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.      

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.     

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .     

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.     

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.     

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.     

Trs130 Participates in Autophagy Through GTPases Ypt31/32 in Saccharomyces cerevisiae

Trs130 is a specific component of the transport protein particle II complex, which functions as a guanine nucleotide exchange factor (GEF) for Rab GTPases Ypt31/32. Ypt31/32 is known to be involved in autophagy, although the precise mechanism has not been thoroughly studied. In this study, we investigated the potential involvement of Trs130 in autophagy and found that both the cytoplasm‐to‐vacuole targeting (Cvt) pathway and starvation‐induced autophagy were defective in a trs130ts (trs130 temperature‐sensitive) mutant. Mutant cells could not transport Atg8 and Atg9 to the pre‐autophagosomal structure/phagophore assembly site (PAS) properly, resulting in multiple Atg8 dots and Atg9 dots dispersed in the cytoplasm. Some dots were trapped in the trans‐Golgi. Genetic studies showed that the effect of the Trs130 mutation was downstream of Atg5 and upstream of Atg1, Atg13, Atg9 and Atg14 on the autophagic pathway. Furthermore, overexpression of Ypt31 or Ypt32, but not of Ypt1, rescued autophagy defects in trs130ts and trs65ts (Trs130‐HA Trs120‐myc trs65Δ) mutants. Our data provide mechanistic insight into how Trs130 participates in autophagy and suggest that vesicular trafficking regulated by GTPases/GEFs is important in the transport of autophagy proteins from the trans‐Golgi to the PAS.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

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.     

Mutational Analysis of the Yeast TRAPP Subunit Trs20p Identifies Roles in Endocytic Recycling and Sporulation

Trs20p is a subunit of the evolutionarily conserved TRAPP (TRAnsport Protein Particle) complex that mediates various aspects of membrane trafficking. Three TRAPP complexes have been identified in yeast with roles in ER-to-Golgi trafficking, post-Golgi and endosomal-to-Golgi transport and in autophagy. The role of Trs20p, which is essential for viability and a component of all three complexes, and how it might function within each TRAPP complex, has not been clarified to date. To begin to address the role of Trs20p we generated different mutants by random mutagenesis but, surprisingly, no defects were observed in diverse anterograde transport pathways or general secretion in Trs20 temperature-sensitive mutants. Instead, mutation of Trs20 led to defects in endocytic recycling and a block in sporulation/meiosis. The phenotypes of different mutants appear to be separable suggesting that the mutations affect the function of Trs20 in different TRAPP complexes.     

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.     

Atg11

Selective macroautophagy uses double-membrane vesicles, termed autophagosomes, to transport cytoplasmic pathogens, organelles and protein complexes to the vacuole for degradation. Autophagosomes are formed de novo by membrane fusion events at the phagophore assembly site (PAS). Therefore, precursor membrane material must be targeted and transported to the PAS. While some autophagy-related (Atg) proteins, such as Atg9 and Atg11, are known to be involved in this process, most of the mechanistic details are not understood. Previous work has also implicated the small Rab-family GTPase Ypt1 in the process, identifying Trs85 as a unique subunit of the TRAPPIII targeting complex and showing that it plays a macroautophagy-specific role; however, the relationship between Ypt1, Atg9 and Atg11 was not clear. Now, a recent report shows that Atg11 is a Trs85-specific effector of the Rab Ypt1, and may act as a classic coiled-coil membrane tether that targets Atg9-containing membranes to the PAS. Here, we review this finding in the context of what is known about Atg11, other Rab-dependent coiled-coil tethers, and other tethering complexes involved in autophagosome formation.