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.      

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.     

The adaptor function of TRAPPC2 in mammalian TRAPPs explains TRAPPC2-associated SEDT and TRAPPC9-associated congenital intellectual disability

Background

The TRAPP (Transport protein particle) complex is a conserved protein complex functioning at various steps in vesicle transport. Although yeast has three functionally and structurally distinct forms, TRAPPI, II and III, emerging evidence suggests that mammalian TRAPP complex may be different. Mutations in the TRAPP complex subunit 2 (TRAPPC2) cause X-linked spondyloepiphyseal dysplasia tarda, while mutations in the TRAPP complex subunit 9 (TRAPPC9) cause postnatal mental retardation with microcephaly. The structural interplay between these subunits found in mammalian equivalent of TRAPPI and those specific to TRAPPII and TRAPPIII remains largely unknown and we undertook the present study to examine the interaction between these subunits. Here, we reveal that the mammalian equivalent of the TRAPPII complex is structurally distinct from the yeast counterpart thus leading to insight into mechanism of disease.

Principal Findings

We analyzed how TRAPPII- or TRAPPIII- specific subunits interact with the six-subunit core complex of TRAPP by co-immunoprecipitation in mammalian cells. TRAPPC2 binds to TRAPPII-specific subunit TRAPPC9, which in turn binds to TRAPPC10. Unexpectedly, TRAPPC2 can also bind to the putative TRAPPIII-specific subunit, TRAPPC8. Endogenous TRAPPC9-positive TRAPPII complex does not contain TRAPPC8, suggesting that TRAPPC2 binds to either TRAPPC9 or TRAPPC8 during the formation of the mammalian equivalents of TRAPPII or TRAPPIII, respectively. Therefore, TRAPPC2 serves as an adaptor for the formation of these complexes. A disease-causing mutation of TRAPPC2, D47Y, failed to interact with either TRAPPC9 or TRAPPC8, suggesting that aspartate 47 in TRAPPC2 is at or near the site of interaction with TRAPPC9 or TRAPPC8, mediating the formation of TRAPPII and/or TRAPPIII. Furthermore, disease-causing deletional mutants of TRAPPC9 all failed to interact with TRAPPC2 and TRAPPC10.

Conclusions

TRAPPC2 serves as an adaptor for the formation of TRAPPII or TRAPPIII in mammalian cells. The mammalian equivalent of TRAPPII is likely different from the yeast TRAPPII structurally.     

Cryo‐EM structure of metazoan TRAPPIII,the multi‐subunit complex that activates the GTPase Rab1

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo‐EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII‐specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease‐causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur‐TRAPP.     

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.     

Biallelic variants in TRAPPC10 cause a microcephalic TRAPPopathy disorder in humans and mice

The highly evolutionarily conserved transport protein particle (TRAPP) complexes (TRAPP II and III) perform fundamental roles in subcellular trafficking pathways. Here we identified biallelic variants in TRAPPC10, a component of the TRAPP II complex, in individuals with a severe microcephalic neurodevelopmental disorder. Molecular studies revealed a weakened interaction between mutant TRAPPC10 and its putative adaptor protein TRAPPC2L. Studies of patient lymphoblastoid cells revealed an absence of TRAPPC10 alongside a concomitant absence of TRAPPC9, another key TRAPP II complex component associated with a clinically overlapping neurodevelopmental disorder. The TRAPPC9/10 reduction phenotype was recapitulated in TRAPPC10^-/- knockout cells, which also displayed a membrane trafficking defect. Notably, both the reduction in TRAPPC9 levels and the trafficking defect in these cells could be rescued by wild type but not mutant TRAPPC10 gene constructs. Moreover, studies of Trappc10^-/- knockout mice revealed neuroanatomical brain defects and microcephaly, paralleling findings seen in the human condition as well as in a Trappc9^-/- mouse model. Together these studies confirm autosomal recessive TRAPPC10 variants as a cause of human disease and define TRAPP-mediated pathomolecular outcomes of importance to TRAPPC9 and TRAPPC10 mediated neurodevelopmental disorders in humans and mice.     

Recessive TRAPPC11 Mutations Cause a Disease Spectrum of Limb Girdle Muscular Dystrophy and Myopathy with Movement Disorder and Intellectual Disability

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.     

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.     

TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane

Background

The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150^Glued, a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown.

Principle Findings

We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150^Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150^Glued via the same carboxyl terminal domain of p150^Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150^Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150^Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane.

Conclusions

These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.     

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.     

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.     

Phagophores evolve from recycling endosomes

The membrane origins of autophagosomes have been a key unresolved question in the field. The earliest morphologically recognizable structure in the macroautophagy/autophagy itinerary is the double-membraned cup-shaped phagophore. Newly formed phosphatidylinositol 3-phosphate (PtdIns3P) on the membranes destined to become phagophores recruits WIPI2, which, in turn, binds ATG16L1 to define the sites of autophagosome formation. Here we review our recent study showing that membrane recruitment of WIPI2 requires coincident detection of PtdIns3P and RAB11A, a protein that marks recycling endosomes. We found that multiple core autophagy proteins are more tightly associated with the recycling endosome compartment than with endoplasmic reticulum (ER)-mitochondrial contact sites. Furthermore, biochemical isolation of the recycling endosomes confirmed that they recruit autophagy proteins. Finally, fixed and live-cell imaging data revealed that recycling endosomes engulf autophagic substrates. Indeed, the sequestration of mitochondria after mitophagy stimulation depends on early autophagy regulators. These data suggest that autophagosomes evolve from the RAB11A compartment.     

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 .     

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

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 .     

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.     

Recombinant protein rVP1 upregulates BECN1-independent autophagy,MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration

The monocyte/macrophage is critical for regulating immune and antitumor responses. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus induces apoptosis and inhibits migration/metastasis of cancer cells. Here, we explored the effects of rVP1 on macrophages. Our results showed that rVP1 increased LC3-related autophagosome formation via WIPI1 and WIPI2 in a BECN1-independent manner. rVP1 treatment increased macrophage migration that was attenuated by knockdown of ATG5, ATG7, WIPI1 or WIPI2 and was abolished when both WIPI1 and WIPI2 were depleted. Treatment of macrophages with rVP1 increased matrix metalloproteinase-9 (MMP9) activity and phosphorylated mitogen-activated protein kinase 1/3 (MAPK1/3), two major mediators of cell migration. Knockdown of WIPI1, WIPI2, ATG5 and ATG7 but not BECN1 attenuated the rVP1-mediated increase in MAPK1/3 phosphorylation and MMP9 activity. These results indicated that rVP1 upregulated autophagy, MAPK1/3 phosphorylation and MMP9 activity to promote macrophage migration, which was dependent on WIPI1, WIPI2, ATG5 and ATG7 but not BECN1.     

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with β-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12–ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12–ATG5-ATG16L1 complex during initiation of autophagy.     

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.     

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.