Multisubunit Tethering Complexes Research Articles

The Architecture of the Multisubunit TRAPP I Complex Suggests a Model for Vesicle Tethering

Transport protein particle (TRAPP) I is a multisubunit vesicle tethering factor composed of seven subunits involved in ER-to-Golgi trafficking. The functional mechanism of the complex and how the subunits interact to form a functional unit are unknown. Here, we have used a multidisciplinary approach that includes X-ray crystallography, electron microscopy, biochemistry, and yeast genetics to elucidate the architecture of TRAPP I. The complex is organized through lateral juxtaposition of the subunits into a flat and elongated particle. We have also localized the site of guanine nucleotide exchange activity to a highly conserved surface encompassing several subunits. We propose that TRAPP I attaches to Golgi membranes with its large flat surface containing many highly conserved residues and forms a platform for protein-protein interactions. This study provides the most comprehensive view of a multisubunit vesicle tethering complex to date, based on which a model for the function of this complex, involving Rab1-GTP and long, coiled-coil tethers, is presented.

MBET3 is required for the organization of the TRAPP complexes

Large tethering complexes mediate the initial interaction of a transport vesicle with its target membrane. There are two forms of the multi-subunit tethering complex called TRAPP (TRAPPI and TRAPPII) that tether transport vesicles in different trafficking steps. Understanding TRAPP complex assembly and the protein–protein interactions among the subunits is an important step in elucidating the function of this tether. Here we have used several different approaches to study the protein–protein interactions among the subunits of the TRAPP complexes in both mammalian cells and yeast. Our studies have revealed that the low molecular weight subunits of TRAPP form two subcomplexes in vitro. One subcomplex contains mammalian BET3 (mBET3), mTRS31 and mTRS20, while mBET5 and mTRS23 form a second subcomplex. Furthermore, mBET3 directly interacts with mBET5 in vitro. Our findings also suggest that the TRAPPII-specific subunit, yTrs120p (yeast Trs120p), binds to the periphery of the TRAPPII complex. Although the non-essential TRAPP subunit yTrs33p interacts with yBet3p, yTrs33p is not required for TRAPP complex assembly. Together our findings indicate that BET3 plays an important role in the organization of the TRAPP complexes in both mammalian cells and yeast.

Concerted Auto-regulation in Yeast Endosomal t-SNAREs

In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated.

Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p.

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN. vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.

New miniature transistor

The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus.