In vivo prenylation analysis of ras and rho proteins

Publisher Summary This chapter discusses the in vivo prenylation analysis of Ras and Rho proteins. Two systems have been used to examine the prenylation of Ras proteins and other smGTPases under different growth or treatment conditions. The first involved heterologous expression of individual proteins in a cellular system; labeling the cells overexpressing the protein with [ 3 H]mevalonate and determining the prenyl group associated with the overexpressed protein by chemical analysis. This analysis provided data on an individual Ras isoform; it did not provide direct information about the endogenous protein under normal expression conditions. A second system was designed to examine protein prenylation of endogenously expressed proteins. This system relied on construction of a human cell line transfected with a mevalonate transporter expression vector to enhance the uptake and incorporation of radiolabeled mevalonate into cellular proteins. Prenyl analysis of endogenous small GTPases is also discussed. This approach is particularly useful for doing dose-response curves for prenylation of Ras proteins in the presence of farnesyltransferase inhibitors.