SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes

Abstract

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in α- but not β-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

“…Subcellular distribution can also limit the interaction between cognate v-and t-SNAREs. For example, in epithelial cells, syntaxin4 is restricted to the basolateral domain of plasma membrane [46], whereas syntaxin1 is delivered to both the apical and basolateral domains [47]. In epithelial cells, VAMP3 can partner with syntaxin4 to deliver cargo proteins to the basolateral side of the plasma membrane.…”

Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

“…The pull-down assay was performed with either plain Sepharose beads or Sepharose beads linked to glutathione S-transferase (GST)-syntaxin 1A. The production of GST-syntaxin 1A and its binding to Sepharose beads were described previously (15). The beads were washed three times with cold PBS before use.…”

Spatial control of exocytosis

“…Evidence for this type of mechanism comes from studies of exocytosis in polarized epithelia. Plasma membrane t-SNARE proteins syntaxin 3 and 4 are confined to the apical and basal-lateral surfaces of polarized epithelial cells, respectively [37,38]. Threedimensional imaging of post-Golgi vesicle traffic reveals that exocytosis of basal-lateral marker proteins occurs exclusively within the upper regions of the lateral membrane [33 •• ].…”