Figure 10.

Ras posttranslational processing and membrane association. (A) The C-terminal 24–25 residues comprise the membrane targeting sequence which is comprised of the C-terminal CAAX box, required for posttranslational lipid modification, and the hypervariable (HV) domain that includes a second membrane targeting sequence element (palmitoylated cysteine(s) or polybasic stretches). (B) CAAX motif-signaled posttranslational processing. The cysteine residue of the CAAX box (red) is modified posttranslationally by cytosolic farnesyltransferase (FTase)-catalyzed covalent addition of a C15 farnesyl isoprenoid. Processing is completed on the cytosolic leaflet of the endoplasmic reticulum (ER) by Rce1 (Ras and a-factor converting enzyme-1)-catalyzed proteolytic removal of the AAX residues and then by Icmt (isoprenylcysteine carboxyl methyltransferase)-catalyzed carboxylmethylation of the now terminal farnesylated cysteine residue. FTase inhibitor (FTI) treatment blocks farnesylation and all subsequent CAAX modifications, rendering Ras cytosolic and inactive. Similarly, a serine substitution of the cysteine residue of the CAAX motif (“SAAX”) also prevents farnesylation and all CAAX-signaled modifications. The X residue of the CAAX motif dictates prenyltransferase specificity. Substitution of the X residue with leucine generates mutants that undergo modification by GGTase-I and addition of a C20 geranylgeranyl lipid instead of a C15 farnesyl isoprenoid. (C) Ras plasma membrane association is dependent on a second targeting element. The CAAX-signaled modifications alone are not sufficient to promote plasma membrane association. Additional sequence elements in the HV provide a second membrane targeting signal. In H-Ras, N-Ras and K-Ras4A, cysteine residues in the HV region are modified posttranslationally by ER-associated protein acyltransferases (PATs) that promote covalent addition of a C16 palmitate fatty acid. In K-Ras4B, lysine-rich polybasic amino acids comprise the second signal, which facilitate association with the negatively charged head groups of phosphatidylserine and phosphatidylinositol in the cytosolic face of the plasma membrane. Whereas H-Ras and N-Ras (and presumably K-Ras4A) traffic through the classical secretory pathway through the Golgi to the plasma membrane, K-Ras4B bypasses the Golgi and transits to the plasma membrane by a poorly characterized mechanism. Additionally, the differing second signals of K-Ras4B and H-Ras dictate localization to distinct plasma membrane subdomains, that may lead to distinct effector signaling.