View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Dispatch R407

Membrane transport: Tethers and TRAPPs Martin Lowe

Transport vesicles are tethered to their target TRAPP was identified using a combination of yeast genet- membrane prior to the interaction of v-SNAREs and ics and biochemistry. The TRAPP subunit Bet3p t-SNAREs across the membrane junction. Recent was first identified by virtue of its genetic interactions with evidence suggests tethering is a complex process the ER-to-Golgi t-SNARE Bet1p [3]. Epitope tagging of requiring multiple components. Bet3p and purification of the tagged protein from yeast lysates resulted in the isolation of the TRAPP complex [4]. Address: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK. TRAPP is highly conserved in evolution, with yeast sub- E-mail: [email protected] units sharing between 29 and 54% identity at the amino- acid level with their human counterparts [4,5]. There is no Current Biology 2000, 10:R407–R409 sequence homology with other known . Genetic 0960-9822/00/$ – see front matter interactions between the TRAPP subunits Bet3p and © 2000 Elsevier Science Ltd. All rights reserved. Bet5p with several ER-to-Golgi SNAREs, Uso1p (a Golgi tethering factor), and Yprt1p (a member of the Rab family Transport along the secretory and endocytic pathways in of small with a demonstrated role in vesicle eukaryotic cells is mediated by vesicular carriers, which docking), strongly suggested that TRAPP was involved in bud from one compartment and fuse with the next com- a late stage in ER-to-Golgi transport [3,6]. This was con- partment in the pathway. Vesicle targeting and fusion firmed by in vitro experiments which showed that TRAPP must be tightly controlled to ensure an ordered flow acts after vesicle budding from the ER but before fusion through the pathway and prevent inappropriate mixing of with the Golgi complex [4]. compartments. Vesicle docking was originally thought to be mediated by ‘SNAREs’ — integral membrane pro- TRAPP has been localised by subcellular fractionation and teins found predominantly on vesicle (v-SNARE) or light microscopy to the Golgi complex in yeast, and unlike target (t-SNARE) membranes [1]. The ability of the ER-to-Golgi SNAREs, it appears not to cycle constitu- SNAREs to form specific v-SNARE–t-SNARE pairs tively through the ER [2]. Furthermore, although TRAPP made them attractive candidates for mediating docking. is a peripheral membrane protein complex, it is tightly asso- However, the finding that some SNAREs can function at ciated with Golgi membranes and is even resistant to more than one step in vivo, the ability of some SNAREs extraction with the non-ionic detergent Triton X-100 [5]. to interact promiscuously in vitro, and the localisation of These findings are consistent with the idea that TRAPP SNAREs to more than one compartment suggested that might determine the site of delivery of ER-derived trans- SNAREs cannot be the sole determinants of docking port vesicles. A role for TRAPP in vesicle tethering was specificity. This made it likely that other components subsequently demonstrated using an in vitro transport assay would impart specificity, and the identification of tether- designed to distinguish between the attachment and fusion ing factors such as the TRAPP complex recently of ER-derived vesicles with Golgi membranes [2]. Inhibi- described by Ferro-Novick and colleagues [2] suggests tion of SNARE assembly prevented fusion but not vesicle this is indeed the case. attachment, while depletion of Bet3p prevented both attachment and fusion. These were restored by addition of Tethering is defined as the initial attachment of a Bet3p-containing cytosol, suggesting Bet3p was the missing transport vesicle with its target membrane, an event that component, but formal proof awaits the re-addition of puri- occurs prior to pairing of SNAREs (the acronym stands for fied native TRAPP, which has so far been unavailable. ‘SNAP receptors’, where SNAP is ‘soluble NSF attach- ment protein’ and NSF is the ATPase ‘N-ethylmaleimide- In addition to TRAPP, two other factors are required for sensitive factor’). Tethering factors have now been tethering of ER-derived vesicles to Golgi membranes in identified for a number of different membrane transport yeast: Uso1p and the Sec34p–Sec35p complex (Figure 1). steps, and although they appear diverse in terms of Uso1p is a parallel homodimer with two globular heads primary structure, they share common characteristics in and an extended coiled-coil tail [7]. Unlike TRAPP, being either elongated coiled-coil proteins or multi- Uso1p is largely cytosolic and is recruited onto membranes subunit complexes. The latter category is typified by during transport in a Ypt1p-dependent manner [8]. Uso1p TRAPP (for ‘transport protein particle’), a complex of ten appears to be the major cytosolic tethering factor for ER- subunits which has recently been identified as an essential derived vesicles, as purified Uso1p alone can substitute for factor for tethering of (ER)-derived total cytosol in an in vitro vesicle tethering assay [8]. While transport vesicles to Golgi membranes [2]. the subunit composition of the approximately 700 kDa R408 Current Biology Vol 10 No 11

