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

ABSTRACT: Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called 'CAAX box' at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

SUBMITTER: Gomez R

PROVIDER: S-EPMC1132125 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Project description:The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.

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

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