Munc18-1 Is Critical for Plasma Membrane Localization of Syntaxin1 but Not of SNAP-25 in PC12 Cells

149

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-...

Section: Munc18mentioning

confidence: 99%

mentioning

confidence: 99%

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

Rathore

Lopez

et al. 2013

149

“…The Munc18 family of proteins has been revealed to play multiple functions, which include both chaperoning cognate syntaxins (14)(15)(16)(17)(18)(19) and priming/stimulating membrane fusion through interaction with the SNARE complex (20-23) (reviewed in ref. 24).…”

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confidence: 99%

“…The chaperoning function of Munc18 contributes to stabilizing the expression level of cognate syntaxins and, in some cases, trafficking them to the appropriate intracellular compartments. For instance, in Munc18-1-deficient neurons, the syntaxin-1 level is reduced by 50-75% (3), whereas in Munc18-1 single-knockdown (KD) and Munc18-1/2 double-knockdown (DKD) pheochromocytoma (PC) 12 cells, not only the reduction of syntaxin-1 expression level but also its localization at the plasma membrane are severely perturbed (14,15). In some patients with FHL5 whose functional Munc18-2 protein is absent, a strong reduction in syntaxin-11 level was found in their immune cells (11,12).…”

mentioning

confidence: 99%

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

Bin

Jung

Piggott

et al. 2013

Self Cite

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.membrane fusion | FHL5

“…Down-regulation or expression of dominant-negative syntabulin reduces but does not abolish membrane delivery of Stx, indicating the existence of other transport mechanisms (6). Moreover, proper intracellular trafficking of Stx and its function in exocytosis depends on Munc18 coexpression (8)(9)(10)(11)(12)(13)(14). Stx trafficking defects were observed in unc-18 knockouts in Caenorhabditis elegans (14), Munc18 knockdowns in PC12 cells (9, 13) but not in mouse Munc18-1 knockouts (15), although in the latter case, a compensation by other Munc18 isoforms cannot be excluded.…”

mentioning

confidence: 99%

Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter

Chua

Butkevich

Worseck

et al. 2012

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans, wild-type but not phosphorylationdeficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kinases. T he formation and maintenance of presynaptic boutons are intricate but highly efficient processes during which the machinery for exocytosis and recycling of synaptic vesicles is assembled from preformed units. These units are delivered to the nascent synapse via molecular motor proteins of the kinesin superfamily (reviewed in Ref. 1).Although Kinesin-3 appears to be the main motor transporting synaptic vesicle precursors, recent evidence suggests that Kinesin-1 (KIF5) is also involved. In Drosophila, deletion of UNC-76/ fasciculation and elongation protein zeta 1 (FEZ1), a specific adaptor for Kinesin-1, left synaptic vesicles stranded in the axon (2, 3), showing that Kinesin-1 is needed at least during later phases of axonal transport. Transport of the synaptic vesicle protein synaptotagmin by the UNC-76/Kinesin-1 complex requires phosphorylation of UNC-76 by the UNC-51/ATG1 kinase, a prerequisite for UNC-76 to bind synaptotagmin (3). Deletion of this kinase phenocopies deletion of UNC-76. Indeed, phosphorylation-regulated interactions between cargo, adaptors, and kinesins have also been observed for other transport complexes such as the kinesin light chain/JIP1 (c-Jun N-terminal kinase-interacting protein 1) complex (4). This suggests that phosphorylation is a common mechanism for the regulation of kinesin-based transport complexes (5).Less is known about the involvement of Kinesin-1 in the transport of other classes of synaptic precursor vesicles. Transport of syntaxin 1a (Stx), an essential component of the exocytotic release apparatus residing in the presynaptic plasma membrane, is clearly distinct from synaptic vesicle precursors and appears to involve a complex between Kinesin-1 and the Stx-binding protein syntabulin (6, 7). Down-regulation or expression of dominant-negative syntabulin reduces but does not abolish membrane delivery of Stx, indicating the existence of other transport mechanisms (6). Moreover, proper intracellular trafficking of Stx and its function in exocytosis depends on Munc18 coexpression (8-14). Stx tra...