Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity

Abstract

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.

Transcriptional Expression of Myelin Basic Protein in Oligodendrocytes Depends on Functional Syntaxin 4: a Potential Correlation with Autocrine Signaling

“…The present study demonstrates that the t-SNAREs syntaxin 3 and 4 are distributed in a polarized fashion in OLGs, in agreement with similar observations reported for more common polarized cells, like epithelial cells (12)(13)(14)(15)(16)(17). Moreover, the localization of syntaxin 3 in the cell body and near the plasma membrane on the one hand and that of syntaxin 4 in the myelin sheet on the other is consistent with the apical-surface-and basolateral-surface-like nature, respectively, of these regions in rat OLGs, as previously established (1-3, 21).…”

“…The SNARE proteins are integral membrane proteins that are present on both the vesicles (v-SNARE) and the target membranes (t-SNARE). Importantly, the apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct t-SNAREs (11), i.e., syntaxins 3 and 4, which are preferentially localized at the apical and basolateral plasma membrane, respectively (12)(13)(14)(15)(16)(17). Both syntaxin 3 and syntaxin 4 are known to be present in OLGs (1,18,19), but their functional role in myelin biogenesis is still largely unknown.…”

Mechanism of polarized lysosome exocytosis in epithelial cells

“…Apical exocytosis of lysosomes has been documented in vivo in hepatocytes (Gross et al, 1989) and the proximal tubule of the kidney (Fujita et al, 1998), both of which lack m1B Schreiner et al, 2010). Indeed, syntaxin 4 is apical in hepatocytes (Fujita et al, 1998) and in LLC-PK1 cells (Reales et al, 2011), providing further support for the role of m1B in restricting syntaxin 4 to the basolateral membrane and regulating the polarity of lysosome fusion.…”

“…However, we were unable to detect a direct interaction between syntaxin 4 and m1A or m1B by yeast two-hybrid analysis, likely due to low binding affinity or the participation of a linker protein. While our studies were underway, it was reported that syntaxin 4 is nonpolar in a porcine proximal tubule cell line, LLCPK1, which lacks m1B (Reales et al, 2011). To further elucidate how AP-1 and syntaxin function in polarized lysosome secretion, we used shRNA-mediated knockdown of endogenous syntaxin 4 (Fig.…”

“…Exocytosis of 'conventional' lysosomes in response to a transient increase in intracellular calcium ([Ca 2+ ] i ) was first observed during cell invasion of Trypanosoma cruzi: binding of the parasite to the cell membrane triggers calcium influx and subsequent fusion of lysosomes with the region of the plasma membrane that surrounds the invading parasite (Andrews, 2002). Lysosome exocytosis has since emerged as an important mechanism in diverse paradigms such as membrane repair after exposure to calcium ionophores and pore-forming toxins (Divangahi et al, 2009;Jaiswal et al, 2002;Rodríguez et al, 1997) and for propagating the Ca 2+ wave in astrocytes to modulate synaptic transmission (Li et al, 2008;Zhang et al, 2007). Molecular machinery implicated in calcium-induced lysosome exocytosis include the lysosomal calcium sensor synaptotagmin VII, the v-SNARE VAMP7 and t-SNAREs SNAP23, syntaxin 2 and syntaxin 4 (Rao et al, 2004).…”