TRAPP stimulates guanine nucleotide exchange on Ypt1p

TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.     

The role of Trs65 in the Ypt/Rab guanine nucleotide exchange factor function of the TRAPP II complex

The conserved modular complex TRAPP is a guanine nucleotide exchanger (GEF) for the yeast Golgi Ypt-GTPase gatekeepers. TRAPP I and TRAPP II share seven subunits and act as GEFs for Ypt1 and Ypt31/32, respectively, which in turn regulate transport into and out of the Golgi. Trs65/Kre11 is one of three TRAPP II-specific subunits. Unlike the other two subunits, Trs120 and Trs130, Trs65 is not essential for viability, is conserved only among some fungi, and its contribution to TRAPP II function is unclear. Here, we provide genetic, biochemical, and cellular evidence for the role of Trs65 in TRAPP II function. First, like Trs130, Trs65 localizes to the trans-Golgi. Second, TRS65 interacts genetically with TRS120 and TRS130. Third, Trs65 interacts physically with Trs120 and Trs130. Finally, trs65 mutant cells have low levels of Trs130 protein, and they are defective in the GEF activity of TRAPP II and the intracellular distribution of Ypt1 and Ypt31/32. Together, these results show that Trs65 plays a role in the Ypt GEF activity of TRAPP II in concert with the two other TRAPP II-specific subunits. Elucidation of the role played by Trs65 in intracellular trafficking is important for understanding how this process is coordinated with two other processes in which Trs65 is implicated: cell wall biogenesis and stress response.     

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.     

Trs65p, a subunit of the Ypt1p GEF TRAPPII, interacts with the Arf1p exchange factor Gea2p to facilitate COPI-mediated vesicle traffic

The TRAPP complexes are multimeric guanine exchange factors (GEFs) for the Rab GTPase Ypt1p. The three complexes (TRAPPI, TRAPPII, and TRAPPIII) share a core of common subunits required for GEF activity, as well as unique subunits (Trs130p, Trs120p, Trs85p, and Trs65p) that redirect the GEF from the endoplasmic reticulum-Golgi pathway to different cellular locations where TRAPP mediates distinct membrane trafficking events. Roles for three of the four unique TRAPP subunits have been described before; however, the role of the TRAPPII-specific subunit Trs65p has remained elusive. Here we demonstrate that Trs65p directly binds to the C-terminus of the Arf1p exchange factor Gea2p and provide in vivo evidence that this interaction is physiologically relevant. Gea2p and TRAPPII also bind to the yeast orthologue of the γ subunit of the COPI coat complex (Sec21p), a known Arf1p effector. These and previous findings reveal that TRAPPII is part of an Arf1p GEF-effector loop that appears to play a role in recruiting or stabilizing TRAPPII to membranes. In support of this proposal, we show that TRAPPII is more soluble in an arf1Δ mutant.     

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.     

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.     

The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.     

TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF

Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.     

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.     

Kinetic Analysis of the Guanine Nucleotide Exchange Activity of TRAPP, a Multimeric Ypt1p Exchange Factor

TRAPP complexes, which are large multimeric assemblies that function in membrane traffic, are guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1p. Here we measured rate and equilibrium constants that define the interaction of Ypt1p with guanine nucleotide (guanosine 5'-diphosphate and guanosine 5'-triphosphate/guanosine 5′-(β,γ-imido)triphosphate) and the core TRAPP subunits required for GEF activity. These parameters allowed us to identify the kinetic and thermodynamic bases by which TRAPP catalyzes nucleotide exchange from Ypt1p. Nucleotide dissociation from Ypt1p is slow (∼ 10^− 4 s^− 1) and accelerated > 1000-fold by TRAPP. Acceleration of nucleotide exchange by TRAPP occurs via a predominantly Mg²⁺-independent pathway. Thermodynamic linkage analysis indicates that TRAPP weakens nucleotide affinity by < 80-fold and vice versa, in contrast to most other characterized GEF systems that weaken nucleotide binding affinities by 4-6 orders of magnitude. The overall net changes in nucleotide binding affinities are small because TRAPP accelerates both nucleotide binding and dissociation from Ypt1p. Weak thermodynamic coupling allows TRAPP, Ypt1p, and nucleotide to exist as a stable ternary complex, analogous to strain-sensing cytoskeleton motors. These results illustrate a novel strategy of guanine nucleotide exchange by TRAPP that is particularly suited for a multifunctional GEF involved in membrane traffic.     

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.     

Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

Background  

Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits.     

