The TRAPP complex: insights into its architecture and function

Vesicle-mediated transport is a process carried out by virtually every cell and is required for the proper targeting and secretion of proteins. As such, there are numerous players involved to ensure that the proteins are properly localized. Overall, transport requires vesicle budding, recognition of the vesicle by the target membrane and fusion of the vesicle with the target membrane resulting in delivery of its contents. The initial interaction between the vesicle and the target membrane has been referred to as tethering. Because this is the first contact between the two membranes, tethering is critical to ensuring that specificity is achieved. It is therefore not surprising that there are numerous 'tethering factors' involved ranging from multisubunit complexes, coiled-coil proteins and Rab guanosine triphosphatases. Of the multisubunit tethering complexes, one of the best studied at the molecular level is the evolutionarily conserved TRAPP complex. There are two forms of this complex: TRAPP I and TRAPP II. In yeast, these complexes function in a number of processes including endoplasmic reticulum-to-Golgi transport (TRAPP I) and an ill-defined step at the trans Golgi (TRAPP II). Because the complex was first reported in 1998 (1), there has been a decade of studies that have clarified some aspects of its function but have also raised further questions. In this review, we will discuss recent advances in our understanding of yeast and mammalian TRAPP at the structural and functional levels and its role in disease while trying to resolve some apparent discrepancies and highlighting areas for future study.     

TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport

TRAPP is a conserved protein complex required early in the secretory pathway. Here, we report two forms of TRAPP, TRAPP I and TRAPP II, that mediate different transport events. Using chemically pure TRAPP I and COPII vesicles, we have reconstituted vesicle targeting in vitro. The binding of COPII vesicles to TRAPP I is specific, blocked by GTPgammaS, and, surprisingly, does not require other tethering factors. Our findings imply that TRAPP I is the receptor on the Golgi for COPII vesicles. Once the vesicle binds to TRAPP I, the small GTP binding protein Ypt1p is activated and other tethering factors are recruited.     

Identification of the Novel TRAPP Associated Protein Tca17

Vesicle tethers are long coiled–coil proteins or multisubunit complexes that provide specificity to the membrane fusion process by linking cargo‐containing vesicles to target membranes. Transport protein particle (TRAPP) is a well‐characterized multisubunit tethering complex that acts as a GTP exchange factor and is present in two cellular forms: a 7 subunit TRAPP I complex required for ER‐to‐Golgi transport, and a 10 subunit TRAPP II complex that mediates post‐Golgi trafficking. In this work, we have identified Tca17, which is encoded by the non‐essential ORF YEL048c, as a novel binding partner of the TRAPP complex. Loss of Tca17 or any of the non‐essential TRAPP subunits (Trs33, Trs65 and Trs85) leads to defects in the Golgi‐endosomal recycling of Snc1. We show that Tca17, a Sedlin_N family member similar to the TRAPP subunit Trs20, interacts with the TRAPP complex in a Trs33‐ and Trs65‐dependent manner. Mutation of TCA17 or TRS33 perturbs the association of Trs65 with the rest of the TRAPP complex and alters the localization of the Rab GTPase Ypt31. These data support a model in which Tca17 acts with Trs33 and Trs65 to promote the assembly and/or stability of the TRAPP complex and regulate its activity in post‐Golgi trafficking events.     

C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking

TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.     

TRAPPC2L is a Novel, Highly Conserved TRAPP-Interacting Protein

Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c . TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.     

Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP

Transport protein particle (TRAPP) is a large multiprotein complex involved in endoplasmic reticulum-to-Golgi and intra-Golgi traffic. TRAPP specifically and persistently resides on Golgi membranes. Neither the mechanism of the subcellular localization nor the function of any of the individual TRAPP components is known. Here, the crystal structure of mouse Bet3p (bet3), a conserved TRAPP component, reveals a dimeric structure with hydrophobic channels. The channel entrances are located on a putative membrane-interacting surface that is distinctively flat, wide and decorated with positively charged residues. Charge-inversion mutations on the flat surface of the highly conserved yeast Bet3p led to conditional lethality, incorrect localization and membrane trafficking defects. A channel-blocking mutation led to similar defects. These data delineate a molecular mechanism of Golgi-specific targeting and anchoring of Bet3p involving the charged surface and insertion of a Golgi-specific hydrophobic moiety into the channels. This essential subunit could then direct other TRAPP components to the Golgi.     

