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.     

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.     

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.     

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.      

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 Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.     

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.     

A Cycling Protein Complex Required for Selective Autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

Atg11 links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway

Proteins are selectively packaged into vesicles at specific sites and then delivered correctly to the various organelles where they function, which is critical to the proper physiology of each organelle. The precursor form of the vacuolar hydrolase aminopeptidase I is a selective cargo molecule of the cytoplasm to vacuole targeting (Cvt) pathway and autophagy. Precursor Ape1 along with its receptor Atg19 forms the Cvt complex, which is transported to the pre-autophagosomal structure (PAS), the putative site of Cvt vesicle formation, in a process dependent on Atg11. Here, we show that this interaction occurs through the Atg11 C terminus; subsequent recruitment of the Cvt complex to the PAS depends on central regions within Atg11. Atg11 was shown to physically link several proteins, although the timing of these interactions and their importance are unknown. Our mapping shows that the Atg11 coiled-coil domains are involved in self-assembly and the interaction with other proteins, including two previously unidentified partners, Atg17 and Atg20. Atg11 mutants defective in the transport of the Cvt complex to the PAS affect the localization of other Atg components, supporting the idea that the cargo facilitates the organization of the PAS in selective autophagy. These findings suggest that Atg11 plays an integral role in connecting cargo molecules with components of the vesicle-forming machinery.     

A cycling protein complex required for selective autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

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.     

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.     

Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid-based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth.     

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.     

Atg9 trafficking in autophagy-related pathways

The origin of the autophagosomal membrane and the lipid delivery mechanism during autophagy remain unsolved mysteries. Some important hints to these questions come from Atg9, which is the only integral membrane protein required for autophagosome formation and considered a membrane carrier in autophagy-related pathways. In S. cerevisiae, Atg9 cycles between peripheral sites and the pre-autophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle. We recently identified a peripheral membrane protein, Atg11, as a binding partner of Atg9, in a yeast two-hybrid screen. Based on our analysis we propose a model for Atg9 cycling. Our model suggests that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS along the actin cytoskeleton, and that this delivery process may serve as a membrane shuttle for vesicle assembly during yeast selective autophagy. Here, we discuss the implications of the model and present additional evidence that extends it with regard to membrane trafficking modes during pexophagy.     

Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.     

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 .     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.     

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins,Arl1 and Ypt6

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.     

Self-interaction is critical for Atg9 transport and function at the phagophore assembly site during autophagy

Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and is proposed to play a key role in membrane transport; the phagophore presumably expands by vesicular addition to form a complete autophagosome. It is not clear through what mechanism Atg9 functions at the phagophore assembly site (PAS). Here we report that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses reveal that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Our results support a model in which Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation.