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Cloning and Characterization of a Mammalian Prenyl Protein-specific Protease

1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês

10.1074/jbc.274.13.8379

1083-351X

James Otto, Edward Kim, Stephen G. Young, Patrick J. Casey,

Monoclonal and Polyclonal Antibodies Research

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Gγ1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins. Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Gγ1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins. A variety of proteins are modified with an isoprenoid lipid at a cysteine that is initially four residues from the C terminus (1Schafer W.R. Rine J. Annu. Rev. Genet. 1992; 26: 209-237Crossref PubMed Scopus (344) Google Scholar, 2Clarke S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (793) Google Scholar, 3Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1738) Google Scholar). Such proteins contain the so-called CAAX motif, in which the "C" is the modified cysteine, the "A" residues are most commonly (but not always) aliphatic amino acids, and the "X" residue can be one of several amino acids. TheX residue determines whether the protein is modified by the 15-carbon farnesyl lipid or the 20-carbon geranylgeranyl. If theX residue is a leucine, the protein will be geranylgeranylated; several other residues (e.g. Met, Ser, Ala, and Gln) direct farnesylation. Following prenylation of the protein, two additional processing steps occur (4Ashby M.N. Curr. Opin. Lipidol. 1998; 9: 99-102Crossref PubMed Scopus (71) Google Scholar, 5Rando R.R. Biochim. Biophys. Acta. 1996; 1300: 5-16Crossref PubMed Scopus (98) Google Scholar). First, a specific protease cleaves the -AAX tripeptide from the protein, leaving the prenylated cysteine as the new C terminus. The carboxyl group of the prenylcysteine is then methylated by a specific methyltransferase. It is well established that protein prenylation plays a vital role in the membrane localization and function of most prenylated proteins (6Glomset J.A. Farnsworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Crossref PubMed Scopus (278) Google Scholar). The role that the proteolysis and methylation of prenyl proteins play in their function, however, is not as well understood. Studies on peptides have demonstrated that each processing step significantly increases the affinity of farnesylated peptides for membranes (7Silvius J.R. l'Heureux F. Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (237) Google Scholar), although the effect of the final two steps is not as great for geranylgeranylated peptides. Proteolysis and methylation also increased the hydrophobicity of Ras proteins processed in an in vitrosystem (8Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar). Prevention of the proteolysis of Ras in cells resulted in a decrease in membrane localization (9Kato K. Cox A.D. Hisaka M.M. Graham S.M. Buss J.E. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6403-6407Crossref PubMed Scopus (555) Google Scholar, 10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar), and in yeast it resulted in at least a partial loss of Ras function (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar). The enzymes responsible for prenylation of CAAX-containing proteins, protein farnesyltransferase and protein geranylgeranyltransferase I, have been cloned and studied in detail (3Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1738) Google Scholar). Additionally, a specific prenyl protein carboxymethyltransferase has been identified in yeast as the product of the STE14gene (11Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar), and a human homologue of the STE14 gene product has recently been described (12Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). These findings have left the prenyl protein protease as the only member of the processing pathway yet to be identified on a molecular level. Recently, an elegant genetic screen in yeast resulted in the identification of two candidate genes for prenyl protein proteases (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar). The first gene, AFC1/STE24, appeared to be primarily involved in the processing of the precursor to