The Sec1p/Munc18 (SM) protein Munc18a binds to the neuronal t-SNARE Syntaxin1A and inhibits SNARE complex assembly. Tomosyn, a cytosolic Syntaxin1A-binding protein, is thought to regulate the interaction between Syntaxin1A and Munc18a, thus acting as a positive regulator of SNARE assembly. This dissertation has investigated the interactions between b-Tomosyn and the adipocyte SNARE complex involving Syntaxin4/SNAP-23A/AMP-2 and the SM protein Munc18c, in vitro, and the potential involvement of Tomosyn in regulating the translocation of GLUT4 containing vesicles, in vivo.
Our data showed that b-Tomosyn is ubiquitously expressed and has several interesting structural features, including a SNARE-like domain, a series of WD-40 repeats and is a putative substrate for phosphorylation by several different kinases. Further, we demonstrated that Tomosyn interacted with the adipocyte t-SNARE Syntaxin4 and formed a high affinity ternary complex with Syntaxin4 and SNAP-23, which was competitively inhibited by VAMP-2. Using a yeast two-hybrid assay we confirmed that the isolated VAMP-2-like domain in Tomosyn facilitated the interaction with Syntaxin4.
Overexpression of b-Tomosyn in 3T3-L1 adipocytes inhibited the translocation of enhanced green fluorescent protein linked GLUT4 to the plasma membrane. The degree of inhibition was similar to that previously reported for overexpression of Munc18c or the cytoplasmic domain of VAMP-2. We verified the intracellular localisation of Tomosyn in adipocytes using a differential centrifugation technique and an immunofluorescence assay. Data showed that although b- Tomosyn was predominantly cytosolic, there was significant amounts of Tomosyn in the plasma membrane and low density microsome enriched fractions. In addition, the plasma membrane associated pool of Tomosyn was sensitive to treatment with the alkylating agent NEM, which suggested that this fraction of Tomosyn was directly or indirectly associated with the SNAREs in intact cells. The NEM data also placed the actions of Tomosyn in the SNARE assembly cycle at the pre-fusion stage, which is consistent with a putative role for Tomosyn in tethering, in t-SNARE activation and as a binary complex chaperone.
Importantly, this study showed that the SM protein Munc18c not only interacted with the cognate t-SNARE Syntaxin4, but also with the Syntaxin4 containing SNARE complexes, including the Syntaxin4/Tomosyn complex. These data have several implications. First, binding of the SM protein to the SNARE complex represents a unified feature in all eukaryotic cells. Second, these data showed that functional characteristics of the mammalian exocytic SM proteins Munc18a and Munc18c may have diverged during the course of evolution, where Munc18a represents a uniquely regulated SM protein in highly specialised cells. Third, because Mun18c and TomosynA/AMP-2 binding to the t-SNARE were not mutually exclusive events, Syntaxin4 may not adopt the closed conformation which prohibits SNARE complex fomnation, thus, Tomosyn is unlikely to function as a lever to selectively remove Munc18c.
We have demonstrated that Munc18c and Syntaxin4 are largely segregated in the adipocyte plasma membrane. While Munc18c is predominantly present in Triton soluble membrane domains, the t-SNAREs are clustered in lipid-raft microdomains. Only a fraction (~ 30%) of Syntaxin4 and Munc18c are found in a stable complex, in vivo, consistent with studies showing several other Syntaxin4- containing complexes, including Synip and Csp, in adipocytes. Our data showed that Munc18c and SNAP-23 binding were mutually exclusive in adipocytes, this was surprising as Munc18c bound to the t-SNARE complex in vitro. However, these data may suggest that Munc18c binds selectively to the cis-SNARE- and t- SNARE-complexes post-fusion, and may suggest a role for Munc18c in the recycling of the individual SNAREs and priming of Syntaxin4.
In conclusion, this dissertation has extended the knowledge of the mammalian SM protein Munc18c in SNARE assembly in adipocytes. Consistent with a recent report concerning the yeast SM protein Vps45p, we propose a role for Munc18c in the priming (NSF-dependent dissociation) of the SNARE complex in adipocytes. We also demonstrated a functional role for Tomosyn in SNARE assembly and insulin-stimulated translocation of GLUT4 vesicles. We hypothesise that Tomosyn acts by coordinating the functions of the SNAREs with that of the polarized secretion machinery, including the cytoskeleton, maybe via its conserved amino terminal domain.