Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

Abstract

Sec1/Munc18 (SM) family proteins are essential for every vesicle fusion pathway. The best-characterized SM protein is the synaptic factor Munc18-1, but it remains unclear whether its functions represent conserved mechanisms of SM proteins or specialized activities in neurotransmitter release. To address this question, we dissected Munc18c, a functionally distinct SM protein involved in nonsynaptic exocytic pathways. We discovered that Munc18c binds to the trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and strongly accelerates the fusion rate. Further analysis suggests that Munc18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulation of fusion by Munc18c is specific to its cognate SNARE isoforms. Because Munc18-1 regulates fusion in a similar manner, we conclude that one conserved function of SM proteins is to bind their cognate trans-SNARE complexes and accelerate fusion kinetics. Munc18c also binds syntaxin-4 monomer but does not block target membrane-associated SNARE assembly, in agreement with our observation that six-to eightfold increases in Munc18c expression do not inhibit insulinstimulated glucose uptake in adipocytes. Thus, the inhibitory "closed" syntaxin binding mode demonstrated for Munc18-1 is not conserved in Munc18c. Unexpectedly, we found that Munc18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways. Together, these comparative studies of two distinct SM proteins reveal conserved as well as divergent mechanisms of SM family proteins in intracellular vesicle fusion. membrane fusion | vesicle transport | exocytosis T he fusion of intracellular vesicles with target membranes requires two classes of conserved proteins: SNAREs and SM (Sec1/Munc18) proteins (1, 2). SNAREs are membrane-associated proteins that contain characteristic stretches of 60-70 amino acids known as core domains or SNARE motifs. Fusion is initiated when the core domains of the vesicle-rooted SNARE (v-SNARE) and the target membrane-associated SNAREs (t-SNAREs) zipper into a four-helix trans-SNARE complex between two apposed bilayers (2-5). N-to C-terminal zippering of the trans-SNARE complex brings the two membranes into close apposition to fuse (6-8).First isolated in genetic screens in yeast and nematodes (9, 10), SM proteins are hydrophilic factors of 60-70 kDa that regulate membrane fusion through binding to their cognate SNAREs (11-13). SM proteins exhibit a similar loss-of-function phenotype as that of SNAREs (i.e., abrogation of fusion) and are essential for every pathway of intracellular vesicle fusion (14-16). Mutations of SM proteins give rise to a number of human diseases, including epilepsy and inflammatory disorders, as well as arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome (17-...

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

“…Furthermore, proteoliposomes reconstituted with SNAREs only support lipid mixing, demonstrating that SNAREs alone can drive membrane fusion (8, 19 -24). Similarly, it was shown that Munc18-1 accelerates SNARE-mediated proteoliposome fusion, supporting the notion that Munc18-1 is part of the minimal fusion machine (12,25).…”

“…The severity of the knock-out phenotype could be explained equally well by the losing of inhibitory capping on syntaxin-1a. Second, Munc18-1 has the capacity of accelerating SNARE-dependent proteoliposome fusion significantly (12,25). However, the caveat here is that Syt1 is not included in those experiments.…”

“…The proteoliposome fusion assay (12,25), on which this proposition relies heavily, did not include synaptotagmin-1 (Syt1), 4 a major Ca 2ϩ sensor for synaptic vesicle fusion (31)(32)(33). Syt1 is a vesicular protein, consisting of tandem Ca 2ϩ -binding C2 domains and a transmembrane helix (34).…”

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

“…Supporting the hypothesis of the importance of the Sx C-terminus, Yu et al (2013) showed that Munc18c positively influenced SNARE-complex formation using a liposomal fusion assay similar to that used by Brandie et al (2008). In these later experiments, the t-SNARE complex consisted of untagged Sx4-TMD and N-terminally His tagged SNAP23 (Yu et al, 2013) and thrombin was not used.…”

“…In these later experiments, the t-SNARE complex consisted of untagged Sx4-TMD and N-terminally His tagged SNAP23 (Yu et al, 2013) and thrombin was not used.…”