a-factor, a farnesylated yeast mating pheromone. AFC1/STE24 catalyzes two cleavage events on thea-factor precursor, the first being the C-terminal proteolysis and the second being a cleavage occurring near the N terminus of the peptide (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar, 13Fujimura-Kamada K. Nouvet F.J. Michaelis S. J. Cell Biol. 1997; 136: 271-285Crossref PubMed Scopus (131) Google Scholar, 14Tam A. Nouvet F.J. Fujimura-Kamada K. Slunt H. Sisodia S.S. Michaelis S. J. Cell Biol. 1998; 142: 635-649Crossref PubMed Scopus (110) Google Scholar). Strong evidence was provided that the second candidate gene, RCE1, was involved in the processing of the yeast Ras proteins, in addition to the C-terminal processing of thea-factor precursor (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar, 15Schmidt W.K. Tam A. Fujimura-Kamada K. Michaelis S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11175-11180Crossref PubMed Scopus (163) Google Scholar). Because RCE1 was linked to the processing of a prenyl protein, it seemed likely that the RCE1 gene product might have a broader range of substrates than AFC1/STE24. We report here the identification, expression, and preliminary characterization of a human homologue of RCE1, termed hRCE1. Evidence is provided that hRCE1 is in fact a prenyl protein protease and that it is involved in the processing of a variety of CAAX-type prenylated proteins, including all known forms of Ras. These findings open the door for molecular studies of this protease and will facilitate studies aimed at determining the roles of the proteolysis and methylation steps in the functions of CAAX-type prenyl proteins. The λgt11 human umbilical vein endothelial cell library (HUVEC) 1The abbreviations used are:HUVEC, human umbilical vein endothelial cell; AdoMet, S-adenosylmethionine; EST, expressed sequence tag; PCR, polymerase chain reaction; bp, base pair(s). (16Sadler J.E. Shelton-Inloes B.B. Sorace J.M. Harlan J.M. Titani K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6394-6398Crossref PubMed Scopus (205) Google Scholar) was provided by John York of this institution. The plasmid pRS315-RCE1 containing the cDNA for yeast RCE1 (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar) was a gift from Jasper Rine (University of California, San Francisco); the pATH-STE14 plasmid containing the cDNA encoding the yeast methyltransferase STE14 (11Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar) was a gift from Susan Michaelis (Johns Hopkins Medical Center); the QE31-N-Ras expression plasmid for N-Ras (17Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) was a gift from Robert Bishop (Schering-Plow Research Institute); the pTrcHis-Rap1b expression plasmid for hexahistidine-tagged Rap1b (18James G.L. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) was a gift from Guy James (University of Texas Health Sciences Center, San Antonio). The reduced farnesyl-peptide analogue RPI (19Ma Y.T. Gilbert B.A. Rando R.R. Biochemistry. 1993; 32: 2386-2393Crossref PubMed Scopus (58) Google Scholar, 20Chen Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar) was a gift from Robert Rando (Harvard Medical School). Oligonucleotide primers were synthesized at the Duke University DNA Core Facility. The Bac-2-Bac Baculovirus Expression System, the GeneTrapper cDNA Positive Selection System, and the pCMV.SPORT 2 human fetal brain cDNA library were purchased from Life Technologies, Inc. S-Adenosylmethionine (AdoMet) was purchased from Research Biochemicals International. [3H-methyl]-S-adenosylmethionine ([3H]AdoMet) was purchased from New England Nuclear. Peptides were synhesized by Princeton Biomolecules. The deduced amino acid sequence of yeast RCE1 was used to conduct a text-based search of the human expressed sequence tag (EST) data base at the National Center for Biotechnology Information. The clone W96411 was identified as a likely homologue. Five additional clones that overlapped W96411 were present in the data base, representing 725 nucleotides of the cDNA sequence. The primer 5′-GGGCTTCAGGCTGGAGGGCATTTT-3′ was chosen from this sequence for use in a GeneTrapper cDNA positive-selection cloning protocol. A partial clone containing the hRCE1 open reading