The Role of Syntaxins in the Specificity of Vesicle Targeting in Polarized Epithelial Cells

Abstract

In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes. INTRODUCTIONEukaryotic cells contain numerous intracellular membranous compartments that are connected by vesicular traffic. After vesicles bud off from a membrane, they must be targeted to and fuse with the correct target membrane. How vesicles are specifically targeted to the correct membrane and avoid fusion with the incorrect membrane remains a paramount question (Mostov et al., 2003;Nelson, 2003;Rodriguez-Boulan et al., 2005). Several classes of molecules may contribute to this specificity and it is possible that specificity is conferred by multiple layers of molecular machinery, which may act sequentially. For instance, at least in larger animal cells, vesicles may first reach the vicinity of their target membrane by using motor proteins and cytoskeletal filaments. Thus, some of the specificity may be achieved by binding and activation of the correct motor protein to the vesicle. In many trafficking steps, vesicles are then brought closer to the membrane by tethering complexes. For instance, the yeast exocyst and its homologous mammalian sec6/8 complex tether vesicles to the specific locations on the plasma membrane (Lipschutz and Mostov, 2002;Novick and Guo, 2002).SNARE proteins act later and may catalyze fusion itself (Sollner, 2003;Ungar and Hughson, 2003;Jahn, 2004). The original formulation of the SNARE hypothesis postulated that specific v-SNARES on the vesicle paired with cognate t-SNAREs on the target, thereby providing specificity to fusion (Sollner et al., 1993). This premise was challenged when SNARE proteins, lacking their membrane anchors, were produced. Soluble versions of v-and t-SNARES paired almost completely promiscuously, suggesting that pairing of specific SNAREs did not contribute ...

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

“…Using an in vitro binding assay, Latham et al (27) reported significant association between Munc18c and a mutant syntaxin 4 that lacked the SNARE motif (H3 domain) and transmembrane domain (syntaxin 4 ⌬H3/TM ). However, our in vivo analysis using a FRET-based assay displayed a loss Munc18c interaction with syntaxin 4 ⌬H3/TM , thereby suggesting that the N-terminal interaction is insufficient to mediate the Munc18c-syntaxin 4 interaction, in parallel to the in vitro findings of Ter Beest et al (42). The necessity of the syntaxin 4 C terminus was additionally confirmed using the I241A mutation of syntaxin 4, a residue predicted to bind to the central cavity of (25).…”

Syntaxin binding mechanism and disease-causing mutations in Munc18-2

“…S3B), similar to the interaction previously reported for Munc18-3 and Stx4 (29), and are consistent with a model in which the N peptide serves as a selection determinant for syntaxin binding (31,32). Our results suggest that if complex formation is initiated by N peptide binding Munc18-2 will bind Stx11 in preference to Stx3 when both syntaxins are present.…”

“…It has been proposed that the N peptide initiates contact between syntaxins and Munc18 proteins and in this way may lead to highaffinity binding of the full-length syntaxin molecule (31). In polarized epithelial cells the N peptide has also been found to determine which Munc18 isoform is bound and where the syntaxin localizes (32). However, nothing is known about the role of the N peptide in the selection of syntaxin binding when two different syntaxins are both able to bind the same Munc18 protein.…”

Regulation of membrane trafficking in polarized epithelial cells

“…Thus, perhaps annexins serve as tethering factors between apical carriers and the apical plasma membrane [15]. Fusion at the apical membrane may then be mediated by the v-SNARE TI-VAMP and the apically localized t-SNARE syntaxin 3 [17][18][19][20].…”