| Plasma membrane (PM) localization is required for Ras proteins to function in signal transduction. This is accomplished in part by the covalent attachment of lipid moieties that target Ras first to the endoplasmic reticulum (ER) and then to the PM, but the mechanism by which this occurs is not known. The classical secretory pathway is the best studied endomembrane protein trafficking route to date, but surprisingly, GFP-Ras2 is still localized to the PM in all secretory pathway mutants (sec mutants) analyzed and in cells treated with brefeldin A (a Golgi disrupting agent). In contrast, deletion of ERF2, which encodes a protein required for efficient palmitoylation of Ras2, causes a severe Ras2 trafficking defect when combined with either sec mutants or brefeldin A. These results provide the first evidence of a novel endomembrane trafficking system utilized by Ras2 protein that is independent of key steps of the classical secretory pathway. To determine how Ras2 interacts with this novel pathway, I mapped the region required to engage the Erf2-dependent trafficking pathway (Erf pathway). Ras proteins from yeast and mammals have highly conserved N-terminal GTPase domain and C-terminal CaaX motif, but otherwise differ in sequence and size through a central hypervariable (HV) domain. I show that deleting the HV domain (amino acids 200--316) of Ras2 results in severe mislocalization of GFP-Ras2 to intracellular membranes. Moreover, the C-terminal 30 amino acids of the HV region (HV2 region) combined with CaaX box and the adjacent cysteine that can be palmitoylated is sufficient to localize GFP to the PM by the Erf pathway. This identifies the region of Ras2 required to engage the Erf pathway to the C-terminal 41 amino acids. I then show that the requirement for HV2 region in Ras2 localization and trafficking is related to palmitoylation. In summary, this work describes the identification of a novel trafficking pathway for yeast Ras2 and the characterization of the sequence elements required for Ras2 to utilize the Erf pathway. |
