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J. Biol. Chem., Vol. 280, Issue 41, 34997-35010, October 14, 2005

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H-Ras Dynamically Interacts with Recycling Endosomes in CHO-K1 Cells

INVOLVEMENT OF Rab5 AND Rab11 IN THE TRAFFICKING OF H-Ras TO THIS PERICENTRIOLAR ENDOCYTIC COMPARTMENT^*

From the Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.

Received for publication, June 8, 2005 , and in revised form, July 26, 2005.

^* This work was supported in part by a grant from SECyT-Universidad Nacional de Córdoba, International Society for Neurochemistry (Special ISN One-Time Fund), PEI of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Grant 6462, Fundación Antorchas Grant 14116-112, Programa de Centros Asociados de Posgrado Brasil/Argentina Grant 001/02, and Agencia Nacional de Promoción Cientifica y Tecnológica Grant 01-13522. 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. 1-4.

¹ Recipient of a fellowship from CONICET (Argentina).

² A Career Investigator of CONICET (Argentina). To whom correspondence should be addressed. Tel.: 54-351-4334171; Fax: 54-351-434074; E-mail: daniotti{at}dqb.fcq.unc.edu.ar.

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