. MALS analysis of exomer in the crystallization solution indicated a size of 191 kDa (Supplementary Figure 5C), consistent with a heterotetramer; therefore, the weak electron density is unlikely to be a result of salt or buffer-dependent disruption of the tetramer. Thus, the heterotetrameric exomer complex can be modelled via this crystal-packing interaction (Figure 3C), though the electron density does not allow modelling of side chains in the Chs5 N-terminal domain. Therefore, precise residue numbers cannot be assigned to this region of the model beyond those indicated by secondary structure prediction (Supplementary Figure 4D). We were initially concerned that the Chs5–Chs5 contact surface area is less than expected for a stable interaction. We therefore considered an alternative model involving domain swapping, in which neighbouring N-termini reach across each other to bind in trans with the opposing Chs6 molecule (Figure 3D and E; Supplementary Movie 2). Domain swapping would account for tetramerization despite the low Chs5–Chs5 surface contact area, because the tetramer would be stabilized by the additional interaction of the Chs5 N-terminus with the opposite Chs6 molecule. The domain swap model is consistent with our finding that deletion of the Chs5 N-terminus disrupted tetramerization (Figure 3B; Supplementary Figure 5B), but did not disrupt the Chs5–Chs6 interaction (Supplementary Figure 4A); this model is also consistent with an alternative interpretation of the asymmetric unit (Supplementary Figure 3). To further test the domain swap model, we generated two point mutants in the N-terminus of Chs5 based on the alignment of predicted secondary structure to the observed main chain density. One mutation, H25D/H26D, we predict to lie in a loop contacting a conserved surface of Chs6; the other mutation, M47P, we predict to disrupt the β-strand mediating the hypothesized domain swap (Figure 3E). Both mutants disrupted exomer tetramerization without disrupting the Chs5/Chs6 interaction (Supplementary Figure 5D–F). These experiments are consistent with the domain swap model; however, we cannot rule out the alternative configuration. Further structural studies are needed to establish the precise atomic details of the tetramerization interface. Our finding that exomer is a heterotetramer is consistent with previous studies (Sanchatjate and Schekman, 2006; Trautwein et al, 2006; Wang et al, 2006), and implies that both mixed exomer heterotetramers (containing two Chs5 molecules and two different ChAPs) and homogeneous exomer heterotetramers (containing two Chs5 molecules and two identical ChAPs) likely exist in vivo. The FBE domain resembles appendage domains Although sharing no obvious sequence homology, the structure of the FBE domain of Chs5 (residues 77–285) resembles ‘appendage’ domains found in the clathrin adaptors and COPI (Figure 4). The appendage domains of these cargo adaptors consist of either an N-terminal β-sandwich subdomain and a C-terminal mixed α/β ‘platform’ subdomain, or just the β-sandwich domain (Owen et al, 1999, 2000; Kent et al, 2002; Collins et al, 2003; Hoffman et al, 2003). The FBE domain also consists of an N-terminal β-sandwich subdomain and a C-terminal mixed α/β subdomain. The precise folds are not identical: the β-sandwich of the clathrin adaptors and COPI consists of an immunoglobulin-like fold, whereas the β-sandwich of Chs5 is a similar but distinct FN3 fold, and the mixed α/β subdomain of Chs5 is a BRCT fold, unlike the platform subdomain of the clathrin adaptor and COPI appendages, which exhibits a much larger β-sheet. The relative orientation of the two subdomains are quite similar in Chs5, the clathrin adaptors and COPI. In particular, an α-helix is projected from the distal end of the FBE domain, and the α2, β2, and γ-COP appendage domains (located at the top of orientation shown in Figure 4). We note that despite the overall resemblance, the FBE domain cannot be classified as a true appendage domain, and indeed has a function distinct from that of appendage domains, as we demonstrate below. Figure 4.The Chs5 FBE domain resembles appendage domains of other cargo adaptors. Structural resemblance of the Chs5 FBE domain (this work) and the appendage domains of the α2 subunit of AP-2 (PDB: 1B9K) (Owen et al, 1999), the β2 subunit of AP-2 (PDB: 1E42) (Owen et al, 2000), the γ-COP subunit of COPI (PDB: 1PZD) (Hoffman et al, 2003), GGA1 (PDB: 1OM9) (Collins et al, 2003), and the γ1 subunit of AP-1 (PDB: 1GYU) (Kent et al, 2002). Download figure Download PowerPoint Chs6 binds directly to membranes and the FBE domain binds directly to Arf1 Previous studies found that exomer localization to the TGN in vivo and robust recruitment to liposome membranes in vitro depend upon Arf1 (Wang et al, 2006). Chs5 localizes correctly to the TGN in the absence of any ChAP proteins (Trautwein et al, 2006), indicating that the primary determinants of exomer localization and Arf1 interaction lie within the Chs5 subunit. To determine which domain(s) of Chs5 mediate the exomer–Arf1 interaction, we used the crystal structure to design and generate stable, well-behaved truncation constructs, based on the observation that the three regions of Chs5 (N-terminus, helix, and FBE) are structurally independent and separated by short linkers (Figure 1D). We assessed the ability of these constructs to be recruited to liposome membranes by Arf1 using a liposome flotation assay (Wang et al, 2006). We confirmed that the Chs5/6 complex was stably recruited to liposomes in an Arf1–GTP-dependent manner (Figure 5A). In contrast, the ‘ΔN’ [Chs5(51–299)/Chs6] construct was more weakly bound to membranes, indicating loss of the robust interaction with Arf1–GTP. The ‘core’ [Chs5(1–80)/Chs6] exomer complex was not detected in the membrane-bound fractions. These results indicate that both the N-terminal and FBE domains of Chs5 play key roles in stable recruitment of exomer to the membrane surface by Arf1. Figure 5.Chs6 binds to membranes and the FBE domain binds to Arf1 to recruit exomer to membranes. (A) Liposome flotation assay comparing Arf1-dependent membrane binding of different exomer constructs. ‘T*’ denotes Arf1 bound to GMPPNP, ‘D’ denotes Arf1 bound to GDP. For simplicity, only the Chs6 protein band is shown for each construct; this is the band that was used for subsequent quantification. (B) Liposome pelleting assay measuring Arf1-independent membrane binding. ‘TGN’, TGN-like liposomes, ‘Folch’, Folch fraction I liposomes. ‘S’, supernatant fractions, ‘P’, pellet fractions. (C) Quantification of Arf1-independent membrane binding. Error bars represent 95% confidence intervals, n=3, with significance determined by one-way ANOVA with post-processing to correct for multiple comparisons. The overall P-value for this statistical model is indicated. Comparisons not labelled with asterisks were not statistically different. (D) Same as (C), except error bars represent s.e.m., n=3. (E) A GST-pulldown was performed to compare binding of purified ΔN17-Arf1 (preloaded with GDP or GTP) to either the GST–FBE domain construct or GST alone. Arf1 was detected with anti-Arf1 antibody and the GST proteins were detected by Ponceau staining. (F) Arf1-dependent liposome pelleting was performed using a different batch of Folch liposomes. Occasionally, liposome batches bound proteins more weakly; we took advantage of one such batch to measure Arf1-dependent binding. Before adding exomer constructs to the ‘+Arf1’ experimental condition, Arf1 was loaded with GTP in the presence