Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells

Abstract

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-Opermeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

Membrane Organization and Dynamics in Cell Polarity

“…Through this interaction, Sec4 controls the assembly of the exocyst complex that is needed for the tethering of secretory vesicles at the plasma membrane. In other eukaryotes, Rab proteins such as Rab11 and Rab8, which are implicated in membrane trafficking to the plasma membrane, were also shown to interact with Sec15 (Zhang et al 2004;Wu et al 2005;Oztan et al 2007). Together, these studies suggest that the exocyst functions as a downstream effector of Rab proteins to mediate vesicle tethering at the plasma membrane in eukaryotic cells.…”

“…In yeast, exocyst mutants accumulate post-Golgi secretory vesicles in the cell (Novick et al 1980;Guo et al 1999a;Zhang et al 2005a;He et al 2007a). In Madin-Darby canine kidney (MDCK) cells, the exocyst has been shown to mediate exocytosis at the basolateral and apical domains (Grindstaff et al 1998;Oztan et al 2007). In adipocytes, the exocyst mediates exocytosis of the glucose transporter Glu4 in response to insulin stimulation (Inoue et al 2003).…”

“…In plants, exocyst subunits are localized at the apex of growing pollen tubes (Hála et al 2008). In mammalian cells, although the exocyst mostly localizes to internal membrane compartments such as Golgi and recycling endosomes Ang et al 2003;Fölsch et al 2003;Oztan et al 2007), it is recruited to designated regions of the plasma membrane where active exocytosis and membrane expansion occur. For example, in neurons undergoing differentiation or synapse formation, the exocyst is enriched at the expanding neurites and discrete puncta along axons where synapses are forming (Hsu et al 1996;Kee et al 1997;Hazuka et al 1999;Vega and Hsu, 2001;Brown et al 2001;Murthy et al 2003).…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Docking of basolateral transporters requires syntaxin 4 (Low et al, 1996), rab 8 (Huber et al, 1993) and a V-SNARE, cellubrevin, which cooperates with AP-1B in basolateral membrane trafficking (Fields et al, 2007), as well as the exocyst (Grindstaff et al, 1998;Shipitsin and Feig, 2004; The EPP: machineries involved and their hijacking by cancer B Tanos and E Rodriguez-Boulan Yeaman et al, 2004b;Langevin et al, 2005), a multiprotein particle that is also a key regulator of yeast's secretory route (Potenza et al, 1992;TerBush and Novick, 1995;TerBush et al, 1996). Interestingly, the exocyst interacts with the clathrin adaptor AP1B (Folsch et al, 2003) and participates not only in biosynthetic delivery, but, also, in postendocytic recycling to both basolateral and apical membranes, the latter in association with the small GTPase Rab11a (Oztan et al, 2007).…”

Active Rab11 and functional recycling endosome are required for E-cadherin trafficking and lumen formation during epithelial morphogenesis

“…Experiments in nonpolarized mammalian cells showed perturbed E-cadherin trafficking in the presence of Rab11 mutants (24). Rab11 also interacts with components of the exocyst complex as part of the trafficking machinery at the RE (32), and loss of function of exocyst in Drosophila epithelial tissues implicated Rab11 in Drosophila epithelial-cadherin (DE-cadherin) trafficking throughout morphogenesis (19).…”