Atg9 Vesicles Recruit Vesicle-tethering Proteins Trs85 and Ypt1 to the Autophagosome Formation Site

Atg9 is a transmembrane protein that is essential for autophagy. In the budding yeast Saccharomyces cerevisiae, it has recently been revealed that Atg9 exists on cytoplasmic small vesicles termed Atg9 vesicles. To identify the components of Atg9 vesicles, we purified the Atg9 vesicles and subjected them to mass spectrometry. We found that their protein composition was distinct from other organellar membranes and that Atg9 and Atg27 in particular are major components of Atg9 vesicles. In addition to these two components, Trs85, a specific subunit of the transport protein particle III (TRAPPIII) complex, and the Rab GTPase Ypt1 were also identified. Trs85 directly interacts with Atg9, and the Trs85-containing TRAPPIII complex facilitates the association of Ypt1 onto Atg9 vesicles. We also showed that Trs85 and Ypt1 are localized to the preautophagosomal structure in an Atg9-dependent manner. Our data suggest that Atg9 vesicles recruit the TRAPPIII complex and Ypt1 to the preautophagosomal structure. The vesicle-tethering machinery consequently acts in the process of autophagosome formation.     

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.     

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.     

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.     

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.     

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.     

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.     

The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes

The multimeric membrane-tethering complexes TRAPPI and TRAPPII share seven subunits, of which four (Bet3p, Bet5p, Trs23p, and Trs31p) are minimally needed to activate the Rab GTPase Ypt1p in an event preceding membrane fusion. Here, we present the structure of a heteropentameric TRAPPI assembly complexed with Ypt1p. We propose that TRAPPI facilitates nucleotide exchange primarily by stabilizing the nucleotide-binding pocket of Ypt1p in an open, solvent-accessible form. Bet3p, Bet5p, and Trs23p interact directly with Ypt1p to stabilize this form, while the C terminus of Bet3p invades the pocket to participate in its remodeling. The Trs31p subunit does not interact directly with the GTPase but allosterically regulates the TRAPPI interface with Ypt1p. Our findings imply that TRAPPII activates Ypt1p by an identical mechanism. This view of a multimeric membrane-tethering assembly complexed with a Rab provides a framework for understanding events preceding membrane fusion at the molecular level.     

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.      

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 .     

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.     

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

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

The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane

Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector–dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.     

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.     

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.      

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.     

Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.     

Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network

Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/trans-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.     

TRAPP complexes in membrane traffic: convergence through a common Rab

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.