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.     

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.     

Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.     

In Sickness and in Health: The Role of TRAPP and Associated Proteins in Disease

Transport protein particle (TRAPP) represents a series of related protein complexes that function in specific stages of inter‐organelle traffic. They share a core of subunits that can activate the GTPase Rab1 through a guanine nucleotide exchange factor (GEF) activity and are distinguished by ‘accessory’ subunits giving each complex its distinct function. The subunits are ubiquitously expressed and, thus, mutations in TRAPP subunits would be expected to be embryonic lethal. However, since its discovery, a number of subunits have been found to be mutated in several diverse human disorders suggesting that some of these subunits may have cell‐ or tissue‐specific functions. Here we review the current state of knowledge with respect to TRAPP subunit mutations in human disease. We suggest ideas to explain their tissue‐specific phenotypes and present avenues for future investigation.      

Multi-component protein complexes and Golgi membrane trafficking

Several large cytosolic protein complexes with multiple components have been proposed to play key roles in mediating or controlling membrane trafficking. Among these complexes, TRAPP, COG and GARP/VFT have been implicated in multiple steps of Golgi membrane trafficking. The importance of these complexes for Golgi function has been established using in vitro biochemical assays and yeast and mammalian somatic cell genetics. Furthermore, mutations in the genes encoding subunits of either TRAPP or the COG complex have been shown to be responsible for human genetic disorders. We here review recent studies exploring the structures and functions of these three oligomeric complexes.     

Trs20 is Required for TRAPP II Assembly

The modular TRAPP complexes act as nucleotide exchangers to activate the Golgi Ypt/Rab GTPases, Ypt1 and Ypt31/Ypt32. In yeast, TRAPP I acts at the cis‐Golgi and its assembly and structure are well characterized. In contrast, TRAPP II acts at the trans‐Golgi and is poorly understood. Especially puzzling is the role of Trs20, an essential TRAPP I/II subunit required neither for the assembly of TRAPP I nor for its Ypt1‐exchange activity. Mutations in Sedlin, the human functional ortholog of Trs20, cause the cartilage‐specific disorder SEDT. Here we show that Trs20 interacts with the TRAPP II‐specific subunit Trs120. Furthermore, the Trs20‐Trs120 interaction is required for assembly of TRAPP II and for its Ypt32‐exchange activity. Finally, Trs20‐D46Y, with a single‐residue substitution equivalent to a SEDT‐causing mutation in Sedlin, interacts with TRAPP I, but the resulting TRAPP complex cannot interact with Trs120 and TRAPP II cannot be assembled. These results indicate that Trs20 is crucial for assembly of TRAPP II, and the defective assembly caused by a SEDT‐linked mutation suggests that this role is conserved .     

Trs20 is Required for TRAPP III Complex Assembly at the PAS and its Function in Autophagy

The modular TRAPP complex acts as a guanine‐nucleotide exchange factor (GEF) for Ypt/Rab GTPases. Whereas TRAPP I and TRAPP II regulate the exocytic pathway, TRAPP III functions in autophagy. The TRAPP subunit Trs20 is not required for assembly of core TRAPP or its Ypt1 GEF activity. Interestingly, mutations in the human functional ortholog of Trs20, Sedlin, cause spondyloepiphyseal dysplasia tarda (SEDT), a cartilage‐specific disorder. We have shown that Trs20 is required for TRAPP II assembly and identified a SEDT‐linked mutation, Trs20‐D46Y, which causes a defect in this process. Here we show that Trs20 is also required for assembly of TRAPP III at the pre‐autophagosomal structure (PAS). First, recombinant Trs85, a TRAPP III‐specific subunit, associates with TRAPP only in the presence of Trs20, but not Trs20‐D46Y mutant protein. Second, a TRAPP complex with Ypt1 GEF activity co‐precipitates with Trs85 from wild type, but not trs20ts mutant, cell lysates. Third, live‐cell colocalization analysis indicates that Trs85 recruits core TRAPP to the PAS via the linker protein Trs20. Finally, trs20ts mutant cells are defective in selective and non‐selective autophagy. Together, our results show that Trs20 plays a role as an adaptor in the assembly of TRAPP II and TRAPP III complexes, and the SEDT‐linked mutation causes a defect in both processes.      

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab‐A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP‐bound) versus wild‐type or constitutively active (GTP‐bound) RAB‐A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab‐A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB‐A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB‐A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF?Rab interactions will be crucial to unravel the co‐ordination of plant membrane traffic.