Cloning and Characterization of a Mammalian Prenyl Protein-specific Protease

Abstract

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 in and at in multiple (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), Y. Goldstein J.L. Casey P.J. Brown M.S. Full Text PDF PubMed Scopus 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 Casey P.J. J. Biol. Chem. 1994; 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 and as described and Rap1b each had an whereas and Gγ1 were and Gγ1 were farnesylated by of 2 protein with farnesyltransferase (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar), farnesyl in A mm 5 2 mm 5 50 mm Tris-HCl, pH for 1 h at and Rap1b were geranylgeranylated by of 2 protein with geranylgeranyltransferase 1 (22Moomaw J.F. Zhang F.L. Casey P.J. Methods Enzymol. 1995; 250: 12-21Crossref PubMed Scopus (25) Google Scholar), geranylgeranyl in A for 1 h at Y. Goldstein J.L. Casey P.J. Brown M.S. Full Text PDF PubMed Scopus Google Scholar, P.J. J.A. J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). and geranylgeranyl-Ki-Ras were from the unprenylated precursor and enzyme by the prenylation to a which was with mm mm 1 mm 5 containing unprenylated and the and then the with containing either or The preparations were diluted in and to an The was with and the was with containing and mm were in and at in multiple The peptides and were prenylated as described previously J.A. J.M. K. Casey P.J. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar) and by high were by the addition of membranes containing the recombinant prenyl protein protease to an mixture containing prenylated proteins in mm pH and 5 in a of 50 were at Following the proteolysis methylation of prenylated protein was by addition of of membranes containing STE14 to the in 5 pH 7.0, mm 1 mm and in a of The addition of EDTA and protease inhibitors both to the proteolysis and to proteases present in the STE14 methylation was at were by with 50 of brain as protein J.A. Casey P.J. Biochem. 1996; PubMed Scopus Google Scholar), and protein was using a Y. Goldstein J.L. Casey P.J. Brown M.S. Full Text PDF PubMed Scopus Google Scholar, P.J. J.A. J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar, J.A. Casey P.J. Biochem. 1996; PubMed Scopus Google Scholar). were also performed on prenylated proteins their In a prenylation was performed as described above, and the of prenylated protein generated in the was The prenylated protein was diluted to the in and a of membranes was to the The then as described The deduced amino acid sequence of yeast RCE1 was used to search a human data base, and the W96411 was identified as a homologue. Five additional were identified a sequence of 725 amino acids. A hRCE1 clone was from a clone from a human fetal brain cDNA which contained the end of the and a clone from a HUVEC cDNA which contained the 5′ end of the The was bp, not including and had an open reading frame that a acid protein 1). The the yeast and human proteins was in a region from to of hRCE1 and that the of the enzyme in this region was also in a homologue of RCE1 in the data base 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 of hRCE1 expression by Northern a of which was expressed in all tissues A was also in tissues. of RCE1 expression in mouse using a probe corresponding to a sequence in the mouse data base, evidence of the of which of RCE1 2 level expression of the yeast carboxymethyltransferase STE14 cells has of a for prenyl protein proteolysis that proteins as substrates and C-terminal proteolysis by In preliminary that expression of the cDNA encoding yeast RCE1 cells by infection with a recombinant baculovirus resulted in a in prenyl protein protease activity in the cells of cell that all of the activity was in the membrane not It was this that the yeast RCE1 gene an prenyl protein protease that to search for and clone hRCE1. to cDNA encoding hRCE1 in insect cells were a in which the first 22 codons of the hRCE1 were was expressed in Sf9 a in prenyl protein protease activity was in the membrane Recombinant hRCE1 produced by expression of the both farnesylated and geranylgeranylated as substrates, with both substrates of and that the enzyme has for farnesylated and geranylgeranylated farnesyl-N-Ras, and were also to be substrates for hRCE1 that the enzyme can a range of CAAX-type prenyl protein studies were performed with peptides corresponding to the C of and of mouse N-Ras to whether the enzyme unprenylated as well as prenylated and that contained farnesylated cysteine residues were to compete for the processing of prenylated whereas the corresponding unprenylated peptides both that hRCE1 is specific for prenylated proteins and that it prenylated peptides Additionally, the activity of a previously identified inhibitor of prenyl protease 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 was an inhibitor of an of 5 The first report of prenyl protein protease activity that proteolysis in a membrane in cells (8Hancock J.F. Cadwallader K. Marshall C.J. EMBO J. 1991; 10: 641-646Crossref PubMed Scopus (249) Google Scholar). in studies have on membrane in of this processing have been The first activity was by membranes C.A. Clarke S. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Y.T. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, M.N. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus (98) Google Scholar, K. Biochemistry. 1993; 32: PubMed Scopus Google Scholar) and only be by Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar, Y. S. K. J. Biochem. 1997; PubMed Scopus Google Scholar, Biochemistry. 1998; PubMed Scopus Google Scholar). The second activity was with membranes and be by a process Y. Biochem. Biophys. 1994; PubMed Scopus Google Scholar). For the of have been described as and 1996; PubMed Scopus Google Scholar). The enzyme an affinity for the prenyl peptide of Y. Ma Y.T. Rando R.R. Biochemistry. 1996; 35: 3227-3237Crossref PubMed Scopus (50) Google Scholar) than the enzyme Y. Biochem. Biophys. 1994; PubMed Scopus Google Scholar). on the enzyme exhibited a for prenylated peptides unprenylated whereas the enzyme had only a 1996; PubMed Scopus Google Scholar). Additionally, only the enzyme was by the 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, 1996; PubMed Scopus Google Scholar). The activity described for hRCE1 is most to the on for for prenylated proteins, and the potent with the RPI The variety of substrates that hRCE1 processed in that it plays a major role in the processing of prenylated proteins in in an study, prepared from mouse in which the RCE1 gene was disrupted to process the Ras proteins, as well as other prenylated proteins E. P. Ashby K. Casey P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the hRCE1 cDNA and the to produce of enzyme in Sf9 cells will several major for First, the high level expression of hRCE1 will of including for specific with of Additionally, the high level production will a for the of the enzyme and will initiation of to in with the of yeast the are now in to generate of processing of prenyl proteins, which be in the that each of the processing steps to the functions of prenylated proteins. We John York and for on cloning We are to Robert Rando for the RPI to Jasper Rine for the and to Susan Michaelis for the yeast STE14 We John and for with protein and Meigs for with Northern and on the