Ubiquitin-RAS peptide extensions as substrates for farnesyl-protein transferase and carboxymethyltransferase.
Using oligonucleotide-mediated 'loop-in' mutagenesis strategies in M13, 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 AR13 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.
Yoo
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Y
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Watts
S
S
Rechsteiner
M
M
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008745:Methylation; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014166:Transferases; D011497:Protein O-Methyltransferase; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D016283:Proto-Oncogene Proteins p21(ras)