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 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.

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 ...

Rab GTPases are key regulators of membrane traffic pathways within eukaryotic cells. They are specifically activated by guanine nucleotide exchange factors (GEFs), which convert them from their "inactive" GDP-bound form to the "active" GTP-bound form. In higher eukaryotes, proteins containing DENN-domains comprise a major GEF family. Here we describe at 2.1-Å resolution the first structure of a DENN-domain protein, DENND1B-S, complexed with its substrate Rab35, providing novel insights as to how DENN-domain GEFs interact with and activate Rabs. DENND1B-S is bi-lobed, and interactions with Rab35 are through conserved surfaces in both lobes. Rab35 binds via switch regions I and II, around the nucleotide-binding pocket. Positional shifts in Rab residues required for nucleotide binding may lower its affinity for bound GDP, and a conformational change in switch I, which makes the nucleotide-binding pocket more solvent accessible, likely also facilitates exchange.I n eukaryotic cells, material is transported between membranebound organelles or the plasma membrane by vesicles. These bud from the membrane of the donor organelle, travel to and are recognized at the target compartment, and finally fuse with it to deliver their cargo. Vesicle traffic is orchestrated by a large number of proteins, including coat proteins that facilitate budding, tethering complexes involved in vesicle recognition, SNARE proteins that drive membrane fusion, and small GTPases in the Rab/Ypt family that have regulatory roles (1). Different subsets of these proteins are involved in different transport pathways.Rab GTPases are key determinants of organelle identity and hence in ensuring that vesicular cargo is delivered to the correct destination (2-4). The Rabs themselves are activated at the appropriate membranes by Rab-specific guanine nucleotide exchange factors (GEFs), which facilitate the conversion of the Rab from its inactive, GDP-bound, to the active, GTP-bound state. The GEF interacts with its Rab substrate to lower its nucleotide-binding affinity, accelerating the departure of bound GDP and allowing GTP, which is 10-fold more abundant in the cell than GDP, to bind (5, 6). In their GTP-bound membrane associated form, Rabs then recruit additional proteins to mediate vesicle recognition, tethering, and fusion events.Mammals have more than 60 different Rabs (7), with GEFs that activate most of these yet to be identified. Recent data suggest that a large subset of mammalian Rabs, or at least 10 different Rab GTPases, are activated by proteins containing a DENN domain (8). In humans, eighteen different DENN-domain proteins have been identified, including members of the DENND1 through DENND5 families, the myotubularin-related proteins MTMR5 and 13, and the MAP-kinase activating death domain MADD (8, 9). Studies of the DENND1 family have shown that the DENN domain itself is sufficient for GEF activity (10, 11), where both DENND1A and 1B activate Rab35 for its role in the endocytic pathway (8, 10, 11). The substrate specificity of DENND1C rema...