The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering

Transport protein particle (TRAPP) I is a multisubunit vesicle tethering factor composed of seven subunits involved in ER-to-Golgi trafficking. The functional mechanism of the complex and how the subunits interact to form a functional unit are unknown. Here, we have used a multidisciplinary approach that includes X-ray crystallography, electron microscopy, biochemistry, and yeast genetics to elucidate the architecture of TRAPP I. The complex is organized through lateral juxtaposition of the subunits into a flat and elongated particle. We have also localized the site of guanine nucleotide exchange activity to a highly conserved surface encompassing several subunits. We propose that TRAPP I attaches to Golgi membranes with its large flat surface containing many highly conserved residues and forms a platform for protein-protein interactions. This study provides the most comprehensive view of a multisubunit vesicle tethering complex to date, based on which a model for the function of this complex, involving Rab1-GTP and long, coiled-coil tethers, is presented.     

细胞内膜泡运输中拴系因子的功能

膜泡运输是不同细胞器间进行物质传递的基本方式,分为4个重要步骤：囊泡的出芽、转运、拴系和融合。在此过程中,有许多相关因子参与调控,如包被蛋白、Rab蛋白、拴系因子、SM蛋白和SNARE等。拴系因子在运输囊泡和靶位膜发生接触的最初阶段起重要调控作用,多数拴系因子形成大的多亚基复合体发挥功能。目前,关于拴系因子的功能已经有了一定的了解,在此,我们对酵母、哺乳动物以及植物细胞中的已知拴系因子的特点和功能进行了概述。     

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.     

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.     

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.     

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.     

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.     

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.     

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.     

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.     

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.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

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.     

An update on transport vesicle tethering

Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.     

Modular TRAPP complexes regulate intracellular protein trafficking through multiple Ypt/Rab GTPases in Saccharomyces cerevisiae

Ypt/Rab are key regulators of intracellular trafficking in all eukaryotic cells. In yeast, Ypt1 is essential for endoplasmic reticulum (ER)-to-Golgi transport, whereas Ypt31/32 regulate Golgi-to-plasma membrane and endosome-to-Golgi transport. TRAPP is a multisubunit complex that acts as an activator of Ypt/Rab GTPases. Trs85 and Trs130 are two subunits specific for TRAPP III and TRAPP II, respectively. Whereas TRAPP III was shown to acts as a Ypt1 activator, it is still controversial whether TRAPP II acts as a Ypt1 or Ypt31/32 activator. Here, we use GFP-Snc1 as a tool to study transport in Ypt and TRAPP mutant cells. First, we show that expression of GFP-Snc1 in trs85Δ mutant cells results in temperature sensitivity. Second, we suggest that in ypt1ts and trs85Δ, but not in ypt31Δ/32ts and trs130ts mutant cells, GFP-Snc1 accumulates in the ER. Third, we show that overexpression of Ypt1, but not Ypt31/32, can suppress both the growth and GFP-Snc1 accumulation phenotypes of trs85Δ mutant cells. In contrast, overexpression of Ypt31, but not Ypt1, suppresses the growth and GFP-Snc1 transport phenotypes of trs130ts mutant cells. These results provide genetic support for functional grouping of Ypt1 with Trs85-containing TRAPP III and Ypt31/32 with Trs130-containing TRAPP II.     

An update on transport vesicle tethering

Abstract

Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.     

TRAPPopathies: An emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)‐associated proteins

The movement of proteins between cellular compartments requires the orchestrated actions of many factors including Rab family GTPases, Soluble NSF Attachment protein REceptors (SNAREs) and so‐called tethering factors. One such tethering factor is called TRAnsport Protein Particle (TRAPP), and in humans, TRAPP proteins are distributed into two related complexes called TRAPP II and III. Although thought to act as a single unit within the complex, in the past few years it has become evident that some TRAPP proteins function independently of the complex. Consistent with this, variations in the genes encoding these proteins result in a spectrum of human diseases with diverse, but partially overlapping, phenotypes. This contrasts with other tethering factors such as COG, where variations in the genes that encode its subunits all result in an identical phenotype. In this review, we present an up‐to‐date summary of all the known disease‐related variations of genes encoding TRAPP‐associated proteins and the disorders linked to these variations which we now call TRAPPopathies.      

Structural analysis of conserved oligomeric Golgi complex subunit 2

The conserved oligomeric Golgi (COG) complex is strongly implicated in retrograde vesicular trafficking within the Golgi apparatus. Although its mechanism of action is poorly understood, it has been proposed to function by mediating the initial physical contact between transport vesicles and their membrane targets. An analogous role in tethering vesicles has been suggested for at least six additional large multisubunit complexes, including the exocyst, a complex essential for trafficking to the plasma membrane. Here we report the solution structure of a large portion of yeast Cog2p, one of eight subunits composing the COG complex. The structure reveals a six-helix bundle with few conserved surface features but a general resemblance to recently determined crystal structures of four different exocyst subunits. This finding provides the first structural evidence that COG, like the exocyst and potentially other tethering complexes, is constructed from helical bundles. These structures may represent platforms for interaction with other trafficking proteins including SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) and Rabs.