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J. Biol. Chem., Vol. 283, Issue 43, 29545-29553, October 24, 2008

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Selective Roles for Cholesterol and Actin in Compartmentalization of Different Proteins in the Golgi and Plasma Membrane of Polarized Cells^*

Stéphanie Lebreton, Simona Paladino, and Chiara Zurzolo¹

From the Unité de Trafic Membranaire et Pathogénèse, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France and the Dipartimento di Biologia e Patologia Cellulare e Moleculare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy

To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.

Received for publication, May 19, 2008 , and in revised form, July 16, 2008.

^* This work was supported by Agence Nationale de la Recherche Grant 05-BLAN 296-01 and Ministero dell' Università e della Ricerca Scientifica e Tecnologica Grant PRIN 2006. 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 Figs. S1-S4.

¹ To whom correspondence should be addressed: Unité de Trafic Membranaire et Pathogénèse, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. Tel.: 33-1-45688277; Fax: 33-1-40613238; E-mail: zurzolo{at}pasteur.fr, zurzolo{at}unina.it.

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