frame but lacking an initiation codon was cloned from a pCMV.SPORT 2 human fetal brain cDNA plasmid library. A probe was generated from the XcmI-PstI fragment of that clone and used to screen a λgt11 HUVEC library (16Sadler J.E. Shelton-Inloes B.B. Sorace J.M. Harlan J.M. Titani K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6394-6398Crossref PubMed Scopus (205) Google Scholar). That screen yielded a clone containing the likely initiation codon, because it contained an upstream stop codon in-frame with the coding sequence. The 5′ end of the HUVEC cDNA containing the initiation codon was removed as a BssHII-XcmI fragment and ligated to the human fetal brain cDNAXcmI-PstI fragment to generate a hRCE1 cDNA containing the entire open reading frame. ThisBssHII-PstI fragment was subcloned into the vector pFASTBAC-1 to produce p-FASTBAC-1-hRCE1. In a second construct, designated pFASTBAC-1-ΔhRCE1, the 5′-untranslated region of hRCE1 was replaced with a portion of the 5′-untranslated region from the baculovirus polyhedron gene (CCTATAAAT), and the codons specifying the first 22 amino acids of hRCE1 were removed (see Fig. 1). A 32P-labeled probe was generated with random hexamer priming from the 1100-bpBssHII-PstI hRCE1 cDNA fragment and hybridized to a human multi-tissue poly(A)+ RNA blot (obtained from CLONTECH); hybridization and washing were performed as described previously (21Glick J.L. Meigs T.E. Miron A. Casey P.J. J. Biol. Chem. 1998; 273: 26008-26013Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The process was repeated using a probe made to the cDNA of β-actin as a control. Expression of RCE1 was also examined using a mouse multiple tissue Northern blot (CLONTECH). The blot was probed with a 223-bp Rce1 cDNA fragment that was amplified from a mouse liver cDNA library (CLONTECH) with oligonucleotide primers 5′-TTGCCCTTGTGACCTGACAGATGG-3′ and 5′-GAGTGGGCAGGTGAACACAGCAGG-3′. Recombinant baculoviruses were produced with the pFASTBAC-1 vector and the protocol provided in the Bac-2-Bac Baculovirus Expression System kit. Recombinant baculoviruses were prepared for the two human RCE1 constructs described above, for yeast RCE1, and for the yeast prenyl protein carboxymethyltransferaseSTE14. For production of recombinant proteins, Sf9 cells in log phase growth were diluted to 1 × 106 cells/ml and infected with recombinant baculoviruses at multiplicities of infection ranging from 2 to 10. Cells producing yeast or human RCE1 were harvested 72 h post-infection and resuspended in 50 mmTris-HCl, pH 7.7. Cells expressing yeast STE14 were harvested 60 h post-infection, and resuspended in 5 mm NaHPO4, pH 7.0, containing 5 mm EDTA (11Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar) and a mixture of protease inhibitors (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar). In all cases, cells were disrupted by sonication, nuclei and debris were removed by centrifugation at 500 ×g for 5 min, and membranes were then pelleted by centrifugation at 200,000 × g for 1.5 h. Membranes from RCE1 producing cells were resuspended in 50 mm Tris-HCl, whereas membranes from cells producing STE14 were resuspended in 5 mm NaHPO4 containing 5 mm EDTA and the protease inhibitor mixture (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar). Final protein concentrations of the membrane suspensions were 10–25 mg/ml. The suspensions were flash-frozen in liquid nitrogen and stored at −80 °C in multiple aliquots. Unprenylated Ki-Ras (18James G.L. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), Ha-Ras (23Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (703) Google Scholar), N-Ras (17Zhang F.L. Kirschmeier P. Carr D. James L. Bond R.W. Wang L. Patton R. Windsor W.T. Syto R. Zhang R. Bishop W.R. J. Biol. Chem. 1997; 272: 10232-10239Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), Gγ1 (24Higgins J.B. Casey P.J. J. Biol. Chem. 1994; 269: 9067-9073Abstract Full Text PDF PubMed Google Scholar), and Rap1b (18James G.L. