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

Abstract

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.

Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes

“…10). This result would be expected given that Vps53 deletion abrogates GARP complex formation (10), whereas the Vps53 C terminus is not important for complex formation (2).…”

“…Interestingly, as for Vps53, the C-terminal helical bundles of Sec6, Cog4, and Dsl1 are not required for complex formation (2,3,15,16). Further, as with Vps53, the C terminus of Dsl1 is thought to interact with transport vesicles (3), and the Sec6 C terminus is thought to be important for binding to the target membrane (22).…”

“…GARP interacts directly with small GTPases that localize to the TGN [Ypt6/Rab6 (12, 13) and Arl1 (14)], and in humans, GARP also interacts with SNARE proteins specific to the retrograde pathway (t-SNARE proteins syntaxin 6, syntaxin 16, and v-SNARE Vamp4) and may facilitate formation of the SNARE complex (2) at the TGN, the site of vesicle fusion. The SNARE interaction requires N-terminal regions in the Vps53 and Vps54 proteins (2). Interaction with transport vesicles additionally requires the C terminus of Vps53, and deletion of that region disrupts retrograde traffic even as it does not affect GARP complex assembly (2).…”

“…The SNARE interaction requires N-terminal regions in the Vps53 and Vps54 proteins (2). Interaction with transport vesicles additionally requires the C terminus of Vps53, and deletion of that region disrupts retrograde traffic even as it does not affect GARP complex assembly (2).…”

“…These complexes may physically link the arriving vesicle and acceptor membrane and may also play a role in facilitating the assembly of the SNARE complexes (2,3), which catalyze vesicle fusion with the target organelle and cargo delivery. Distinct tethering complexes function in distinct transport pathways.…”