Intracellular Redirection of Plasma Membrane Trafficking after Loss of Epithelial Cell Polarity

Abstract

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity. INTRODUCTIONTraffic between membranous compartments is mediated by the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) machinery in virtually all membrane traffic pathways investigated so far (Rothman and Warren, 1994;Hanson et al., 1997;Hay and Scheller, 1997;Nichols and Pelham, 1998). During vesicle docking, membrane proteins on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs) bind to each other to form a complex that ultimately leads to fusion of the lipid bilayers. One aspect of the SNARE hypothesis is that successful membrane fusion requires the binding of matching combinations of v-and t-SNAREs, thereby ensuring the necessary specificity of vesicle fusion. Accordingly, each membrane organelle and each class of transport vesicles should be defined by a certain set of t-and v-SNARE isoforms. Many SNAREs have been identified to date, and protein sequence analysis has shown that v-and t-SNAREs of the currently known SNARE subfamilies are evolutionarily related to each other and belong to a common superfamily (Weimbs et al., 1997b(Weimbs et al., , 1998. It is conceivable that the specificity of vesicle fusion is not directly determined by t-SNARE/v-SNARE interactions per se but rather by interactions involving larger complexes, including SNAREs and their regulatory proteins, such as those of the rab and sec1 protein families (Christoforidis et al., 1999;Gonzalez and Scheller, 1999;Pfeffer, 1999).Epithelial cells display an additional layer of complexity in that they are typically polarized and possess two distinct plasma membrane domains (Louvard et al., 1992;Simons et al., 1992;Le Gall et al., 1995;Drubin and Nelson, 1996;Yeaman et al., 1999). The apical and basolateral plasma membrane domains have different protein and lipid compositions that reflect the different functions of these domains. This plasma membrane polarity is established and maintained by protein sorting and specific vesicle trafficking routes in th...

Nonpolarized Cells Selectively Sort Apical Proteins from Cell Surface to a Novel Compartment, but Lack Apical Retention Mechanisms

“…The apical compartment identified in this study is not the vacuolar apical compartment (VAC) that was previously identified in nonpolarized epithelial cells (Vega-Salas et al, 1987Gilbert and Rodriguez-Boulan, 1991;Low et al, 2000). The apical structures we have described occur in normally growing cells that do not polarize (Fao and Clone 9) or in WIF-B cells before they become polarized.…”

Cutting Edge: Selective Requirement for the Wiskott-Aldrich Syndrome Protein in Cytokine, but Not Chemokine, Secretion by CD4+ T Cells

“…Mechanistically, this linkage may reflect a requirement for WASp in the redistribution of essential soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery (SNARE) (30) to the effector synapse for cytokine-containing vesicle release. Indeed, loss of cellular polarity in epithelial cells leads to redistribution of target SNAREs to intracellular locations (31).…”

Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution

“…Thus, the evidence suggests that these genes also diverged similarly in function in each lineage, indicative of convergent evolution. Because these examples involve basic cellular machinery, they differ from gene duplications involving specialized tissuespecific machinery (e.g., syntaxins 1, 2, 3, and 4 and their plant equivalents, syp 111, 121, 131, and relatives) (48,49) and also from cases such as SSO1 and SSO2 in yeast, which encode plasma-membrane syntaxins with no discernable difference in function (50).…”