cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.

* Part of this work was supported by National Science Foundation American Hearthsociation (both to P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. residue is leucine (1,2). Known targets of GGTase-I include the y subunits of brain heterotrimeric G proteins and Ras-related small GTP-binding proteins such as Racl, Rac2, RaplA, and RaplB (3)(4)(5). Additionally, short peptides encompassing the CAAX motif of these substrates can also be recognized by the enzyme (4)(5)(6). Immobilization of one such peptide for use as a n affinity matrix has led to the isolation of GGTase-I from bovine brain (7). The purified enzyme contains two subunits with molecular masses of 48 kDa and 43 kDa, which have been designated, respectively, as a and p (henceforth designated &GI).
GGTase-I is dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence required prolonged incubation against, or purification in the presence of, a chelating agent. This property led to the designation of GGTase-I as a zinc metalloenzyme (7).
The properties of GGTase-I are similar to those of a related enzyme, protein farnesyltransferase (FTase). FTase transfers the prenyl moiety from farnesyl diphosphate to the cysteine residue of substrate proteins. FTase protein substrates, like those for GGTase-I, possess a carboxyl-terminal CAAX motif.
The "X" residue of mammalian FTase substrates, however, is generally methionine, serine, or glutamine as opposed to leucine for GGTase-I substrates (5, 7). Substrates for FTase include p2lrU8 protein, lamin B, and several proteins involved in visual signal transduction (1). Like GGTase-I, FTase is dependent upon Mg2' and Zn2+ ions for optimal activity (8). Purified mammalian FTase is composed of two nonidentical subunits, a and (henceforth designated PF), with apparent molecular masses of approximately 48 kDa and 46 kDa, respectively, on SDS-PAGE. (9). cDNA clones encoding the FTase a and PF subunits have been isolated, and their deduced amino acid sequences are homologous to the Saccharomyces cerevisiae proteins Ram2 and Dprl/Raml, respectively, which encode the subunits of yeast FTase (5,(10)(11)(12).
The 48-kDa a subunits of mammalian GGTase-I and FTase have been shown to be immunologically cross-reactive, suggesting that these two enzymes share a common a subunit (7, 11,13). Similarly, a mutation in the S. cerevisiae gene R A M 2 , which encodes the d i k e subunit of yeast FTase, results in a strain with a small, but significant, defect in GGTase-I activity in addition to a drastic reduction in FTase activity (5,11). Further confirmation of a common subunit for yeast FTase and GGTase-I came from the bacterial co-expression of Ram2 with Ram1 that resulted in FTase activity (12), while co-expression of Ram2 with Cdc43Eall resulted in GGTase-I activity (14). Since the S. cerevisiae Cdc43Kall subunit of GGTase-I shows amino acid similarity to yeast RamlDprl and mammalian pF, it may be the yeast homolog of mammalian PGGI (15).
GGTase-I differs from the related enzyme Rab geranylger-3175 anyltransferase, also called protein geranylgeranyltransferase type-I1 (designated GGTase-111, which attaches a geranylgeranyl group to the COOH-terminal cysteines in small GTP-binding proteins that terminate in Cys-Cys or Cys-X-Cys motifs (5, 16,17). Target proteins of GGTase-I1 include RablA, which resides in the endoplasmic reticulum and Golgi complex, and Rab 3A, a component of synaptic vesicles (18). GGTase-I1 does not recognize COOH-terminal peptides corresponding to its target proteins, rather, substrate recognition appears to involve additional determinants on the Rab proteins (5,19). GGTase-I1 consists of three protein components. One component, the 95-kDa Rab escort protein, appears to bind substrate proteins and presents them to the catalytic subunits of the enzyme (20). The catalytic component of GGTase-I1 contains two tightly associated polypeptides with apparent molecular masses of 60 kDa and 38 kDa that, respectively, show similarity to the a and PF subunits of FTase (21).
In the current study, we obtained peptide sequence from both the a and pmI subunits of purified bovine GGTase-I. The three peptide sequences from the a subunit of GGTase-I showed 100% identity to the amino acid sequence deduced from a cDNA encoding the a subunit of bovine FTase (ll), providing direct evidence to support the hypothesis that mammalian FTase and GGTase-I share a common a subunit. Peptide sequences from the subunit were used to design probes to screen rat and human cDNA libraries. Overlapping cDNA clones from both human and rat cDNAlibraries were isolated that encode nearly identical proteins with 377 amino acids. The deduced protein sequences show similarity to yeast Cdc43 as well as to the p subunits of mammalian FTase and GGTase-11. Co-expression in Escherichia coli of the human pcGI subunit with the a subunit of human FTase produced a heterodimeric enzyme with the catalytic properties of bovine brain GGTase-I, confirming that the cDNAs obtained encode the pmI subunit of mammalian GGTase-I.

