Dual Roles of the Mammalian GARP Complex in Tethering and SNARE Complex Assembly at the trans-Golgi Network

Tethering factors and SNAREs control the last two steps of vesicular trafficking: the initial interaction and the fusion, respectively, of transport vesicles with target membranes. The Golgi-associated retrograde protein (GARP) complex regulates retrograde transport from endosomes to the trans-Golgi network (TGN). Although GARP has been proposed to function as a tethering factor at the TGN, direct evidence for such a role is still lacking. Herein we report novel and specific interactions of the mammalian GARP complex with SNAREs that participate in endosome-to-TGN transport, namely, syntaxin 6, syntaxin 16, and Vamp4. These interactions depend on the N-terminal regions of Vps53 and Vps54 and the SNARE motif of the SNAREs. We show that GARP functions upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. However, interactions of GARP with SNAREs are insufficient to promote retrograde transport, because deletion of the C-terminal region of Vps53 precludes GARP function without affecting GARP-SNARE interactions. Finally, we present in vitro data consistent with a tethering role for GARP, which is disrupted by deletion of the Vps53 C-terminal region. These findings indicate that GARP orchestrates retrograde transport from endosomes to the TGN by promoting vesicle tethering and assembly of SNARE complexes in consecutive, independent steps.Conveyance of cargo among organelles of the secretory and endosomal-lysosomal pathways is mediated by transport vesicles that bud from a donor compartment and fuse with an acceptor compartment in a specific and regulated manner (2, 25, 42). The accuracy and efficiency of vesicle fusion with the target compartment are provided by the concomitant actions of at least three protein families: tethers, small GTPases, and SNAREs. The general view is that a transport vesicle first finds its target organelle through interaction with tethering factors and then fuses with it through assembly of SNARE proteins while small GTPases of the Rab and Arl subfamilies orchestrate multiple steps of the overall process (1, 38, 44). The mechanistic details, however, are far from being completely understood and might vary depending on the transport pathway considered.Tethering represents the first step in the interaction between a transport vesicle and its target membrane and results in the formation of physical links between two membranes that are bound to fuse. Two types of tethering factor, long coiled-coil proteins (e.g., p115, GCC185, and GM-130) and multisubunit complexes (e.g., HOPS/Vps-C, exocyst, COG, and GARP/VFT) have been implicated in nearly all vesicular transport routes (19, 38), although their direct role in connecting two opposing membranes has been documented for only a few (7, 40). Fusion is triggered by the assembly of SNAREs on the transport vesicle (v-SNAREs) with their cognate SNAREs on the target membranes (t-SNAREs) to form a SNARE pin or SNARE complex (12, 35). SNARE complex assembly involves the formation of a four-helix bundle that drives fusion of the two lipid bilayers (10, 14). Small GTPases participate in the initial recruitment of tethering factors and other peripherally associated effectors to specific locations on membranes, as well as in the subsequent fusion events (21). For example, the long coiled-coil protein GCC185 binds different GTPases, Rab9 on transport vesicles through the middle part and Rab6 and Arl1 at the trans-Golgi network (TGN) through the C-terminal part, thereby facilitating the recognition and connection of both membrane-bound compartments (11, 33). Other coiled-coil tethers have the ability to bind several different Rabs through domains that are not required for Golgi apparatus targeting. This supports a general model for a tentacular Golgi complex in which coiled-coil proteins capture and retain Rab-containing vesicles (33).In addition to bringing together transport vesicles with target organelles, tethers may also regulate SNARE complex assembly, thus coordinating these two steps of vesicular transport. Several examples of tether-SNARE interactions have been reported, but no consensus for a mechanism of interaction or functional significance has yet emerged. For example, the HOPS complex associates with v- and t-SNARE complexes on Saccharomyces cerevisiae vacuoles both before and after fusion (37). Sec6p, a member of the exocyst complex, binds to the plasma membrane t-SNARE Sec9p, preventing its interaction with the cognate t-SNARE Sso1p (34). The COG complex binds the Golgi t-SNARE syntaxin 5 and enhances intra-Golgi SNARE complex stability (29). The long coiled-coil protein p115 also stimulates SNARE complex assembly (30).The Golgi-associated retrograde protein (GARP) complex, also named the Vps fifty-three (VFT) complex, together with COG and the exocyst, belongs to the quatrefoil family of multisubunit tethering complexes (43), a structurally diverse group of peripheral membrane protein assemblies. Defects in the GARP, COG, or exocyst complexes cause accumulation of untethered vesicles that are scattered throughout the cytoplasm and contain different cargo proteins (18, 20, 45, 47). Direct proof of a tethering function for the GARP complex is still lacking, although its inactivation leads to defects consistent with a prominent role in the fusion of endosome-derived transport intermediates with the TGN (4-6, 20, 31). The yeast GARP complex is composed of four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Mutations in any of these subunits impair the retrieval of the secretory vesicle v-SNARE Snc1p and the carboxypeptidase Y receptor, Vps10p, from endosomes (5, 23, 32). The mammalian GARP complex also comprises Vps52, Vps53, and Vps54 subunits, but no Vps51 subunit has been identified to date (13). Depletion of the mammalian GARP complex prevents the delivery of Shiga toxin B subunit and the retrieval of TGN-localized proteins, such as TGN46, from endosomes to the TGN (20). Moreover, GARP depletion blocks the recycling of the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the TGN, leading to missorting of the CI-MPR cargo, lysosomal hydrolases, into the extracellular space (20). The essential nature of mammalian GARP function in endosome-to-TGN transport is highlighted by the embryonic lethality of mice with ablation of the Vps54 subunit gene (27) and the motor neuron degeneration of Wobbler mice bearing a Vps54 hypomorphic mutation (27).In yeast, the GARP subunit Vps51p specifically binds to the conserved N-terminal regulatory domain of the t-SNARE Tlg1p (5, 32). This finding led to the proposal that GARP tethers endosome-derived vesicles through its interaction with Tlg1p. However, deletions or point mutations that eliminate the binding of Vps51p to Tlg1p do not show any functional phenotype in vivo (8). Binding of Tlg1p to Vps51p is thus not essential for GARP-mediated vesicle tethering. In this work, we set out to study the possible link between the mammalian GARP complex and SNAREs. We found that GARP specifically and directly interacts with SNAREs that participate in the endosome-to-TGN retrograde route (i.e., syntaxin 6 [Stx6], Stx16, and Vamp4). These interactions depend on the fusion-inducing SNARE “motif” of the SNAREs and the N-terminal regions of Vps53 and Vps54. Functional analyses place the GARP complex upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. In addition, we demonstrate that the GARP complex has a vesicle tethering function independent of its interaction with the SNAREs.