Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart.
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.
Gomez
R
R
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Goodman
L E
LE
Tripathy
S K
SK
O'Rourke
E
E
Manne
V
V
Tamanoi
F
F
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002412:Cations; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D002412:Cations; D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; C070038:RAM1 protein, S cerevisiae