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Vol. 18, Issue 10, 3978-3992, October 2007

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

Asli Oztan^*,, Mark Silvis, Ora A. Weisz^*,, Neil A. Bradbury, Shu-Chan Hsu, James R. Goldenring^||, Charles Yeaman^¶, and Gerard Apodaca^*,

*Laboratory of Epithelial Cell Biology/Renal Electrolyte Division of the Department of Medicine and Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261; Department of Physiology and Biophysics, Chicago Medical School, Chicago, IL 60064; Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854; ^||Department of Surgery and Cell and Developmental Biology, Vanderbilt University and the Nashville Veterans Affairs Medical Center, Nashville, TN 37212; and ^¶Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242

Submitted February 2, 2007; Revised July 24, 2007; Accepted July 26, 2007
Monitoring Editor: Keith Mostov

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-O–permeabilized 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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Gerard Apodaca (gla6{at}pitt.edu)

Abbreviations used: AEE, apical early endosome; ARE, apical recycling endosome; BEE, basolateral early endosome; CRE, common recycling endosomes; GFP, enhanced green fluorescent protein; MDCK, Madin-Darby canine kidney; pIgR, polymeric immunoglobulin receptor; PNS, postnuclear supernatant; SLO, streptolysin O; Tf, transferrin; TGN, trans-Golgi network.

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