SM proteins and tethering complexes are two separate families of proteins known to be intimately involved in the final stages of membrane trafficking, through both their recognition of vesicle and target membranes and their interactions with the membrane-fusing SNARE proteins. However, their mechanisms of action have been enigmatic (Carr and Rizo, 2010
; Yu and Hughson, 2010
), possibly because these proteins have been studied independently.
Here we uncover a molecular link between these families—the exocyst subunit Sec6 directly binds the exocytic SM protein Sec1. Importantly, Sec6 is the exocyst subunit that binds the t-SNARE Sec9, thus inhibiting Sec9 from forming SNARE complexes with its partner syntaxin Sso1 in vitro (Sivaram et al., 2005
). Sec6–Sec1 is compatible with Sec6–exocyst interactions, whereas Sec6 and Sec9 interact in the absence of the other exocyst subunits (). Furthermore, the N-terminal domain of Sec6 is required for both Sec6–Sec1 and Sec6–Sec9 (Sivaram et al., 2005
; ), suggesting that the binding sites on Sec6 may be overlapping for Sec1 and Sec9. Consistent with these data, we were unable to detect a Sec6-Sec1-Sec9 ternary complex () or any Sec6–Sec1 complexes in the presence of Sec9 (). These data suggest a model in which Sec6–exocyst coordinates with Sec1 to regulate SNARE complex assembly.
Sec1 had appeared to be an outlier of the SM protein family; unlike other SM proteins, it did not seem to interact with individual SNARE proteins (Carr et al., 1999
; Scott et al., 2004
; Togneri et al., 2006
), but instead bound ternary Sso1-Sec9-Snc2 complexes (Togneri et al., 2006
). In fact, Sec1 and Sso1 lack the conserved binding sites observed for SM proteins bound to the N-peptides of their cognate syntaxins (Bracher and Weissenhorn, 2002
; Peng and Gallwitz, 2004
; Arac et al., 2005
; Carpp et al., 2006
; Hu et al., 2007
; Burkhardt et al., 2008
; Furgason et al., 2009
; Xu et al., 2010
). These data suggested that Sec1 has a role downstream of SNARE complex assembly, perhaps driving SNARE-mediated membrane fusion (Scott et al., 2004
). However, characterization of novel Sec1 mutants recently uncovered a role for Sec1 prior to SNARE complex assembly (Hashizume et al., 2009
). Our in vitro binding assays revealed weak interactions with both Sso1 and Sec9 (Kd
> 10 μM; ), supporting the idea that Sec1 and Sec6–exocyst may work together to facilitate proper SNARE complex assembly.
We propose that the Sec6–Sec1 interaction is critical for targeting Sec1 to sites of polarized secretion. The weak interactions between Sec1 and the individual SNARE proteins are not likely to recruit Sec1; furthermore, neither Sso1 nor Sec9 localizes specifically to sites of secretion on the plasma membrane (Brennwald et al., 1994
). In contrast, Sec6 is critical for anchoring the exocyst complex at sites of secretion on the plasma membrane (Songer and Munson, 2009
). We propose that exocyst-bound Sec6 recruits Sec1 to sites of secretion, where it is handed off to newly formed ternary SNARE complexes for membrane fusion (Scott et al., 2004
). Consistent with this idea, destabilization or mislocalization of the exocyst complex, for example, in the sec6-4
mutant, leads to loss of Sec1 localization (Grote et al., 2000
In contrast to Sec1 recruitment, Sec6 is not a likely candidate to be the factor that recruits Sec9 to the plasma membrane. Unlike its homologue SNAP-25, Sec9 has no lipid modifications to anchor it and thus requires a binding partner on the plasma membrane. Moreover, Sec9 is dispersed throughout the plasma membrane, whereas Sec6 and Sec1 are specifically polarized to sites of secretion. Instead, we propose that Sec6–Sec9 holds Sec9 in an inactive state (perhaps an assembly intermediate) at sites of secretion, where Sso1 becomes activated, to prevent premature or inappropriate SNARE assembly and vesicle fusion. The small amount of Sec6 that is Sec9 bound () is consistent with the low abundance of Sec9 localized to sites of exocytosis.
What releases Sec6 from Sec9 to drive SNARE complex assembly? At sites of secretion, a small amount of unbound Sso1 and Sec9 would be present. The factor that opens Sso1 would also be localized to these sites; any SNARE complex assembly caused by premature opening of Sso1 at these sites would be blocked by the Sec6–Sec9 interaction. Vesicle arrival would then trigger exocyst assembly (Boyd et al., 2004
), leading to the release of Sec6 from Sec9, concomitant with Sso1 opening. These changes may also occur concurrently with Sec1 recruitment to provide coordinated regulation of vesicle arrival, tethering, and production of fusion-ready SNARE complexes. Individually, the Sec6–exocyst interactions are weak and do not compete with Sec9 for binding Sec6; however, the multivalent combination of exocyst subunits would likely be strong enough to release Sec9. Furthermore, it is possible that in the cell, Sec1 may exist at a high enough local concentration at sites of secretion to drive Sec9 release, likely in conjunction with exocyst assembly.
The exocyst and Sec1 are also likely to interact with the Sso1 opener, whose identity is unknown. Evidence suggests that Caenorhabditis elegans
Unc13 and the mammalian Munc13 may provide this function for Munc18-bound syntaxin-1 (Betz et al., 1997
; Richmond et al., 2001
; Guan et al., 2008
; Ma et al., 2011
). There is no obvious homologue for Munc13 in yeast; the most likely candidate(s) are members of the exocyst complex, which were recently shown to be structurally homologous to the MUN domain of Munc13 (Li et al., 2011
). However, no opening activity has been directly observed for any exocyst subunit or the complex.
The cooperation between Sec1 and the exocyst represents a general phenomenon for SM proteins and their partner tethering complexes. For example, the vacuolar SM protein Vps33 is an essential component of the class C/HOPS complex (Sato et al., 2000
; Seals et al., 2000
). Sly1, the SM protein that regulates traffic between the endoplasmic reticulum and Golgi, was recently shown to interact with the assembled COG complex (Laufman et al., 2009
). Interactions between tethering complexes and SM proteins have been shown to be important for proofreading SNARE complexes and/or for SNARE complex formation (Koumandou et al., 2007
; Starai et al., 2008
; Laufman et al., 2009
), suggesting that Sec1-Sec6-exocyst may add further regulation and specificity to the fusion process.
Elucidation of these interactions is a critical step toward understanding the molecular mechanism(s) underpinning the regulation of specific SNARE complex assembly. Future studies will require reconstitution and testing of assembled exocyst complexes and generation of novel separation-of-function mutants to further characterize these events. In addition, tethering complexes and SM proteins are not the only regulators at these sites; in yeast, the tomosyn homologue Sro7 interacts with Sec9 and the exocyst to form a parallel regulatory pathway (Lehman et al., 1999
; Zhang et al., 2005
; Grosshans et al., 2006
), and a fungal-specific Sec1 cofactor, Mso1, may bridge Sec1 and Sso1 to facilitate SM recruitment and SNARE regulation (Castillo-Flores et al., 2005
; Weber et al., 2010
; Weber-Boyvat et al., 2011
). Their precise role in these processes and their potential cooperation with the exocyst and Sec1 require further study. Identification of the factor responsible for triggering the open conformation of Sso1, in combination with the known regulatory proteins and SNAREs, will lead to a more complete picture of how SNAREs are inhibited and then released at the proper time and place for specific vesicle docking and fusion.