Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Abstract

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.

Prenylation of the Floral Transcription Factor APETALA1 Modulates Its Function

“…GST-ap1mL was also labeled weakly with 3 H-FPP but not with 3 H-GGPP, consistent with earlier reports that plant FTase can farnesylate GGTase-I-like substrates but cannot utilize GGPP (Yalovsky et al, 1997). Such complementation would be similar to the promiscuity of GGTase-I observed in yeast and mammalian cells (Trueblood et al, 1993;Dalton et al, 1995;James et al, 1995).…”

“…GST-ap1mL was also labeled weakly with 3 H-FPP but not with 3 H-GGPP, consistent with earlier reports that plant FTase can farnesylate GGTase-I-like substrates but cannot utilize GGPP (Yalovsky et al, 1997). Such complementation would be similar to the promiscuity of GGTase-I observed in yeast and mammalian cells (Trueblood et al, 1993;Dalton et al, 1995;James et al, 1995).To confirm that AP1 is prenylated in vivo, we expressed full-length AP1 or ap1mS in Nicotiana benthamiana plants, using a potato virus X (PVX) vector (Figure 3). Proteins were prepared from infected leaves labeled with 3 H-mevalonic acid and separated on SDS-polyacrylamide gels, which were then used either for immunoblot analysis with polyclonal AP1 antibodies ( Figure 3A) or for fluorography to detect labeled AP1 ( Figure 3B).…”

“…The protein was not labeled in an extract prepared from yeast ram1 ⌬ cells, which lack FTase activity (Trueblood et al, 1993;Yalovsky et al, 1997) (Figure 2A). Labeling was also substantially reduced in a protein extract from mock-infected Sf 9 cells containing only endogenous FTase.…”

Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.

“…As we have shown above, the FTase becomes essential when it is needed to substitute for the GGTase I and prenylate Rholp and Cdc42p (Figure 1 Trueblood et al, 1993). Ras2p is likely the most important normal FTase substrate, because overproduction of Ras2p suppresses the cdc43-2 Adprl synthetic lethality.…”

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

“…This result confirmed that Chs4p is prenylated in vivo. Since prenylation is an irreversible process, we assume that if Chs4p is preferentially farnesylated, inactivation of endogenous FTase will increase the amount of unmodified Chs4p available as a substrate for reaction in vitro, even if crossspecificity between FTase and GGTase I are observed (29,31,41). To prove the involvement of FTase in modification of Chs4p, we immobilized TAP-Chs4p expressed in wt yeast cells or cells lacking the catalytic subunit of FTase (Ram1p) on IgG-Sepharose beads and used the resin-Chs4p as a substrate for FTase assays in vitro.…”