Figure 1

Transport vesicle

Coiled-coil tethering protein v-SNARE (Uso1p) Sec34p–Sec35p

?

Target specifier (TRAPP) Rab-GTP (Ypt1p) Sly1p t-SNARE Target membrane Pre-tethering Tethering SNARE pairing Current Biology

A model for vesicle docking using ER-to-Golgi transport in yeast as Sec34p—Sec35p complex would also somehow participate in this an example. The site of vesicle docking would be specified by a tethering event. Tethering of the vesicle would trigger release of the tethering complex restricted to that location (TRAPP). Binding of a inhibitory t-SNARE-binding protein Sly1p, making the t-SNARE GTP-bound Rab protein (Ypt1p) to that site would stimulate competent for binding to a v-SNARE. Reorganisation of the recruitment of an elongated coiled-coil tethering protein (Uso1p). tethering complex would then facilitate v-SNARE—t-SNARE pairing This in turn would facilitate capture of the transport vesicle, forming across the membrane junction, which would lead either directly or a bridge between the vesicle and target membrane. The indirectly to membrane fusion.

Sec34p–Sec35p complex is not clear [9,10], it is known to Tethering of post-Golgi secretory vesicles to the plasma peripherally associate with membranes [9–11] and deple- membrane in yeast requires the heptameric ‘Exocyst’ tion of either Sec34p or Sec35p prevents tethering of ER complex, which is reminiscent of TRAPP [16]. The vesicles in the same in vitro tethering assay used to show ‘Exocyst’ subunit Sec3p is restricted to sites of polarised the tethering function of Uso1p [9,11]. Neither Sec34p or secretion, suggesting that it marks plasma membrane Sec35p share any similarity with TRAPP subunits, indi- docking sites for secretory vesicles [17]. Similarly, the cating these are distinct complexes. The three ER-to- mammalian ‘Exocyst’ homologue — the Sec6–Sec8 Golgi tethering factors interact genetically, consistent with complex — is recruited to cell-cell contacts, where it spec- their role being at the same stage in transport, but whether ifies delivery of vesicles to the basolateral membrane of any physical association exists is unclear, as is the underly- epithelial cells [18]. Electron microscopy studies have ing mechanism by which the three factors co-operate in shown that the Sec6–Sec8 complex has a 30 nanometre the tethering reaction. elongated structure, with two arms projecting outwards from one end, suggestive of a linker function [19]. Does tethering of ER-derived vesicles to Golgi mem- Whether the Sec6–Sec8 complex acts in conjunction with branes share common features with tethering events in a coiled-coil protein is not known, but a physical associa- other membrane transport steps? One common feature is tion with septin filaments has been shown [19] suggesting the requirement for a Rab protein, with genetic and/or these two structures may co-operate in vesicle tethering at physical interaction between the tethering factor and a the plasma membrane. Rab protein demonstrated in nearly all cases. Another feature common to most transport steps is the involve- The identification of three different tethering complexes ment of an elongated coiled-coil protein structurally operating at one transport step suggests that tethering is similar to Uso1p [12–15]. These are predominantly cytoso- more complex than a simple cross-bridging of two mem- lic proteins which are recruited onto membranes in a Rab branes. Tethering might rather be thought of as a series of protein-dependent manner. None of the coiled-coil events that leads to pairing of v-SNAREs and t-SNAREs tethers (apart from Uso1p) have so far been shown to act at the target membrane (Figure 1). Important steps might in conjunction with other oligomeric tethering complexes include: first, specification of the vesicle delivery site; (analogous to TRAPP or Sec34p–Sec35p). second, the recruitment of components capable of Dispatch R409

