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J. Biol. Chem., Vol. 281, Issue 22, 15376-15384, June 2, 2006

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Phosphatidylinositol 5-Kinase Stimulates Apical Biosynthetic Delivery via an Arp2/3-dependent Mechanism^*

From the Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

The mechanisms by which polarized epithelial cells target distinct carriers enriched in newly synthesized proteins to the apical or basolateral membrane remain largely unknown. Here we investigated the effect of phosphatidylinositol metabolism and modulation of the actin cytoskeleton, two regulatory mechanisms that have individually been suggested to function in biosynthetic traffic, on polarized traffic in Madin-Darby canine kidney cells. Overexpression of phosphatidylinositol 5-kinase (PI5K) increased actin comet frequency in Madin-Darby canine kidney cells and concomitantly stimulated trans-Golgi network (TGN) to apical membrane delivery of the raft-associated protein influenza hemagglutinin (HA), but did not affect delivery of a non-raft-associated apical protein or a basolateral marker. Modulation of actin comet formation by pharmacologic means, by overexpression of the TGN-localized inositol polyphosphate 5-phosphatase Ocrl, or by blockade of Arp2/3 function had parallel effects on the rate of apical delivery of HA. Moreover, HA released from a TGN block was colocalized in transport carriers in association with PI5K and actin comets. Inhibition of Arp2/3 function in combination with microtubule depolymerization led to a virtual block in HA delivery, suggesting synergistic coordination of these cytoskeletal assemblies in membrane transport. Our results suggest a previously unidentified role for actin comet-mediated propulsion in the biosynthetic delivery of a subset of apical proteins.

Received for publication, February 8, 2006 , and in revised form, April 6, 2006.

^* This work was supported in part by National Institutes of Health Grants DK054407 and DK064613 and the Commonwealth of Pennsylvania (to O. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movie 1.

¹ Supported in part by National Institutes of Health Grant T32-DK61296.

² To whom correspondence should be addressed: 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8891; Fax: 412-383-8956; E-mail: weisz{at}pitt.edu.

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