Studies of protein farnesylation in yeast

Abstract

Protein farnesylation has emerged as one of the important classes of posttranslational modification of proteins [84, 109]. Farnesylation is the first step in a three-step series of protein processing and involves the addition of a farnesyl group to a cysteine located in a C-terminal motif called the CaaX motif (C is cysteine, a is aliphatic amino acid, and X is the C-terminal amino acid that is usually methionine, cysteine, glutamine, alanine, or serine). These proteins subsequently undergo proteolytic cleavage and carboxylmethylation (Figure 1). Significance of protein farnesylation in human cancer has been underscored by the findings that many proteins undergoing farnesylation play roles in signal transduction. Of these, Ras proteins are particularly noteworthy. Protein farnesylation is catalyzed by protein farnesyltransferase (FTase) that is conserved from yeast to human [84, 109]. This enzyme recognizes the CaaX motif and transfers a farnesyl group from farnesyl pyrophosphate (FPP), an intermediate in cholesterol biosynthesis, resulting in the formation of a thioether bond. The catalytic mechanism of action of the FTase has been elucidated [91]. The enzyme contains one molecule of a tightly bound zinc ion (Zn2+) that participates in the catalytic reaction [51, 91, 97]. With yeast FTase, the reaction proceeds by an ordered mechanism with FPP binding first. In the case of mammalian enzyme, it has been suggested from kinetic and structural studies that the bound FPP forms part of the binding surface of the protein substrate [36]. Three-dimensional structures of the rat and human enzymes have been determined with and without bound substrates [65, 82, 95]. The structure mainly consists of ?-helices with the β-subunit forming a barrel like structure and the ?-subunit wrapping around the β-subunit from one side of the barrel. FTase belongs to a family of protein prenyltransferases which includes protein geranylgeranyltransferase type I (GGTase I) and type II (GGTase II). GGTase I catalyzes geranylgeranylation of proteins such as RhoA, Cdc42, or Rac proteins [24, 39, 86]. The modification involves a 20-carbon geranylgeranyl group added to a cysteine in the CaaL motif (similar to the CaaX motif except that the C-terminal amino acid is leucine or phenylalanine) [74, 108]. GGTase I shares a common ?-subunit with FTase, while its β-subunit shares about 30% homology with the βsubunit of FTase. GGTase II catalyzes the addition of a geranylgeranyl group to both cysteines within the CC or the CXC motif that are found in a number of Rab protein [88]. In addition to α - and β-subunits that share homology with their counterparts in FTase, GGTase II contains an additional component-Rep (Rab escort protein) [96]. Recently, anticancer drugs based on the inhibition of protein farnesylation have been developed. These small molecular weight compounds called farnesyltransferase inhibitors (FTIs) selectively inhibit farnesyltransferase [7, 38, 84, 94]. A variety of compounds including peptidomimetic inhibitors, farnesyl pyrophosphate analogues, bisubstrate inhibitors, and natural compounds have been identified. FTIs have been shown to reverse rasmediated phenotypes in ras-transformed cells. FTIs inhibit the growth of tumors or even regress tumors in animal model systems [62]. Currently, FTIs are being evaluated in a variety of clinical trials [57]. In this review, we discuss how yeast studies contributed to the overall study of protein farnesylation. We will first describe yeast studies concerning FTase as well as yeast-based assays that led to the identification of one of the first generation FTI compounds. We will then focus on identification and characterization of farnesylated proteins. Yeast has provided a comprehensive analysis of farnesylated proteins that was instrumental in the identification of a number of novel farnesylated proteins. Of particular interest is Rheb, a novel family of the Ras-superfamily G-proteins. This protein is highly conserved and plays a role in the regulation of cell cycle at the G1/S phase. We will summarize the current understanding of Rheb in S. cerevisiae and S. pombe. © 2007 Springer.