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.

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.utophagy is a catabolic process in which damaged or superfluous cytoplasmic components are degraded in response to stress conditions; it is evolutionarily conserved in eukaryotes and is integrally involved in development and physiology (1, 2). The morphological hallmark of autophagy is the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasm. The autophagosomes then fuse with the lysosome, resulting in the degradation of the cargo. The mechanism of autophagosome formation is distinct from that used for vesicle formation in the secretory or endocytic pathways and is said to be de novo in that it does not occur by direct budding from a preexisting organelle. Instead, a nucleating structure, the phagophore, appears to expand by the addition of membrane possibly through vesicular fusion. One consequence of this mechanism is that it allows the sequestration of essentially any sized cargo, including intact organelles or invasive microbes, and this capability is critical to autophagic function. When autophagy is induced there is a substantial demand for membrane, and a major question in the field concerns the membrane origin; nearly every organelle has been implicated in this role (3). The early secretory pathway is likely one such membrane source for autophagy (4, 5).Rab GTPases are key regulators of membrane traffic that mediate multiple events including vesicle tethering and membrane fusion. These molecular switches cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The yeast Rab Ypt1, which is essential for ER-Golgi and Golgi traffic (6), is activated by the multimeric guanine nucleotide exchange factor (GEF) called TRAPP (7,8). Two forms of the TRAPP complexes have been identified (9). These two complexes share several subunits, including four (Bet3, Bet5, Trs23, and Trs31) that are essential to activate Ypt1. How each of th...

The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.

How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum. Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ ortholog), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.

The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagyspecific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the domeshaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.M acroautophagy is a highly conserved catabolic process that uses a specialized membrane trafficking pathway to target proteins and organelles for degradation (1). Defects in this process have been linked to a variety of human diseases, including neurodegenerative diseases such as Parkinson's disease (2). Macroautophagy is induced by a variety of physiological stresses and begins with the expansion of a cup-shaped nucleating membrane called the phagophore, or isolation membrane. As the phagophore expands, it engulfs intracellular proteins and membranes that are marked for degradation. This expanding membrane eventually closes to become an autophagosome, a double-membrane structure that seals its contents from the cytosol and delivers it to the lysosome or vacuole for degradation. A central unanswered question in the autophagy field is the mechanism by which the phagophore forms and matures into an autophagosome. Although it was once thought that the phagophore assembles de novo, recent evidence suggests it forms from a preexisting compartment. Compartments on the secretory pathway, including the endoplasmatic reticulum (ER) and Golgi complex, have been invoked in phagophore assembly (3, 4).A collection of ATG (autophagy-related) genes, the products of which regulate autophagy, were identified in the yeast Saccharomyces cerevisiae (1). Many of the Atg proteins needed for macroautophagy in yeast are shared with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that transports certain hydrolases into the vacuole. Both pathways require the sequestration of cargo within a double-membrane structure; however, only the macroautophagy pathway is conserved in higher eukaryotes (5). When autophagy is induced, ATG gene products assemble at the phagophore assembly site (PAS) in a hierarchical manner. The scaffold protein complex that organizes this site is the Atg17 complex (6, 7).Previous studies have shown that the transport protein particle (TRAPP) III complex, an autophagy-specic guanine nucleotide exchange factor (GEF) for ...

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 gamma1COP, 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.

When macroautophagy, a catabolic process that rids the cells of unwanted proteins, is initiated, 30-60 nm Atg9 vesicles move from the Golgi to the preautophagosomal structure (PAS) to initiate autophagosome formation. The Rab GTPase Ypt1 and its mammalian homolog Rab1 regulate macroautophagy and two other trafficking events: endoplasmic reticulum-Golgi and intra-Golgi traffic. How a Rab, which localizes to three distinct cellular locations, achieves specificity is unknown. Here we show that transport protein particle III (TRAPPIII), a conserved autophagy-specific guanine nucleotide exchange factor for Ypt1/Rab1, is recruited to the PAS by Atg17. We also show that activated Ypt1 recruits the putative membrane curvature sensor Atg1 to the PAS, bringing it into proximity to its binding partner Atg17. Since Atg17 resides at the PAS, these events ensure that Atg1 will specifically localize to the PAS and not to the other compartments where Ypt1 resides. We propose that Ypt1 regulates Atg9 vesicle tethering by modulating the delivery of Atg1 to the PAS. These events appear to be conserved in higher cells.GEF | membrane tethering

Ypt1 directly recruits the kinase Hrr25 to COPII vesicles to activate it in two different pathways: ER to Golgi and the catabolic macroautophagy pathway induced in response to cell stress.