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Molecular and Cellular Biology, August 2004, p. 6799-6810, Vol. 24, No. 15
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.15.6799-6810.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Three Separable Domains Regulate GTP-Dependent Association of H-ras with the Plasma Membrane

Barak Rotblat,1 Ian A. Prior,2 Cornelia Muncke,3 Robert G. Parton,3,4 Yoel Kloog,1 Yoav I. Henis,1* and John F. Hancock3*

Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel,1 The Physiological Laboratory, University of Liverpool, Liverpool, England,2 Institute for Molecular Bioscience,3 Centre for Microscopy and Microanalysis and School of Biomedical Sciences, University of Queensland, Brisbane, Australia4

Received 8 April 2004/ Accepted 10 May 2004

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

* Corresponding author. Mailing address for John F. Hancock: Institute for Molecular Bioscience, 306 Carmody Rd., University of Queensland, Brisbane 4072, Australia. Phone: 61 7 3346 2033. Fax: 61 7 3346 2101. E-mail: j.hancock{at}imb.uq.edu.au. Mailing address for Yoav I. Henis: Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-640-9053. Fax: 972-3-640-7643. E-mail: henis{at}post.tau.ac.il.

Molecular and Cellular Biology, August 2004, p. 6799-6810, Vol. 24, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.15.6799-6810.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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