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 6221-6226Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) were expressed in Escherichia coli and purified essentially as described previously. Ki-Ras, N-Ras, and Rap1b each had an N-terminal hexahistidine tag, whereas Ha-Ras and Gγ1 were unmodified. Ki-Ras, Ha-Ras, N-Ras, and Gγ1 were farnesylated by incubation of 2 μm protein with 20 μg/ml purified farnesyltransferase (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar), 6 μm farnesyl diphosphate in buffer A (5 mm MgCl2, 5 μm ZnCl2, 2 mm dithiothreitol, 5 μm GDP, 50 mm Tris-HCl, pH 7.7) for 1 h at 37 °C. Ki-Ras and Rap1b were geranylgeranylated by incubation of 2 μm protein with 20 μg/ml purified geranylgeranyltransferase 1 (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar), 6 μm geranylgeranyl diphosphate in buffer A for 1 h at 37 °C (23Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (703) Google Scholar, 25Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar). Farnesyl-Ki-Ras and geranylgeranyl-Ki-Ras were resolved from the unprenylated precursor and modifying enzyme by applying the prenylation reactions to a heptyl-Sepharose column, which was washed with buffer B (50 mm Hepes, 3 mm MgCl2, 1 mm dithiothreitol, 5 μm GDP) containing 0.1% lubrol (removing unprenylated Ki-Ras and the prenyltransferase), and then eluting the purified prenyl-Ki-Ras with buffer B containing either 2% sodium cholate (farnesyl-Ki-Ras) or 4% sodium cholate (geranylgeranyl-Ki-Ras). The eluted prenyl-Ki-Ras preparations were diluted 20-fold in buffer B and applied to an S-Sepharose column. The column was washed with buffer B, and the prenyl-Ki-Ras was eluted with buffer B containing 0.1% octylglucoside and 750 mm NaCl. Purified prenyl-Ki-Ras stocks were flash-frozen in liquid nitrogen and stored at −80 °C in multiple aliquots. The peptides CVIM and GSPCVLM were prenylated chemically as described previously (26Thissen J.A. Gross J.M. Subramanian K. Meyer T. Casey P.J. J. Biol. Chem. 1997; 272: 30362-30370Crossref PubMed Scopus (102) Google Scholar) and purified by high pressure liquid chromatography. Protease reactions were initiated by the addition of membranes containing the recombinant prenyl protein protease to an assay mixture containing prenylated proteins in 100 mm Hepes, pH 7.4, and 5 mmMgCl2 in a total volume of 50 μl; reactions were conducted at 37 °C. Following the proteolysis reaction, methylation of proteolysed prenylated protein was initiated by addition of 20 μg of membranes containing STE14 to the reaction in 17.5 μm[3H]AdoMet (2000 Ci/mmol), 5 mmNaHPO4, pH 7.0, 62.5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 10 mm 1,10-phenanthroline, and 300 μm N-tosyl-l-phenylalanine chlorophenyl ketone in a total volume of 20 μl. The addition of EDTA and protease inhibitors served both to quench the proteolysis reaction and to inhibit proteases present in the STE14 membranes. Stoichiometric methylation was achieved within 20 min at 37 °C. Reactions were terminated by adding 0.5 ml 4% SDS along with 50 μg of bovine brain cytosol as carrier protein (27Thissen J.A. Casey P.J. Anal. Biochem. 1996; 243: 80-85Crossref PubMed Scopus (16) Google Scholar), and 3H-methylated protein was quantitated using a filter assay (23Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (703) Google Scholar, 25Casey P.J. Thissen J.A. Moomaw J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8631-8635Crossref PubMed Scopus (156) Google Scholar, 27Thissen J.A. Casey P.J. Anal. Biochem. 1996; 243: 80-85Crossref PubMed Scopus (16) Google Scholar). Protease assays were also performed directly on prenylated proteins following their prenylation. In these assays, a 45-μl prenylation reaction was performed as described above, and the concentration of prenylated protein generated in the reaction was determined. The prenylated protein was diluted to the desired concentration in buffer A, and a source of membranes was added to the mixture. The assay then proceeded as described above. The deduced amino acid sequence of yeast RCE1 was used to search a human EST data base, and the entry W96411 was identified as a potential homologue. Five additional overlapping ESTs were identified giving a composite sequence of 725 amino acids. A full-length hRCE1 clone was assembled from a clone isolated from a human fetal brain cDNA library, which contained the 3′ end of the cDNA, and a clone isolated from a HUVEC cDNA library, which contained the 5′ end of the cDNA. The transcript was 1500 bp, not including polyadenylation, and had an open reading frame that encoded a 329-amino acid protein (Fig. 1). The strongest homology between the yeast and human proteins was found in a region from Arg166 to Pro270 of hRCE1 (42% identity and 68% similarity), suggesting that the active site of the enzyme could reside in this region. This region was also highly conserved in a potential homologue of RCE1 found in the Caenorhabditis elegans genome data base (CEF48F5). As reported for yeast RCE1 (10Boyartchuk V.L. Ashby M.N. Rine J. Science. 