Munc18-2, a Functional Partner of Syntaxin 3, Controls Apical Membrane Trafficking in Epithelial Cells

Abstract

The Sec1-related proteins bind to syntaxin family tSNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.It is generally accepted that intracellular membrane trafficking requires compartment-specific membrane-anchored proteins denoted collectively as soluble NSF attachment protein receptors (SNAREs), 1 as well as the general factors N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs), or their homologues (1, 2). The SNARE proteins present on the transport vesicles (v-SNAREs; related to the neuronal synaptobrevin/VAMP proteins) and the target membranes (t-SNAREs; homologues of the neuronal syntaxin and SNAP-25 proteins) assemble into stable core complexes through formation of coiled coil helix bundles, a process which is closely connected to the fusion of membrane bilayers (3-5).Proteins of the Sec1 family (reviewed in Ref. 6) are suggested to play a crucial role in the control of SNARE complex assembly. These proteins bind with high affinity to specific syntaxins, thus modulating the capability of these t-SNAREs to interact with their cognate SNARE partners. In vitro binding assays or in vivo overexpression of Sec1 homologues have provided evidence for an inhibitory role of Sec1 proteins in SNARE complex formation and membrane trafficking (7-12). On the other hand, a wealth of evidence shows that Sec1 action is required for normal physiological function of the intracellular trafficking pathways: loss-of-function mutations in the Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster Sec1-related proteins have been demonstrated to lead to specific blocks in vesicle transport, the phenotypic effects often implying disturbance in consumption of transport vesicles (13)(14)(15)(16)(17)(18)(19)(20)...

Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

“…2 A, B). The NV mutants of both isoforms were less abundant than the corresponding WT variants, indicating that the NV mutation decreases the stability of Munc18, as reported previously for Munc18-2 (Riento et al, 2000). Nevertheless, all Munc18 variants were expressed in excess of native Munc18-1 in chromaffin cells.…”

“…Munc18-2 fully restored docking in chromaffin cells, although the cognate syntaxin partner for Munc18-2 is syntaxin3 (Riento et al, 2000). This observation provides an opportunity for investigating whether there are additional aspects to the function of Munc18 proteins beyond vesicle docking.…”

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

“…Previous studies have suggested that Munc18-2 can interact with the monomeric Syntaxins STX3 (39,40), STX2 (41), and STX11 (13,(33)(34)(35). However, other SM proteins have distinct binding modes in which they engage either monomeric syntaxins or SNARE complexes (42-45).…”

1-Benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside Blocks the Apical Biosynthetic Pathway in Polarized HT-29 Cells

“…Proteins of SNARE complex are known to play a role in the regulation of the direct apical biosynthetic route: t-SNARE syntaxin-3 and v-SNARE Ti-VAMP (36,37,44,45). In addition, the sec1-related protein Munc18-2 modulates the interaction of syntaxin-3 with other SNARE partners, and controls the vesicular apical transport (38). Annexin XIIIb has been also previously described to play a role in apical delivery through an association with rafts (40).…”

“…The t-SNAREs syntaxin-3 and -4 were, respectively, localized at the apical and basolateral membranes of polarized MDCK and Caco-2 cells (36, 37). Munc18-2/Munc18b is a Sec1 homologue, which interacts with the apical t-SNARE syntaxin-3 (38). Annexin XIIIb is an N-myristoylated protein, which was reported to participate in the apical transport via an association with the raft lipid microdomains (39,40).…”