Vesicular traffic between different organelles in the secretory and endocytic pathways involves the highly regulated docking and fusion of a transport vesicle with a specific target membrane. Current models suggest that at least some of the specificity of the docking and fusion process is provided by the recognition and pairing of small membrane-anchored proteins called SNAREs (Chen and Scheller, 2001
). As a single v-SNARE on the vesicle membrane assembles with three t-SNAREs on the target membrane, opposing membranes are drawn together, resulting in bilayer mixing and fusion. However, additional factors implicated in vesicle docking such as Rab GTPases and multisubunit tethering complexes may act at a step that precedes SNARE pairing and assembly (Whyte and Munro, 2002
). Although much debate has centered on which of these factors mediate the primary membrane recognition event, specificity may be determined not by a single component, but instead by a combination of SNAREs, Rabs, and tethers that together uniquely define a given transport step. Defining the nature of these interactions remains a major challenge.
The tethering factors identified to date are a diverse collection of long coiled-coil proteins and multisubunit complexes that are presumed to link vesicles to their target membrane in a step that promotes subsequent SNARE complex formation (Whyte and Munro, 2002
). Many if not all of these tethering factors interact with specific Rab proteins and therefore may couple membrane recognition to the activation of the Rab GTPase. What is less clear is whether tethering factors promote fusion simply by increasing the local concentration of vesicles at the correct target membrane, or if they act directly on the SNAREs to activate their assembly.
SNARE proteins have a membrane proximal coiled-coil SNARE motif that assembles to form a stable four-stranded helical bundle referred to as the core complex (Fernandez et al., 1998
). In addition, some SNARE proteins have an independently folded N-terminal domain that binds the C-terminal SNARE motif, thus preventing its assembly with other SNAREs (Dulubova et al., 1999
). Removal of the N-terminal domain of syntaxin accelerates SNARE complex formation in vitro (Parlati et al., 1999
). Therefore, an attractive model is that regulatory factors interact with the N-terminal domain of syntaxin-like t-SNAREs to modulate fusion.
Sec1p inhibits assembly of SNAREs at the plasma membrane by binding to syntaxin and holding it in the closed conformation (Dulubova et al., 1999
). Other Sec1 family members bind to the N-terminal regions of SNARE proteins but do not prevent SNARE formation and instead may contribute to its specificity (Peng and Gallwitz, 2002
; Dulubova et al., 2002
; Yamaguchi et al., 2002
). Furthermore, the N-terminal domains of many SNARE proteins are structurally related to that of syntaxin but not all of these form closed conformations in vitro (Dulubova et al., 2001
; Antonin et al., 2002
). Understanding the precise role of SNARE N-terminal domains in the fusion process will require a more detailed picture of their interactions with regulatory proteins.
In a few instances, tethering complexes unrelated to Sec1p have been shown to interact directly with t-SNARE N-terminal domains, and it may be that more such associations will be found as more of these complexes are identified and characterized. Whereas Rabs, SNAREs, and Sec1-like proteins are members of conserved families, tethering factors are somewhat more diverse and have been identified in functional studies rather than by sequence similarity. Candidate tethering factors exist for many but not all fusion events, and the subunit structure/components of these complexes are still being identified. The COG (conserved oligomeric Golgi) complex, also known as the Sec34/35 complex, is localized to the cis
-Golgi where it has been shown to have a tethering function in an in vitro assay (Morsomme and Riezman, 2002
; Ungar et al., 2002
). The recent identification of six new subunits of the COG complex led to the recognition that at least some of these components are related at the sequence level to components of two other large, multisubunit tethering factors: the exocyst and Vps52/53/54 complexes (Whyte and Munro, 2001
). COG has now been shown to be an effector of the Rab protein Ypt1p and to bind the cis
-Golgi syntaxin Sed5p (Suvorova et al., 2002
). The exocyst, an eight-subunit complex that directs the fusion of secretory vesicles with the plasma membrane, is the best studied of the three. An effector of the Sec4p Rab protein, the exocyst localizes to sites of polarized secretion where exocytosis takes place (Guo et al., 1999
), though no interaction with t-SNAREs has yet been reported.
Although the exocyst and COG complexes each have eight subunits, only three components of the Vps52/53/54 complex have previously been identified (Conibear and Stevens, 2000
). Vps52p is related to the exocyst component Sec3p, whereas Vps53p and Vps54p both contain an N-terminal domain that is shared by subunits of the other two docking complexes (Whyte and Munro, 2001
). Vps52p, Vps53p, and Vps54p are large coiled-coil containing proteins that are tightly associated in a stoichiometric complex localized to the late Golgi. The sorting defects of these mutants suggest that the Vps52/53/54 complex is required for the retrograde trafficking of vesicles from the late endosomal/prevacuolar compartment back to the late Golgi. The Vps52/53/54 complex has been identified as an effector of the Rab protein Ypt6p and also binds the N-terminal region of the TGN t-SNARE Tlg1p, further supporting the idea that this complex is a tethering factor for vesicle fusion at the TGN (Siniossoglou and Pelham, 2001
). Sequence relationships between components of these multisubunit tethering complexes suggest that all three may share a common mechanism of action. Therefore, understanding the interactions that link tethering complexes to SNARE proteins will be important for understanding both the mechanism of vesicle docking and the regulation of SNARE complex formation.
Here, we identify a fourth component of the Vps52/53/53 complex, Vps51p, that regulates its association with the N-terminal domain of the t-SNARE Tlg1p. In addition to a role in retrograde transport from the late endosomal/prevacuolar compartment, we find that all four subunits of this complex are also required for trafficking on a distinct transport pathway, from early endosomes back to the late Golgi. We have named this tetrameric complex the GARP (Golgi-associated retrograde protein) complex to reflect its role in the docking and fusion of multiple classes of endosome-derived vesicles with the late Golgi membrane.