EXPERIMENTAL PROCEDURES
General Methods-Standard molecular biology techniques were used (22, 23). Enzymes were obtained from New England Biolabs or Boehringer Mannheim. cDNA clones were subcloned into pGEM-4Z or pUC18 and sequenced by the dideoxy chain termination method (24) using universal primers or specific internal primers. All c D N h and PCR products were sequenced on both strands. 32P-Labeled DNA probes were synthesized using a random primer labeling kit (Bethesda Research Laboratories or Boehringer Mannheim) and [32P1dNTPs (Amersham). Total cellular RNA was isolated from tissue by the guanidinium thiocyanate/CsCl centrifugation procedure (25). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography (22).
Protein Sequence Determination of GGTase-I-Approximately 1 nmol of GGTase-I purified from bovine brain as described (7) was subjected to electrophoresis on an 11% SDS-polyacrylamide gel and transferred to nitrocellulose paper. The nitrocellulose paper was then stained with Ponceau S to localize the subunit polypeptides. The 48-kDa and 43-kDa bands were excised from the nitrocellulose and sent to Harvard Microchem (Cambridge, MA) for processing. Briefly, this involved digestion in situ with trypsin, isolation of peptides produced by microbore HPLC, and sequence determination (26). High-confidence sequences of three a subunit peptides and five pal subunit peptides were obtained (Table I).

AT(A/CPT)-CCN-TT(T/C)-AA(T/C)-CC) encoding part of the peptide
GSSYLGIPFNPSK. The PCR fragment was cleaved with EcoRI and BamHI (restriction sites in the PCR oligos) and cloned into pUC18 creating pRD548.
To isolate human cDNh encoding the pwI subunit, a 300-bp EcoRI-Hind111 fragment containing the amino-terminal portion of the coding sequence in pRD548 was 32P-labeled and used to screen approximately lo6 plaques each from both a human placenta cDNA library in hgtll (Clontech) and a human kidney cDNA library in hmaxl (Clontech) as previously described (29). Six cDNA clones were isolated from the human placenta cDNA library, and seven were isolated from the human kidney cDNA library. Phage from the hgtll library were isolated, and the cDNA inserts were subcloned into pUC18 as EcoRI fragments. cDNA inserts from clones from the hmaxl library were excised as phagemids. A plasmid containing the 1.55-kb cDNA from clone 3 from the human placenta cDNA library was designated pRD550. The insert in pRD550 contains all but the amino-terminal 36 codons for pwI. The phagemid containing the 0.7-kb cDNA from clone 27 from the human kidney cDNA library was designated pRD558. The insert in pRD558 encodes the amino-terminal 123 amino acids of pwI. Aplasmid with the complete human pwI coding sequence was constructed as follows. PCR was performed on pRD558 placing a BamHI and ScaI site upstream of the pGcI start codon. This DNA was cleaved with BamHI and XhoI, which cleaves within the p c C I coding sequence, creating Fragment 1 of 0.13 kb. Fragment 2 was a 1.52-kb XhoI-EcoRI fragment from pRD550 that contained the coding sequence downstream of the XhoI site. Fragments 1 and 2 were cloned into BamHI-EcoRI-digested pUC18 creating pRD566 which contains the complete coding sequence for human and 3"untranslated sequences.
To isolate rat cDNh encoding the par subunit, the PCR probe from pRD548 was labeled and used to screen a rat brain 5"stretch cDNA To isolate the amino-terminal coding sequence for rat the 5'the 5'-end of clone 22 (29). Three primers were prepared based on the 5'-end sequences of clone 22. First strand cDNA was synthesized from rat brain mRNA with one of the primers, and the cDNA was then tailed with dATP using terminal transferase. Two rounds of PCR reactions were performed using the two nested primers and an oligo(dT) primer. The RACE products obtained were cloned into pGEM-4Z. Twelve clones from different PCR reactions were sequenced on both strands, and all yielded identical sequences. The DNA sequence of these clones contained an in-frame ATG codon 3 base pairs upstream of the 5'-end of the cDNA in clone 22. This ATG codon is in the same position as the initiation codon found in the cDNA sequence of human pwI (Fig. l).
