Molecular architecture of the TRAPPII complex and implications for vesicle tethering

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 10/2010; 17(11):1298-304. DOI: 10.1038/nsmb.1914
Source: PubMed


Multisubunit tethering complexes participate in the process of vesicle tethering--the initial interaction between transport vesicles and their acceptor compartments. TRAPPII (named for transport protein particle II) is a highly conserved tethering complex that functions in the late Golgi apparatus and consists of all of the subunits of TRAPPI and three additional, specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle EM. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer, which is responsible for dimerization. TRAPPII binds the Ypt1 GTPase and probably uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss the implications of the structure of TRAPPII for coat interaction and TRAPPII-associated human pathologies.

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Available from: Calvin Yip, Mar 17, 2015
    • "This idea is based on two types of evidence. First, negative biochemical evidence for lack of GEF activity and co-precipitation of Ypt31/Ypt32 with TRAPP II (Cai et al., 2008; Yip et al., 2010). However, both these studies used GST-tagged Ypt31/ Ypt32, whereas all our studies that show activity and co-precipitation used untagged Ypts (Jones et al., 2000; Morozova et al., 2006). "
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    ABSTRACT: Abstract Ypt/Rab GTPases are key regulators of all membrane trafficking events in eukaryotic cells. They act as molecular switches that attach to membranes via lipid tails to recruit their multiple downstream effectors, which mediate vesicular transport. Originally discovered in yeast as Ypts, they were later shown to be conserved from yeast to humans, where Rabs are relevant to a wide array of diseases. Major principles learned from our past studies in yeast are currently accepted in the Ypt/Rab field including: (i) Ypt/Rabs are not transport-step specific, but are rather compartment specific, (ii) stimulation by nucleotide exchangers, GEFs, is critical to their function, whereas GTP hydrolysis plays a role in their cycling between membranes and the cytoplasm for multiple rounds of action, (iii) they mediate diverse functions ranging from vesicle formation to vesicle fusion and (iv) they act in GTPase cascades to regulate intracellular trafficking pathways. Our recent studies on Ypt1 and Ypt31/Ypt32 and their modular GEF complex TRAPP raise three exciting novel paradigms for Ypt/Rab function: (a) coordination of vesicular transport substeps, (b) integration of individual transport steps into pathways and (c) coordination of different transport pathways. In addition to its amenability to genetic analysis, yeast provides a superior model system for future studies on the role of Ypt/Rabs in traffic coordination due to the smaller proteome that results in a simpler traffic grid. We propose that different types of coordination are important also in human cells for fine-tuning of intracellular trafficking, and that coordination defects could result in disease.
    Critical Reviews in Biochemistry and Molecular Biology 02/2015; 50(3):1-9. DOI:10.3109/10409238.2015.1014023 · 7.71 Impact Factor
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    • "TRAPPI mediates ER to early Golgi trafficking whereas the TRAPPII complex performs late Golgi vesicle tethering for a diverse group of membrane proteins [17]. Despite a wealth of functional insight, and although the overall architecture of the TRAPPII complex has been determined [49], the molecular structure of individual TRAPPII complex subunits and the biochemical basis for how they recognize their targets remain unknown. "
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    ABSTRACT: Background Assembly of primary cilia relies on vesicular trafficking towards the cilium base and intraflagellar transport (IFT) between the base and distal tip of the cilium. Recent studies have identified several key regulators of these processes, including Rab GTPases such as Rab8 and Rab11, the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances, the exact molecular mechanisms by which these proteins interact and target to the basal body to promote ciliogenesis are not fully understood. Results We surveyed the human proteome for novel ASPM, SPD-2, Hydin (ASH) domain-containing proteins. We identified the TRAPP complex subunits TRAPPC8, -9, -10, -11, and -13 as novel ASH domain-containing proteins. In addition to a C-terminal ASH domain region, we predict that the N-terminus of TRAPPC8, -9, -10, and -11, as well as their yeast counterparts, consists of an α-solenoid bearing stretches of multiple tetratricopeptide (TPR) repeats. Immunofluorescence microscopy analysis of cultured mammalian cells revealed that exogenously expressed ASH domains, as well as endogenous TRAPPC8, localize to the centrosome/basal body. Further, depletion of TRAPPC8 impaired ciliogenesis and GFP-Rabin8 centrosome targeting. Conclusions Our results suggest that ASH domains confer targeting to the centrosome and cilia, and that TRAPPC8 has cilia-related functions. Further, we propose that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery.
    Cilia 06/2014; 3(1):6. DOI:10.1186/2046-2530-3-6
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    • "Furthermore, most methods for labeling termini are not well suited for labeling internal sites because the labels are necessarily large globular domains (e.g., GFP) and need to fold independently of the protein in which they are inserted without disrupting the overall tertiary/quaternary structure of the subject particle. An additional limitation of most labeling methods is that terminal labels tend to be flexible (Lees et al., 2010; Shanks et al., 2010; Yip et al., 2010), and thus, the attachment site can be difficult to precisely locate by EM. Flexibility stems from unstructured elements commonly located at protein termini and also because the label is tethered to only one point in the subject protein. "
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    ABSTRACT: Single-particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition of individual protein domains. Labels that can be visualized by EM have been developed for protein termini, but tagging internal domains remains a challenge. We describe a robust strategy for determining the position of internal sites within EM maps, termed domain localization by RCT sampling (DOLORS). DOLORS uses monovalent streptavidin added posttranslationally to tagged sites in the target protein. Internal labels generally display less conformational flexibility than terminal labels, providing more precise positional information. Automated methods are used to rapidly generate assemblies of unique 3D models allowing the attachment sites of labeled domains to be accurately identified and thus provide an overall architectural map of the molecule.
    Structure 12/2012; 20(12):1995-2002. DOI:10.1016/j.str.2012.10.019 · 5.62 Impact Factor
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