Replacement of the H-Ras farnesyl group by lipid analogues: implications for downstream processing and effector...

Ras proteins must undergo a series of posttranslational lipidation steps before they become biologically functional. While the fact that farnesylation is required for subsequent processing steps and indispensable for Ras function has been established, the significance of the isoprenoid structure per se in the context of fully processed Ras is unknown. Here, we describe a novel approach for studying the isoprenoid structure-function relationship in vivo by replacing the H-Ras farnesyl group with synthetic analogues and analyzing their biological functions following microinjection into Xenopus oocytes. We show that the H-Ras farnesyl group can be stripped of most of its isoprenoid features that distinguish it from a fatty acid without any apparent effect on its ability to induce oocyte maturation and activation of mitogen-activated protein kinase. In contrast, replacement by the less hydrophobic isoprenoid geranyl causes severely delayed oocyte activation. Analysis of posttranslational processing reveals a striking correlation between the kinetics of processing, membrane binding, and the onset of biological activity regardless of lipid structure and suggests that slow C-terminal proteolysis and/or methylation can become rate-limiting for H-Ras function. Thus, while our results suggest no stringent requirement for the H-Ras farnesyl structure for effector activation in Xenopus oocytes, they reveal an important role for the lipid present at the farnesylation site in promoting efficient proteolysis and/or methylation which allows rapid palmitoylation, membrane localization, and biological activity. Xenopus oocytes provide a useful in vivo system for the kinetic analysis of the function of the protein of interest present at the physiological dose, which is required for accurate determination of structure-function relationships.