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J. Biol. Chem., Vol. 280, Issue 38, 32768-32774, September 23, 2005

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Differential Membrane Localization of ERas and Rheb, Two Ras-related Proteins Involved in the Phosphatidylinositol 3-Kinase/mTOR Pathway^*

Kazutoshi Takahashi, Masato Nakagawa, Stephen G. Young, and Shinya Yamanaka1

From the Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University and CREST, Japan Science and Technology Agency, Kyoto 606-8507, Japan and the Department of Medicine, University of California, Los Angeles, California 90095

Two Ras-related proteins, ERas and Rheb, which are involved in the phosphatidylinositol 3-kinase pathway, display high GTP affinity and have atypical CAAX motifs. The factors governing the intracellular localization of ERas and Rheb are incompletely understood. In the current study, we show by confocal microscopy that ERas is localized to the plasma membrane, whereas Rheb is confined to the endomembranes. Membrane localization of the two proteins was abolished by mutation of the cysteine of the CAAX motif. Membrane targeting was also abolished by a farnesyltransferase inhibitor but not by a geranylgeranyltransferase inhibitor. In mouse fibroblasts deficient in either Rce1 (Ras converting enzyme 1) or Icmt (isoprenylcysteine carboxyl methyltransferase), ERas was mislocalized mainly to the Golgi apparatus, whereas Rheb showed diffuse localization. Mutation of cysteines in the hypervariable region of ERas prevented the plasma membrane localization of ERas, very strongly suggesting that palmitoylation of the cysteines is essential for membrane targeting. The hypervariable region of Rheb does not contain cysteines or polybasic residues, and when it was replaced with the hypervariable region of H-Ras, Rheb displayed plasma membrane localization. These data indicate that ERas shares the same posttranslational modifications with H-Ras and N-Ras and is localized at the plasma membrane. Rheb also shares the same membrane-targeting pathway but because of the absence of palmitoylation is located on endomembranes.

Received for publication, June 9, 2005 , and in revised form, July 7, 2005.

^* This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, The Uehara Memorial Foundation, The Naito Foundation, The Sumitomo Research Foundation, The Mitsubishi Foundation, and a Toray Science and Technology Grant (to S. Y.). This work was also supported in part by a Grant-in-Aid for 21st Century Center of Excellence Research from the Ministry of Education, Culture, Sports, Science and Technology. This work was also supported in part by National Institutes of Health Grants CA099506 and CA103999–01 (to S. G. Y.). 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.

¹ To whom correspondence should be addressed: Dept. of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan 606-8507. Tel.: 81-75-751-3839; Fax: 81-75-751-4632; E-mail: yamanaka{at}frontier.kyoto-u.ac.jp.

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