Farnesylation and proteolysis are sequential, but distinct steps in the CaaX box modification pathway.
Membrane localization of Ras proteins requires posttranslational modification of a conserved C-terminal sequence motif known as the CaaX box (C is Cys, a is any aliphatic amino acid, and X is the carboxyl terminal residue). The modification steps include farnesylation, removal of the three C-terminal amino acids, carboxyl-methylation, and palmitoylation. In yeast, the farnesyltransferase (FTase) is encoded by the RAM1(DPR1) and RAM2 genes, and the methyltransferase is the product of STE14. The gene encoding the protease(s) that is responsible for modification of the CaaX has not been identified. We have used in vitro-synthesized Ras2p and synthetic peptide substrates to investigate the relationship between farnesylation and proteolysis. Addition of yeast cytosolic extracts to rabbit reticulocyte extracts programmed to synthesize Ras2p led to prenylation of Ras2p and a change in electrophoretic mobility similar to that observed during Ras maturation in vivo. However, it was not possible to determine if the mobility shift is the result of prenylation, proteolysis or a combination of both steps. Therefore, we examined the relationship between farnesylation and proteolysis directly using extracts prepared from bacteria overexpressing the genes for the yeast FTase (RAM1 and RAM2) and synthetic CaaX box peptides. Extracts from bacteria expressing RAM1/RAM2 efficiently prenylate CaaX box peptides, but do not proteolyze the -aaX residues. However, addition of yeast extracts from wild type, ram1, or ste14 mutants resulted in the removal of the -aaX residues from prenylated CaaX box peptides.
Farh
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Department of Biochemistry, University of Iowa, Iowa City 52242, USA.
Mitchell
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DA
Deschenes
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RJ
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051231:Farnesyltranstransferase; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D010455:Peptides; C077378:RAM2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; C070038:RAM1 protein, S cerevisiae; D018631:ras Proteins