initiating vesicle capture; third, the formation of a bridge 18. Grindstaff KK, Yeaman C, Anandasabapathy N, Hsu SC, Rodriguez-Boulan E, Scheller RH, Nelson WJ: Sec6/8 complex is between the vesicle and target membrane; fourth, a recruited to cell-cell contacts and specifies transport vesicle conformational change to allow the v-SNAREs and delivery to the basal-lateral membrane in epithelial cells. Cell t-SNAREs to come into close enough proximity to inter- 1998, 93:731-740. 19. Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH: act; and fifth, break-up of tethers to prime the tethering Subunit composition, protein interactions, and structures of the proteins for another round of transport. mammalian brain sec6/8 complex and septin filaments. Neuron 1998, 20:1111-1122. The use of multiple tethering factors is likely to ensure the high selectivity and spatial and temporal regulation of membrane targeting. Precisely how multiple tethering factors co-operate to ensure proper targeting awaits the further characterisation of known tethering proteins, iden- tification of other proteins with which they interact, and the development of more refined functional assays to study the functions of these proteins in greater detail.

References 1. Söllner T, Whitehart SW, Brunner M, Erdjumentbromage H, Geromanos S, Tempst P, Rothman JE: SNAP receptors implicated in vesicle targeting and fusion. Nature 1993, 362:318-324. 2. Barrowman J, Sacher M, Ferro-Novick S: TRAPP stable associates with the Golgi and is required for vesicle docking. EMBO J 2000, 19:862-869. 3. Rossi G, Kolstad K, Stone S, Palluault F, Ferro-Novick S: BET3 encodes a novel hydrophilic protein that acts in conjunction with yeast SNAREs. Mol Biol Cell 1995, 6:1769-1780. 4. Sacher M, Jiang Y, Barrowman J, Scarpa A, Burston J, Zhang L: TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J 1998, 17:2494-2503. 5. Sacher M, Barrowman J, Schieltz D, Yates III, JR, Ferro-Novick: Identification of five new subunits of TRAPP. Eur J Cell Biol 2000, 79:71-80. 6. Jiang Y, Scarpa A, Zhang L, Stone S, Feliciano E, Ferro-Novick S: A high copy suppressor screen reveals genetic interactions between BET3 and a new gene. Evidence for a novel complex in ER-to-Golgi transport. Genetics 1998, 149:833-841. 7. Yamakawa H, Seog DH, Yoda K, Yamasaki M, Wakabayashi T: Uso1 protein is a dimer with 2 globular heads and a long coiled-coil tail. J Struct Biol 1996, 116:356-365. 8. Cao X, Ballew N, Barlowe C: Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J 1998, 17:2156-2165. 9. VanRheenen SM, Cao X, Sapperstein SK, Chiang EC, Lupashin VV, Barlowe C, Waters MG: Sec34p, a protein required for vesicle tethering to the yeast , is in a complex with Sec35p. J Cell Biol 1999, 147:729-742. 10. Kim DW, Sacher M, Scarpa A, Quinn AM, Ferro-Novick S: High-copy suppressor analysis reveals a physical interaction between Sec34p and Sec35p, a protein implicated in vesicle docking. Mol Biol Cell 1999, 10:3317-3329. 11. VanRheenen SM, Cao X, Lupashin VV, Barlowe C, Waters MG: Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking. J Cell Biol 1998, 141:1107-1119. 12. Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G: The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell 1997, 89:445-455. 13. Barr FA: A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr Biol 1999, 9:381-384. 14. Christoforidis S, McBride HM, Burgoyne RD, Zerial M: The Rab5 effector EEA1 is a core component of endosome docking. Nature 1999, 397:621-625. 15. Peterson MR, Burd CG, Emr SD: Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome. Curr Biol 1999, 9:159-162. 16. TerBush DR, Maurice T, Roth D, Novick P: The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 1996, 15:6483-6494. 17. Finger FP, Hughes TE, Novick P: Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 1998, 92:559-571.