K-ras4B and prenylated proteins lacking "second signals" associate dynamically with cellular membranes.
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.
Silvius
John R
JR
Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada. john.r.silvius@mcgill.ca
Bhagatji
Pinkesh
P
Leventis
Rania
R
Terrone
Donato
D
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D008928:Mitochondria / Q000378:metabolism*; D019814:Myristic Acid / Q000378:metabolism; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology
D019814:Myristic Acid; D016283:Proto-Oncogene Proteins p21(ras); D020123:Sirolimus