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

The protein farnesyltransferase (PFT) 13-subunit gene of Saccharomyces cerevisiae, DPRI, was randomly mutagenized by PCR to construct a mutant DPRI gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) 18-subunit gene, CALl. A mutant DPRI gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a call disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I 1B subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dprl disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the 18 subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 ofDPRI product is critical for its substrate specificity. Farnesylation plays a critical role in the localization and function of proteins such as Ras (1, 2). This modification is a crucial step in a series of C-terminal modification events that facilitate membrane association. Because membrane association is important for the function of Ras protein including its transforming capability, protein farnesyltransferase (PFT), which catalyzes the reaction, has been studied extensively as a possible target of antitumor drugs (3, 4). PFT is a heterodimer composed of a and 13 subunits (5, 6). The a subunit is shared by another member of protein prenyltransferases, protein geranylgeranyltransferase type I (PGGT-I). PFT transfers a C15 farnesyl group from farnesyl diphosphate (FPP) to proteins ending with CAAX (C is cysteine, A is usually an aliphatic amino acid, and X is the C-terminal amino acid, which is predominantly methionine, serine, cysteine, alanine, or glutamine), whereas PGGT-I transfers a C20 geranylgeranyl group from geranylgeranyl diphosphate (GGPP) to a protein ending with CAAL (L is leucine, but phenylalanine also can function). The 13 subunit of PGGT-I is distinct from the 13 subunit of PFT, but significant sequence similarity is found between the two (7). Thus, the substrate specificity appears to be conferred by the 13 subunit. Consistent with this idea is the suggestion by cross-linking studies that both FPP (8) and protein substrates (9, 10) bind The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. to the X3 subunit of PFT. When two different-sized photoaffinity-labeling peptide substrates were used, a shorter peptide substrate cross-linked to the (3 subunit, whereas a longer peptide substrate cross-linked to both a and (3 subunits (10), L which raises the possibility that the peptide binding site is at the interface between the two subunits. To gain information concerning the region of PFT (3 subunit involved in substrate specificity, we have used the yeast Saccharomyces cerevisiae system in which genetic screens can be applied. Yeast PFT ,B subunit and PGGT-I 13 subunit are encoded by DPR1/RAM1 (referred to as DPR1 throughout) and CAL1/CDC43, respectively (11-13). PFT and PGGT-I share an a subunit encoded by RAM2 (12, 13). We have attempted to isolate mutant PFT (3 subunit that can substitute for PGGT-I (3 subunit by screening randomly mutagenized DPR1 genes for suppression of a temperature-sensitive mutation in the CAL] gene. We reasoned from the following observations that such mutant DPR1 genes could be obtained: (i) DPR1 and CAL1 products share a significant sequence similarity (14), and (ii) like mammalian PFT (15), yeast enzyme shows low but significant cross-specificity to substrates for PGGT-I (16-18). In this paper, we describe isolation and characterization of mutant PFT, which can function in place of PGGT-I in vivo. The mutant enzyme has increased affinity to a PGGT-I substrate, while exhibiting reduced affinity for its own substrate. Interestingly, a single amino acid alteration is responsible for this change in substrate specificity. MATERIALS AND METHODS Yeast Strains, Media, and Transformation. S. cerevisiae strains used in this study were the following: YOT559-3C (MATa call-i leu2 trpl ura3 ade2), YOT359-12C (MATa Acall::ura3 lys2 leu2 trpl ura3 ade2 his3 pRB1601) (16), KMY200-sgp2-No.2-12A [MATTa dprl(sgp2)::URA3 ade8 trpl ura3 his3] (19), and CJ198-2B (MATa cdc43-2 trpl ura3 gal2) (13). Complex medium (YPD; yeast extract/peptone/dextrose) was prepared as described (20). Synthetic complete media lacking tryptophan (SC-Trp) were prepared by supplementing the synthetic minimal medium (20) with 0.5% Casamino acids and uracil and/or adenine. The yeast transformation was carried out by a modified lithium acetate method (21). Plasmids. pSK(DPR1) was constructed by ligating a 2.4-kb Sal I/Xba I DPRI fragment (22) into the Sal I/Xba I site of pBluescript SK+ (Stratagene). The 1.3-kb Pst I/Hpa I region ofDPRJ (Fig. 1A) on pSK(DPR1) was amplified by PCR using Abbreviations: PFT, protein farnesyltransferase; PGGT-I, protein geranylgeranyltransferase type I; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; GST-CIIS, glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein; GSTCIIL, the same as GST-CIIS except the C-terminal serine is changed to leucine. *To whom reprint requests should be addressed.