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.

Molecular and Biochemical Characterization of Tomato Farnesyl-Protein Transferase

“…6B). This result establishes that LeFTB is the component of a 100-kD LeFTase in plants and confirms that LeFTase has a molecular mass of approximately 100 kD, which is similar to that of the mammalian and yeast FTases (Reiss et al, 1990;Gomez et al, 1993). .…”

Mutant farnesyltransferase beta subunit of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit.

“…Wild-type and mutant PFTs were partially purified from KMY200-sgp2-No.2-12A carrying pYO324(DPR1)BA or 1-2N (chimeric plasmid containing 5' portion of the mutant gene shown in Fig. lC) as described (11). Briefly, extracts of yeast cells grown in 5 liters of SC-Trp medium at 30°C were subjected to ammonium sulfate fractionation (30-60% saturation), and the sample was applied to a Mono Q column.…”

“…Yeast PFT ,B subunit and PGGT-I 13 subunit are encoded by DPR1/RAM1 (referred to as DPR1 throughout) and CAL1/CDC43, respectively (11)(12)(13). PFT and PGGT-I share an a subunit encoded by RAM2 (12,13).…”

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

“…FTase is a conserved heterodimeric enzyme in mammalian cells, yeast, and plants (Reiss et al, 1991a(Reiss et al, , 1991bCrowell et al, 1993;Gomez et al, 1993;Trueblood et al, 1993; results) that recognizes a conserved C-terminal CaaX motif (C = Cys, a = aliphatic amino acid, X = variable) (Reiss et al, 1991(Reiss et al, , 1991bSchafer and Rine, 1992). The heterodimeric GGTase type I, or GGTase I, shares the a subunit with FTase but has a distinct p subunit (Seabra et al, 1991;Mayer et al, 1992;Trueblood et al, 1993).…”