RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.

Abstract

In the yeast Saccharomyccs cerevwise, qiutations in either of two unlinked genes, RAM)-1or RAM2, abolish the farnesyltrnsferae activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that thefunction of RAM) antd RAM2 genme is required for the membrane laliation ofRas proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein.We examined tie functional interaction ofRAM2 andRAMf by expressing the genes in Escherchia colt. Extracts derived from an E. coil strain that coexpressed RAM] and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. colN strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferaSe activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Raml and Ram2 polypeptides. Although Raml is a component of the enzyme, disruption of the RAM) gene in yeast was not lethal indicating that the Raml-Ram2 farnesyltserase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Raml in protein farnesylation.The post-translational modification of proteins by the covalent attachment of isoprenoid groups plays an important role in the membrane targeting of various proteins (1-5). Three classes of prenylated proteins in eukaryotic cells have been described. The first class, represented by certain fungal mating pheromones, Ras proteins, and nuclear lamins, is initially synthesized with a C-terminal CAAX sequence, where X = Ser, Cys, Met, or Ala, and is modified by thioether linkage of a C15 farnesyl group to the cysteine residue (6-9). The second class of prenylated proteins, represented by the y subunits of certain heterotrimeric G proteins and various Ras-like proteins, is initially synthesized with different C-terminal CAAX sequences, in which X = Leu or Phe, and is modified by the attachment of a C20 geranylgeranyl group to the cysteine residue (10-12). Members of both of these classes of prenylated proteins are subsequently processed by proteolytic removal of the three terminal amino acids and methylation of the newly exposed carboxyl group of the prenyl-cysteine (6, 10-15). The third class of prenylated proteins, represented by the Yptl/Sec4 (Rab) family of Ras-like GTPases that terminate with the sequence CC or CXC, is also geranylgeranylated at a C-terminal cysteine residue(s) (16)(17)(18).

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

“…43 kDa [24]. The RAM2 gene encodes a protein of 316 amino acid residues [21]. These genes were placed on multicopy plasmids and transfected into wild-type yeast cells, either together or individually.…”

“…The rabbit sera containing the DPR1 antibody was used to probe the purified yeast FTase. As shown in Figure 9( [2,18]; (ii) DPR1 protein purified from E. coli can reconstitute FTase when added to the extracts prepared from dprl mutant cells [18]; (iii) sequences of DPR1 and RAM2 proteins share a low, but significant, similarity with ,-and a-subunits respectively of the mammalian FTase [19,20]; (iv) extracts of E. coli cells expressing both DPRI and RAM2 exhibit FTase activity [21]. What has been lacking in these studies is the direct demonstration of [10] reported similar findings using the mammalian FTase and peptide substrates.…”

“…detected after simultaneous expression of DPRI and RAM2 One of the interests in FTase is its potential as a target for genes in Escherichia coli [21]. Although these results point to the drugs which inhibit the action of ras proteins [12,13].…”

Protein Farnesyltransferase in Plants (Molecular Cloning and Expression of a Homolog of the [beta] Subunit from the Garden Pea)

“…cDNA clones encoding FTase a and P subunits from rat have recently been sequenced (Chen et al, 1991a(Chen et al, , 1991b. The Saccharomyces cerevisiae counterparts, the proteins encoded by DPRlIRAMI and RAMZ, share 37 and 30% identities with the predicted amino acid sequences of the rat subunits, respectively (Goodman et al, 1988(Goodman et al, , 1990Chen et al, 1991b;He et al, 1991). The yeast CAL2 gene, which is identical to CDC43, encodes the p subunit of GGTase I, which shows 24% identity with the protein encoded by R A M l (Finegold et al, 1991;Kohl et al, 1991;Ohya et al, 1991).…”

“…Yeast DPRlIRAMl mutants are defective in both cell division and mating (Goodman et al, 1990), and mutations in RAM2 are lethal (He et al, 1991). Mutation in CDC43ICALl results in loss of the establishment of cell polarity in budding yeast (Johnson et al, 1990).…”