1997; 275: 1796-1800Crossref PubMed Scopus (307) Google Scholar), the deduced amino acid sequence of hRCE1 contained multiple predicted transmembrane domains (not shown). Examination of hRCE1 expression by Northern blotting revealed a primary band of 1.8 kilobases, which was expressed in all tissues tested (Fig.2 A). A faint 2.4-kilobase band was also evident in some tissues. Examination of RCE1 expression in mouse tissues, using a probe corresponding to a sequence found in the mouse data base, gave stronger evidence of the presence of larger transcripts, which may represent alternatively spliced variants of RCE1 (Fig. 2 B). High level expression of the yeast carboxymethyltransferase STE14 inSf9 cells has allowed development of a coupled assay for prenyl protein proteolysis that utilizes proteins prenylatedin vitro as substrates and quantitates C-terminal proteolysis by stoichiometric methylation. In preliminary experiments, we found that expression of the cDNA encoding yeast RCE1 inSf9 cells by infection with a recombinant baculovirus resulted in a dramatic increase (>1000-fold) in prenyl protein protease activity in the cells (Fig. 3); subfractionation of cell extracts revealed that all of the activity was found in the membrane fraction (data not shown). It was this demonstration that the yeast RCE1 gene encoded an authentic prenyl protein protease that compelled us to search for and ultimately clone hRCE1. Although attempts to express full-length cDNA encoding hRCE1 in insect cells were unsuccessful, when a construct in which the first 22 codons of the hRCE1 were deleted (designated ΔhRCE1) was expressed in Sf9 cells, a dramatic (>1000-fold) increase in prenyl protein protease activity was observed in the membrane fraction (Fig. 3). Recombinant hRCE1 produced by expression of the ΔhRCE1 construct utilized both farnesylated and geranylgeranylated Ki-Ras as substrates, with both substrates exhibiting apparent K m values of approximately 0.5 μm (Fig.4 A) and similark cat values, indicating that the enzyme has similar catalytic efficiencies for farnesylated and geranylgeranylated substrates. Farnesyl-Ha-Ras, farnesyl-N-Ras, farnesyl-Gγ1, and geranylgeranyl-Rap1b were also found to be substrates for hRCE1 (Fig. 4 B), demonstrating that the enzyme can utilize a broad range of CAAX-type prenyl protein substrates. Competition studies were performed with peptides corresponding to the C termini of Ki-Ras (CVIM) and of mouse N-Ras (GSPCVLM) to determine whether the enzyme could recognize unprenylated as well as prenylated substrates. GSPCVLM and CVIM that contained farnesylated cysteine residues were able to compete for the processing of prenylated Ki-Ras, whereas the corresponding unprenylated peptides could not, demonstrating both that hRCE1 is specific for prenylated proteins and that it recognizes short prenylated peptides (Fig.4 C). Additionally, the activity of a previously identified inhibitor of prenyl protease activity, a reduced farnesyl-peptide analogue termed RPI (19Ma Y.T. Gilbert B.A. Rando R.R. Biochemistry. 1993; 32: 2386-2393Crossref PubMed Scopus (58) Google Scholar, 20Chen Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar), was examined. The RPI compound was indeed an extremely effective inhibitor of hRCE1, exhibiting an IC50 of approximately 5 nm (Fig.4 D). The first report of prenyl protein protease activity indicated that proteolysis (and methylation) occurs in a membrane compartment in cells (8Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar). Subsequent in vitro studies have focused on membrane fractions in analysis of this processing step. Two distinct activities have been characterized. The first activity was tightly bound by membranes (28Hrycyna C.A. Clarke S. J. Biol. Chem. 1992; 267: 10457-10464Abstract Full Text PDF PubMed Google Scholar, 29Ma Y.T. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6275-6279Crossref PubMed Scopus (78) Google Scholar, 30Ashby M.N. King D.S. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4613-4617Crossref PubMed Scopus (98) Google Scholar, 31Jang G.F. Yokoyama K. Gelb M.H. Biochemistry. 1993; 32: 9500-9507Crossref PubMed Scopus (37) Google Scholar) and could only be released by detergents (20Chen Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar, 32Nishii W. Muramatsu T. Kuchino Y. Yokoyama S. Takahashi K. J. Biochem. (Tokyo). 1997; 122: 402-408Crossref PubMed Scopus (11) Google Scholar, 33Jang G.F. Gelb M.H. Biochemistry. 1998; 37: 4473-4481Crossref PubMed Scopus (22) Google Scholar). The second activity was loosely associated with membranes and could be solubilized by a freeze-thaw process (34Akopyan T.N. Couedel Y. Orlowski M. Fournie-Zaluski M.C. Roques B.P. Biochem. Biophys. Res. Commun. 1994; 198: 787-794Crossref PubMed Scopus (15) Google Scholar). For the purpose of comparison, these activities have been described as microsomal and soluble (35Hitz A.M. Georgopapadakou N.H. FEBS Lett. 1996; 391: 310-312Crossref PubMed Scopus (2) Google Scholar). The microsomal enzyme exhibits an apparently higher affinity for the prenyl peptide CVIM (K m of 0.65 μm) (20Chen Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar) than the soluble enzyme (K mof 32 μm) (34Akopyan T.N. Couedel Y. Orlowski M. Fournie-Zaluski M.C. Roques B.P. Biochem. Biophys. Res. Commun. 1994; 198: 787-794Crossref PubMed Scopus (15) Google Scholar). Based on competition-type assays, the microsomal enzyme exhibited a 250-fold specificity for prenylated peptides over unprenylated peptides, whereas the soluble enzyme reportedly had only a 5-fold specificity (35Hitz A.M. Georgopapadakou N.H. FEBS Lett. 1996; 391: 310-312Crossref PubMed Scopus (2) Google Scholar). Additionally, only the microsomal enzyme was inhibited by the RPI compound (19Ma Y.T. Gilbert B.A. Rando R.R. Biochemistry. 1993; 32: 2386-2393Crossref PubMed Scopus (58) Google Scholar, 20Chen Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar, 35Hitz A.M. Georgopapadakou N.H. FEBS Lett. 1996; 391: 310-312Crossref PubMed Scopus (2) Google Scholar). The activity described herein for hRCE1 is most similar to the above-mentioned microsomal activity, based on its apparentK m for farnesyl-Ki-Ras (∼0.5 μm), its specificity for prenylated proteins, and the potent inhibition observed with the RPI compound. The variety of substrates that hRCE1 processed in vitrosuggests that it plays a major role in the processing of prenylated proteins in cells. Indeed, in an independent study, fibroblasts prepared from mouse embryos in which the RCE1 gene was disrupted failed to process the Ras proteins, as well as other prenylated proteins (36Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Identification of the hRCE1 cDNA and the ability to produce substantial quantities of enzyme in Sf9 cells will provide several major advantages for its study. First, the high level expression of hRCE1 will allow detailed examination of its activity, including screening for specific inhibitors, with minimal concerns of background. Additionally, the high level production system will provide a platform for the purification of the enzyme and will allow initiation of structural approaches to its study. Finally, in combination with the overexpression of yeast carboxymethyltransferase, the tools are now in place to generate large amounts of processing intermediates of prenyl proteins, which should be quite useful in examining the properties that each of the processing steps import to the functions of prenylated proteins. We thank John York and Michael Howell for discussions on cloning strategies. We are indebted to Robert Rando for providing the RPI compound, to Jasper Rine for providing the yeastRCE1 cDNA, and to Susan Michaelis for providing the yeast STE14 cDNA. We thank John Moomaw, Carolyn Weinbaum, and Ying Chen for assistance with protein purification and Ted Meigs for assistance with Northern blotting and comments on the manuscript.