Identification and characterization of five new subunits of TRAPP

TRAPP (transport protein particle), a multiprotein complex containing ten subunits, plays a key role in the late stages of endoplasmic reticulum to Golgi traffic in the yeast Saccharomyces cerevisiae. We previously described the identification of five TRAPP subunits (Bet5p, Trs20p, Bet3p, Trs23p and Trs33p). Now we report the identification of the remaining five subunits (Trs31p, Trs65p, Trs85p, Trs120p and Trs130p) as well as an initial characterization of the yeast complex and its human homologue. We find that three of the subunits are dispensable for growth and a novel sequence motif is found in Bet3p, Trs31p and Trs33p. Furthermore, biochemical characterization of both yeast and human TRAPP suggests that this complex is anchored to a Triton X-100 resistant fraction of the Golgi. Differences between yeast and human TRAPP as well as the relationship of TRAPP subunits to other docking/tethering factors are discussed.     

Structural insights into assembly of TRAPPII and its activation of Rab11/Ypt32

Transport protein particle (TRAPP) complexes belong to the multisubunit tethering complex. They are guanine nucleotide exchange factors (GEFs) that play essential roles in secretory and endocytic recycling pathway and autophagy. There are two major forms of TRAPP complexes, TRAPPII and TRAPPIII, which share a core set of small subunits. TRAPPIII activates Rab1, while TRAPPII primarily activates Rab11. A steric gating mechanism has been proposed to control the substrate selection in vivo. However, the detailed mechanisms underlying the transition from TRAPPIII's GEF activity for Rab1 to TRAPPII's GEF activity for Rab11 and the roles of the complex-specific subunits in this transition are insufficiently understood. In this review, we discuss recent advances in understanding the mechanism of specific activation of Rab11/Ypt32 by TRAPPII, with a particular focus on new findings from structural studies.     

The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex

The TRAPP (transport protein particle) complexes are tethering complexes that have an important role at the different steps of vesicle transport. Recently, the crystal structures of the TRAPP subunits SEDL and BET3 have been determined, and we present here the 1.7 Angstroms crystal structure of human TPC6, a third TRAPP subunit. The protein adopts an alpha/beta-plait topology and forms a dimer. In spite of low sequence similarity, the structure of TPC6 strikingly resembles that of BET3. The similarity is especially prominent at the dimerization interfaces of the proteins. This suggests heterodimerization of TPC6 and BET3, which is shown by in vitro and in vivo association studies. Together with TPC5, another TRAPP subunit, TPC6 and BET3 are supposed to constitute a family of paralogous proteins with closely similar three-dimensional structures but little sequence similarity among its members.     

Structure of the Bet3-Tpc6B core of TRAPP: two Tpc6 paralogs form trimeric complexes with Bet3 and Mum2

The transport protein particle (TRAPP) complexes are involved in the tethering process at different trafficking steps of vesicle transport. We here present the crystal structure of a human Bet3-Tpc6B heterodimer, which represents a core sub-complex in the assembly of TRAPP. We describe a conserved patch of Tpc6 with uncharged pockets, forming a putative interaction interface for an anchoring moiety at the Golgi. The structural and functional comparison of the two paralogs Tpc6A and Tpc6B, only found in some organisms, indicates redundancy and added complexity of TRAPP architecture and function. Both iso-complexes, Bet3-Tpc6A and Bet3-Tpc6B, are able to recruit Mum2, a further TRAPP subunit, and we identify the alpha1-alpha2 loop regions as a binding site for Mum2. Our study reveals similar stability of the iso-complexes and similar expression patterns of the tpc6 variants in different mouse organs. These findings raise the possibility that the Tpc6 paralogs might contribute to the formation of two distinct TRAPP complexes that differ in function.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

TRAPP II Complex Assembly Requires Trs33 or Trs65

TRAPP is a multi-subunit complex that acts as a Ypt/Rab activator at the Golgi apparatus. TRAPP exists in two forms: TRAPP I is comprised of five essential and conserved subunits and TRAPP II contains two additional essential and conserved subunits, Trs120 and Trs130. Previously, we have shown that Trs65, a nonessential fungi-specific TRAPP subunit, plays a role in TRAPP II assembly. TRS33 encodes another nonessential but conserved TRAPP subunit whose function is not known. Here, we show that one of these two subunits, nonessential individually, is required for TRAPP II assembly. Trs33 and Trs65 share sequence, intracellular localization and interaction similarities. Specifically, Trs33 interacts genetically with both Trs120 and Trs130 and physically with Trs120. In addition, trs33 mutant cells contain lower levels of TRAPP II and exhibit aberrant localization of the Golgi Ypts. Together, our results indicate that in yeast, TRAPP II assembly is an essential process that can be accomplished by either of two related TRAPP subunits. Moreover, because humans express two Trs33 homologues, we propose that the requirement of Trs33 for TRAPP II assembly is conserved from yeast to humans.     

