Endogenous syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression at the hepatocyte plasma membrane

Abstract

To investigate the mechanisms regulating polarized vesicle delivery to the cell surface in hepatocytes, we have characterized the endogenous plasma membrane (PM)-associated syntaxins. These integral membrane proteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, the PM is divided into two domains, the apical and basolateral. If syntaxins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain expresses one syntaxin isoform. However, we report that rat hepatocytes express three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the syntaxins exhibit distinct, but overlapping patterns of expression among the PM domains. Syntaxin 4 is primarily expressed at the basolateral surface while syntaxins 2 and 3 are enriched at the apical PM. The immunolocalization of syntaxins 2 and 4 in rat hepatocytes and PM sheets revealed similarly complex patterns of PM expression with enhanced apical staining for both. A significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles. We have used quantitative immunoblotting to determine that the syntaxins are relatively abundant PM molecules (11-260 nM) in rat liver, spleen and kidney. Also, we determined that the syntaxin binding protein, Munc-18, is present at concentrations from 1.5-20 nM in the same tissues. Although this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntaxin function, but also for predicting the specific mechanisms that regulate vesicle targeting in hepatocytes and other tissues.

Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines

“…They are in distinct cellular locations in epithelial cells [21,24,45]. Whereas HIT and islet beta cells contain both syntaxins [26], non-neuronal cells more often contain abundant Syn-3 (and Syn-2 and Syn-4) but Syn-1 was undetectable [24,25,43,44]. Syn-3 was also present in the cytoplasm of the HITcells which overlaps with the insulin labelling.…”

“…However, it would be difficult to precisely determine this action of Syn-3 in the present study because of the inhibition on insulin biosynthesis. In MDCK cells [24] and hepatocytes [44], Syn-3 is more abundant on apical plasma membranes. In these non-neuronal cells, Syn-3 is thought to be the putative syntaxin mediating apical exocytosis.…”

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

“…Proteins are sent to the correct surface either by direct delivery from the TGN, or by delivery to one surface and then endocytosis followed by transcytosis to the other surface. Syntaxin 3 (syx3) is located exclusively at the AP domain, as well as intracellularly in endosomes and lysosomes (Gaisano et al, 1996;Low et al, 1996;Fujita et al, 1998). In contrast, the highly homologous syntaxin 4 (syx4) is located entirely at the BL surface.…”

TFE3, HSP47, and CREB3 Pathways of the Mammalian Golgi Stress Response

“…STX3A is member of the syntaxin family and is important in establishing and maintaining polarity necessary for protein trafficking as well as in vesicle fusion and apical exocytosis (Vogel et al, 2015). Most STX3A is localized to the apical plasma membrane, whereas a significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles (Fujita et al, 1998). WIPI49 (also called WIPI1 and ATG18) is a WD40 repeat protein that interacts with phosphoinositides and is localized to both trans-Golgi and endosomal membranes, where it regulates membrane traffic (Jeffries et al, 2004).…”