Ubiquitin-RAS peptide extensions as substrates for farnesyl-protein transferase and carboxymethyltransferase

Abstract

Using oligonucleotide-mediated 'loop-in' mutagenesis strategies in Ml 3, a heat-inducible ubiquitin (Ub) gene was extended by sequences coding for the C-terminal 11 amino acids of Ha-RAS. The resulting gene was transformed into AR1 3 and production of the Ub-peptide extension was induced by heat treatment. After one-step purification, the fusion protein (Ub-cRAS) was used as a substrate for farnesyl-protein transferase. Ub-cRAS was farnesylated on incubation in Xenopus egg extract or rabbit reticulocyte lysate. In contrast, when serine was substituted for the last cysteine in the RAS extension, transfer of the [3H]farnesyl group from [3H] farnesyl pyrophosphate to the modified Ub-cRAS was not observed. Farnesylation of Ub-cRAS permitted us to develop an easy membrane-binding assay for farnesyl-protein transferase enzyme activity. Using this assay, we partially purified the enzyme from rabbit reticulocyte lysate. We also detected methylation of the farnesylated Ub-cRAS terminus in Xenopus egg extract. INTRODUCTIONAmong the enzymes left to be discovered and characterized, many catalyse reactions in which proteins are the substrate. This class of enzymes includes the ever-expanding families of protein kinases [1,2], enzymes that add lipids to proteins [3,4] and a wide variety of enzymes that modify specific amino acid side chains on cellular and secreted proteins [5][6][7]. Analysis of such enzymes requires peptides, either as potential substrates for measuring kinetic features of these catalysts or in the preparation of affinity matrices for purifying them. Recently, we described a method for synthesizing peptides as ubiquitin (Ub) extensions [8]. We have also demonstrated that Ub-peptide extensions can be used as substrates for a specific protein kinase [9]. In this regard, they offer distinct advantages over smaller peptide substrates commonly used to assay phosphotransferases.The RAS proteins, members of a large family of small guanine nucleotide-binding proteins, are present in all eukaryotic organisms. In order to function, they have to be localized at the plasma membrane [10]. This localization requires a series of posttranslational modifications which include the farnesylation of cysteine (the fourth residue from the C-terminus), proteolytic cleavage of the C-terminal three amino acids and the methylation of exposed COOH of cysteine [3,[11][12]. These modifications seem to require only the C-terminal consensus motif Cys-A-A-X (A, aliphatic amino acid; X, any amino acid) which is referred to as a CAAX box [13].Exploring further the utility of Ub-peptides as enzyme substrates, we have constructed a ubiquitin molecule extended by 11 amino acids from the C-terminus of Ha-RAS (PGCMSCKCVLS in the one-letter code). Here we describe the preparation and use of the Ub-peptide extension (Ub-cRAS) as a substrate for farnesyl-protein transferase (FPT) and carboxymethyltransferase. We also report on the partial purification and characterization of FPT from rabbit reticulocyte lysate.