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Vol. 17, Issue 1, 192-202, January 2006

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K-ras4B and Prenylated Proteins Lacking "Second Signals" Associate Dynamically with Cellular Membranes

Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada

Submitted May 9, 2005; Revised October 7, 2005; Accepted October 12, 2005
Monitoring Editor: Robert Parton

We have used fluorescence microscopy and the technique of rapamycin-regulated protein heterodimerization to examine the dynamics of the subcellular localizations of fluorescent proteins fused to lipid-modified protein sequences and to wild-type and mutated forms of full-length K-ras4B. Singly prenylated or myristoylated fluorescent protein derivatives lacking a "second signal" to direct them to specific subcellular destinations, but incorporating a rapamycin-dependent heterodimerization module, rapidly translocate to mitochondria upon rapamycin addition to bind to a mitochondrial outer membrane protein incorporating a complementary heterodimerization module. Under the same conditions analogous constructs anchored to the plasma membrane by multiply lipid-modified sequences, or by a transmembrane helix, show very slow or no transfer to mitochondria, respectively. Interestingly, however, fluorescent protein constructs incorporating either full-length K-ras4B or its plasma membrane-targeting sequence alone undergo rapamycin-induced transfer from the plasma membrane to mitochondria on a time scale of minutes, demonstrating the rapidly reversible nature of K-ras4B binding to the plasma membrane. The dynamic nature of the plasma membrane targeting of K-ras4B could contribute to K-ras4B function by facilitating redistribution of the protein between subcellular compartments under particular conditions.

Abbreviations used: CFP, Cerulean variant of cyan fluorescent protein; ER, endoplasmic reticulum; FKBP, rapamycin-binding heterodimerization domain of human FKBP12; FRB, rapamycin-binding heterodimerization domain of mTOR/FRAP; (m)RFP, monomeric red fluorescent protein; YFP, monomeric Citrine variant of yellow fluorescent protein.

Address correspondence to: John R. Silvius (john.silvius{at}mcgill.ca).

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