Expression and Purification of Recombinant Human GGTase-I and FTaSe-lb express human GGTase-I in E. coli, the cloned human pw1 subunit cDNA and the previously cloned human Fl'ase-a subunit cDNA (28) were co-expressed from a plasmid in which their expression was translationally coupled. In E. coli, the plasmid pT5T-hFFTase-a expresses the human a subunit protein with a carboxyl-terminal Glu-Glu-Phe epitope tag from a bacteriophage T7 promoter (28). The coding sequence for the human par protein was cloned downstream of the a subunit coding sequence in pT5T-hFFTase-a as follows. Fragment 1, a 0.5-kb SpeI-XhoI fragment, containing the sequence CT between the carboxyl terminus of a and the amino terminus of the subunit coding sequences was made by recombinant PCR using pT5T-hFP-Tase-a and pRD566 as templates (30). Fragment 2, a 1.52-kb XhoI-EcoRI fragment from pRD566 contained the part of the p a r coding sequence not in fragment 1. Fragment 3 was a 6.2-kb SpeI (partial digestion)-EcoRI fragment from pT5T-hFPTase-a that contained the portion of the a coding sequence not in fragment 1 and the vector and promoter sequences from pT5T-hFPTase-a. Fragments 1,2, and 3 were ligated together to create pRD577 which has the following structure:  The purified 48-kDa a subunit and 43-kDa PcGI subunits of bovine brain GGTase-I were digested with trypsin and the resulting peptides were purified by HPLC and sequenced (see "Experimental Procedures"). The GGTase-I a subunit peptides are shown compared to peptides deduced from a cDNAencoding the bovine Fl'ase a subunit (11). aa, amino acid. To express human GGTase-I, pRD577 was transformed into E. coli BL21(DE3), grown, and induced with isopropyl-P-o-thiogalactoside as described (28). Recombinant, human GGTase-I was purified from the cells essentially as described for human FTase using a YL1/2 antibody column, which binds the Giu-Glu-Phe epitope tag on the a subunit and a subsequent Mono Q HR 515 column (28). The GGTase-I eluted from the Mono Q column at approximately 0.25 M NaCI.
Recombinant human Fl'ase was expressed in E. coli and purified as described (28).
Antibodies and Immunoblotting-A fragment from pRD548 encoding amino acids 49-146 of pal was cloned as an EcoRI-Hind111 fragment into pMAL-cRI (New England Biolabs) in order to produce a maltose binding protein-fusion protein. The maltose binding protein-PGoI fusion protein was isolated as previously described (31) and sent to Hazelton Research Products (Hazelton, PA) to immunize rabbits. Immunoblotting using the immune antisera was performed as previously described (11). GGTase-I and muse Assays-GGTase-I and Fl'ase activities were assayed as previously described (5). Ras protein substrates used were

Protein Sequencing
of GGTase-I Subunit Polypeptides-Affinity chromatography of GGTase-I yielded sufficient quantities of purified protein from bovine brain to obtain the internal sequences of both the 48-kDa a and 43-kDa pGGI polypeptide components. A number of clearly resolved peptides were obtained from HPLC purification of tryptic digests from each polypeptide (results not shown), and their sequences were determined (Table I). A sequence comparison between the peptides obtained from the 48-kDa a subunit of bovine GGTase-I and corresponding regions of bovine FTase-a as deduced from a cDNA clone (11) are shown (Table I). Three different peptides from the a subunit of GGTase-I were sequenced and found to be 100% identical with regions of FTase-a. This sequence identity provides direct evidence that both GGTase-I and FTase share a common subunit. Verification of this hypothesis comes from the finding that co-expression of cDNA encoding FTase-a and GGTase-I p w I produce a fully active GGTase-I enzyme (see below). product was identified that hybridized to a degenerate oligonucleotide probe for pmI peptide 2. DNA sequencing of the PCR fragment indicated that it contained an open reading frame coding for peptides 1, 2, and 3, suggesting that it coded for about two-thirds of the pGGI subunit (data not shown). Overlapping cDNAs encoding the complete rat and human pGGI subunits of GGTase-I were obtained by a combination of screening with the 730-bp PCR probe and RACE techniques as described under "Experimental Procedures." The nucleotide sequence of the overlapping cDNAs encoding the pwI subunit of human GGTase-I is shown in Fig. 1. The sequence contains a 312-bp 5"untranslated region followed by a coding region of 1131 bp and a 3"untranslated region of 526 bp. "he cDNA sequence for the pCcI subunit of rat GGTase-I (not shown) contains a 9-bp 5"untranslated region followed by an 1131-bp coding region and a 420-bp 3"untranslated region. Both cDNAs encode a protein of 377 amino acids that contains all five peptides whose sequences were obtained from tryptic digestion of the purified p m~ subunit (Table I). The predicted molecular mass of the cloned rat and human P~I polypeptides, 42.4 and 42.5 kDa, respectively, is very similar to the 43 kDa observed on SDS-PAGE for the pccI subunit of the enzyme purified from bovine brain (7). In addition, the human cDNA clone contains an in-frame termination