Organization and assembly of the TRAPPII complex

Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals.     

Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN. vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.     

The Plant Exocyst

The exocyst is an octameric vesicle tethering complex that functions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. All proteins in the complex have been conserved during evolution, and genes that encode the exocyst subunits are present in the genomes of all plants investigated to date. Although the plant exocyst has not been studied in great detail, it is likely that the basic function of the exocyst in vesicle tethering is conserved. Nevertheless, genomic and genetic studie...     

Remote Homology between Munc13 MUN Domain and Vesicle Tethering Complexes

Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca²⁺-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.     

Are All Multisubunit Tethering Complexes Bona Fide Tethers?

Since the late 1990s, a number of multisubunit tethering complexes (MTCs) have been described that function in membrane trafficking events: TRAPP I, TRAPP II, TRAPP III, COG, HOPS, CORVET, Dsl1, GARP and exocyst. On the basis of structural and sequence similarities, they have been categorized as complexes associated with tethering containing helical rods (CATCHR) (Dsl1, COG, GARP and exocyst) or non‐CATCHR (TRAPP I, II and III, HOPS and CORVET) complexes (Yu IM, Hughson FM. Tethering factors as organizers of intracellular vesicular traffic. Annu Rev Cell Dev Biol 2010;26:137–156). Both acronyms (CATCHR and MTC) imply these complexes tether opposing membranes to facilitate fusion. The main question we will address is: have these complexes been formally demonstrated to function as tethers? If the answer is no, then is it premature or even correct to refer to them as tethers? In this commentary, we will argue that the vast majority of MTCs have not been demonstrated to act as a tether. We propose that a distinction between the terms tether and tethering factor be considered to address this issue.     

Biochemical and crystallographic studies reveal a specific interaction between TRAPP subunits Trs33p and Bet3p

Transport protein particle (TRAPP) comprises a family of two highly related multiprotein complexes, with seven common subunits, that serve to target different classes of transport vesicles to their appropriate compartments. Defining the architecture of the complexes will advance our understanding of the functional differences between these highly related molecular machines. Genetic analyses in yeast suggested a specific interaction between the TRAPP subunits Bet3p and Trs33p. A mammalian bet3-trs33 complex was crystallized, and the structure was solved to 2.2 angstroms resolution. Intriguingly, the overall fold of the bet3 and trs33 monomers was similar, although the proteins had little overall sequence identity. In vitro experiments using yeast TRAPP subunits indicated that Bet3p binding to Trs33p facilitates the interaction between Bet3p and another TRAPP subunit, Bet5p. Mutational analysis suggests that yeast Trs33p facilitates other Bet3p protein-protein interactions. Furthermore, we show that Trs33p can increase the Golgi-localized pool of a mutated Bet3 protein normally found in the cytosol. We propose that one of the roles of Trs33p is to facilitate the incorporation of the Bet3p subunit into assembling TRAPP complexes.     

mTrs130 Is a Component of a Mammalian TRAPPII Complex,a Rab1 GEF That Binds to COPI-coated Vesicles

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.     

A mouse TRAPP-related protein is involved in pigmentation

We identified a new spontaneous recessive mutation in the mouse, mhyp (mosaic hypopigmentation), in a screen for novel proviral integration sites in a multiple ecotropic provirus mapping stock. Integration of an 8.4-kb retrovirus results in mosaic loss of coat pigment in mhyp homozygotes. Patchy loss of pigmentation in the retinal pigmented epithelial layer of the eye with abnormal melanosomes is also evident. We mapped mhyp to mouse chromosome 7 and cloned the underlying gene. mhyp is a defect in the Trappc6a gene. Expression of Trappc6a is markedly diminished in mhyp homozygotes. The normal protein, TRAPPC6A, is a subunit of the TRAPP (transport protein particle) I and II complexes. While TRAPP complexes are essential for ER-to-Golgi and intra-Golgi vesicle trafficking in yeast, TRAPP subunits participate in additional, including post-Golgi, transport events in mammals. The data implicate mammalian TRAPPC6A in vesicle trafficking during melanosome biogenesis.