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

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 (F N3–B RCT of e xomer), 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.     

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes

We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.     

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

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.     

Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface

Kidney anion exchanger 1 (kAE1) plays an important role in acid–base homeostasis by mediating chloride/bicarbornate (Cl^?/HCO₃^?) exchange at the basolateral membrane of α‐intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease – distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans‐Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral‐related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non‐polarized kidney cells. By using RNA interference, co‐immunoprecipitation, yellow fluorescent protein‐based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin (but not AP‐1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral‐related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid‐secreting α‐intercalated cells.      

Oligomerization of the chitin synthase Chs3 is monitored at the Golgi and affects its endocytic recycling

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP‐1‐dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N‐terminal cytosolic region. However, the truncated ^Δ126Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI‐ and Rer1‐dependent manner. A subset of the oligomerization‐deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N‐terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly.     

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Distinct functions for Arf guanine nucleotide exchange factors at the Golgi complex: GBF1 and BIGs are required for assembly and maintenance of the Golgi stack and trans-Golgi network, respectively

We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, whereas BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the endoplasmic reticulum (ER), but caused its accumulation into peripheral vesiculotubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, brefeldin A-inhibited guanine nucleotide exchange factors (BIGs) knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, whereas GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization, and cargo progression to the cell surface.     

Overexpression of trans‐Golgi network t‐SNAREs rescues vacuolar trafficking and TGN morphology defects in a putative tethering factor mutant

The trans‐Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN‐localized coiled‐coil protein TNO1 is a putative tethering factor that interacts with the TGN t‐SNARE SYP41 and is required for correct localization of the SYP61 t‐SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41–SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41–SYP61 t‐SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.     

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.     

Arabidopsis mu A-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1

In receptor‐mediated transport pathways in mammalian cells, clathrin‐coated vesicle (CCV) µ‐adaptins are the main binding partners for the tyrosine sorting/internalization motif (YXXØ). We have analyzed the function of the µA‐adaptin, one of the five µ‐adaptins from Arabidopsis thaliana, by pull‐down assays and plasmon resonance measurements using its receptor‐binding domain (RBD) fused to a histidine tag. We show that this adaptin is able to bind the consensus tyrosine motif YXXØ from the pea vacuolar sorting receptor (VSR)‐PS1, as well as from the mammalian trans‐Golgi network (TGN)38 protein. Moreover, the tyrosine residue was revealed to be crucial for binding of the complete cytoplasmic tail of VSR‐PS1 to the plant µA‐adaptin. The trans‐Golgi localization of the µA‐adaptin strongly suggests its involvement in Golgi‐ to vacuole‐trafficking events.     

Identification of sorting motifs of AtβFruct4 for trafficking from the ER to the vacuole through the Golgi and PVC

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtβFructosidase 4 (AtβFruct4), to the central vacuole in protoplasts. AtβFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtβFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtβFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.     

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor,specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF-GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

In situ localization and in vitro induction of plant COPI-coated vesicles

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.     

DKWSLLL,a Versatile DXXXLL‐Type Signal with Distinct Roles in the Cu+‐Regulated Trafficking of ATP7B

In the liver, the P‐type ATPase and membrane pump ATP7B plays a crucial role in Cu⁺ donation to cuproenzymes and in the elimination of excess Cu⁺. ATP7B is endowed with a COOH‐cytoplasmic (DE)XXXLL‐type traffic signal. We find that accessory (Lys ?3, Trp ?2, Ser ?1 and Leu +2) and canonical (D ?4, Leu 0 and Leu +1) residues confer the DKWSLLL signal with the versatility required for the Cu⁺‐regulated cycling of ATP7B between the trans‐Golgi network (TGN) and the plasma membrane (PM). The separate mutation of these residues caused a disruption of the signal, resulting in different ATP7B distribution phenotypes. These phenotypes indicate the key roles of specific residues at separate steps of ATP7B trafficking, including sorting at the TGN, transport from the TGN to the PM and its endocytosis, and recycling to the TGN and PM. The distinct roles of ATP7B in the TGN and PM and the variety of phenotypes caused by the mutation of the canonical and accessory residues of the DKWSLLL signal can explain the separate or joined presentation of Wilson's cuprotoxicosis and the dysfunction of the cuproenzymes that accept Cu⁺ at the TGN.      

The Sec7 Arf-GEF is recruited to the trans-Golgi network by positive feedback

Arf GTPases are key regulators of both retrograde and anterograde traffic at the Golgi complex. The Golgi-localized Arf activators, Arf-GEFs (guanine exchange factor) of the BIG/GBF family, are poorly understood in terms of both their regulatory and localization mechanisms. We have performed a detailed kinetic characterization of a functional Golgi Arf-GEF, the trans-Golgi network (TGN)-localized Sec7 protein from yeast. We demonstrate that Sec7 is regulated by both autoinhibition and positive feedback. We show that positive feedback arises through the stable recruitment of Sec7 to membranes via its HDS1 domain by interaction with its product, activated Arf1. This interaction mediates localization of Sec7 to the TGN, because deletion of the HDS1 domain or mutation of the HDS1 domain in combination with deletion of Arf1 significantly increases cytoplasmic localization of Sec7. Our results lead us to propose a model in which Arf-GEF recruitment is linked to Golgi maturation via Arf1 activation.     

Arf6 Regulation of Gyrating‐Clathrin

‘Gyrating‐’ or ‘G’‐clathrin are coated endocytic structures located near peripheral sorting endosomes (SEs), which exhibit highly dynamic but localized movements when visualized by live‐cell microscopy. They have been implicated in recycling of transferrin from the sorting endosome directly to the cell surface, but there is no information about their formation or regulation. We show here that G‐clathrin comprise a minority of clathrin‐coated structures in the cell periphery and are brefeldin A (BFA)‐resistant. Arf6‐GTP substantially increases G‐clathrin levels, probably by lengthening coated bud lifetimes as suggested by photobleaching and photoactivation results, and an Arf6(Q67L)‐GTP mutant bearing an internal GFP tag can be directly visualized in G‐clathrin structures in live cells. Upon siRNA‐mediated depletion of Arf6 or expression of Arf6(T27N), G‐clathrin levels rise and are primarily Arf1‐dependent, yet still BFA‐resistant. However, BFA‐sensitive increased G‐clathrin levels are observed upon acute incubation with cytohesin inhibitor SecinH3, indicating a shift in GEF usage. Depletion of both Arf6 and Arf1 abolishes G‐clathrin, and results in partial inhibition of fast transferrin recycling consistent with the latter's participation in this pathway. Collectively, these results demonstrate that the dynamics of G‐clathrin primarily requires completion of the Arf6 guanine nucleotide cycle, but can be regulated by multiple Arf and GEF proteins, reflecting both overlapping mechanisms operative in their regulation and the complexity of processes involved in endosomal sorting.     

ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells

Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.     

The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins –major histocompatibility complex II (MHCII), CD86 and CD97 –from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.