codon 24 bp upstream of the initial  I1 ( a -1 1 ) . Sequence comparisons were performed with the CLUSTAL program of the DNA-STAR sofiware package. By this analysis, rGG-I shows 30.2% identity and 53.5% similarity with Cdc43,30% identity and 55% similarity with rGG-11, and 28% identity and 51% similarity with rFTase. An alignment of the amino acid sequences of the rat and human GGTase-I p m I subunits together with other protein prenyltransferase p subunits is shown in Fig. 2 and 43 kDa as expected for GGTase-I (Fig. 3, lane 1). Confirmation that the 43-kDa protein was in fact pmr was done using an antibody directed to a fusion protein of part of the cloned pGGI sequence. This antibody reacted with the 43-kDa peptide in the purified, recombinant human GGTase-I and to the identically migrating peptide of GGTase-I purified from bovine brain ( Fig. 3, lanes 3 and 4 ) . This antibody did not react with the pF subunit of human FTase (Fig. 3, lane 5). Preimmune sera from the same rabbit did not react with any of these proteins (data not shown). Enzymatic Properties ofRecombinant GGZbse-I-Purified recombinant human GGTase-I was assayed to see if its enzymatic properties were similar to GGTase-I isolated from a natural source. The results in Table I1 show that recombinant GGTase-I, as previously reported for GGTase-I isolated from bovine brain, preferentially utilizes Ras-CAIL and geranylgeranyl diphosphate as substrates (5)(6)(7)131. In comparison, the substrate preferences of FTase are the opposite of GGTase-I in that it preferentially utilizes Ras-CVLS over Ras-CAIL and farnesyl diphosphate instead of geranylgeranyl diphosphate. The specific activity of the recombinant human GGTase-I using Ras-CAIL and geranylgeranyl diphosphate as substrates was 1060 nmol.h".mgl which is similar to that previously determined for GGTase-I isolated from bovine brain (7). The combination of substrate specificity of the recombinant human GGTase-I along with its specific activity indicate that recombi-anti-PGG Briefly, 100 ng of GGTase-I or FTase was incubated in a 300-pl reaction at 30 "C, 50-pl aliquots were removed after 2,4,6,8, and 10 min, and aciaethanol-insoluble radioactivity was determined. Each assay was performed in duplicate, and the rates of incorporation (nmol.h-'.mg") were determined. The mean value of the duplicates is shown. nant human GGTase-I has the same catalytic properties as the enzyme isolated from bovine brain (5, 7). DISCUSSION We have cloned cDNAs encoding the complete subunits of rat and human GGTase-I that contain all five peptides isolated from the purified protein ( Figs. 1 and 2). The proteins encoded by each of these cDNAs are 377 amino acids in length ( Figs. 1 and 2). Human paI, when expressed in E. coli, comigrates with the paI subunit of GGTase-I isolated from bovine brain, and both are recognized by antisera raised to the recombinant protein (Fig. 3).
The similarity among the amino acid sequences of the rat and human GGTase-I paI subunit and the S. cereuisiue CDC43 gene product indicates that the Cdc43 protein is the yeast equivalent of the pal subunit of mammalian GGTase-I (Fig. 2). This finding is consistent with previous studies showing that point mutations in the yeast CDC43 gene decrease GGTase-I activity and that co-expression of Cdc43 and Ram2 in bacteria produces GGTase-I activity (5, 14,35). The deduced polypeptide sequences of the rat and human Par subunit of GGTase-I also show homology to the sequences of other known protein prenyltransferase /3 subunits (Fig. 2). The sequences of the three peptides isolated from the a subunit of bovine GGTase-I are identical with corresponding regions of the deduced amino acid sequence of the a subunit of bovine FTase (Table I). We have cloned numerous cDNAs for the a subunit of bovine and human FTase (11, 28h2 DNA sequence and restriction endonuclease analysis of these clones have shown no evidence for the existence of multiple genes. Additionally, the 48-kDa a subunits of GGTase-I and FTase purified from bovine brain or fractionated from rat brain co-migrate on SDS-PAGE (7,13) This information, combined with the demonstration that co-expression of FTase-a with GGTase-I p a I resulted in GGTase-I enzyme activity (Fig. 3, Table 11), strongly indicates that mammalian FTase and GGTase-I share an identical a subunit. A similar situation has been demonstrated in yeast with Ram2 being a common subunit for FTase and GGTase-I (12,14).
Since both FTase and GGTase-I have an identical a subunit, the P subunits of these enzymes must determine which prenyl diphosphate and protein substrate they use. Consistent with this idea, both Ras and FPP substrates cross-link to the OF subunit of FTase (8,28). cDNA cloning of the pGCI subunit of GGTase-I will allow construction of chimeras between the P subunits of FTase and GGTase-I. Such experiments might define regions that are responsible for the substrate specificities of the two enzymes. Furthermore, overexpression systems for GGTase-I similar to those recently described for FTase will provide a route for detailed structural and functional studies of GGTase-I (28,36).