ENDOGENOUS SYNTAXIN-2, SYNTAXIN-3 AND SYNTAXIN-4 EXHIBIT DISTINCT BUTOVERLAPPING PATTERNS OF EXPRESSION AT THE HEPATOCYTE PLASMA-MEMBRANE

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 pr oteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, t he PM is divided into two domains, the apical and basolateral. If synt axins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain exp resses one syntaxin isoform. However, we report that rat hepatocytes e xpress three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the synta xins exhibit distinct, but overlapping patterns-of expression among th e PM domains. Syntaxin 4 is primarily expressed at the basolateral sur face while syntaxins 2 and 3 are enriched at the apical PM. The immuno localization of syntaxins 2 and 4 in rat hepatocytes and PM sheets rev ealed 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 ha ve used quantitative immunoblotting to determine that the syntaxins ar e relatively abundant PM molecules (11-260 nM) in rat liver, spleen an d kidney. Also, we determined that the syntaxin binding protein, Munc- 18, is present at concentrations from 1.5-20 nM in the same tissues. A lthough this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntax in function, but also for predicting the specific mechanisms that regu late vesicle targeting in hepatocytes and other tissues.