Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4613-4617, May 1992
Biochemistry
Endoproteolytic processing of a farnesylated peptide in vitro (CAAX motifs/prenylation/pttranslational procsing/carboxyl-terminal protease)
MATTHEW N. ASHBY*, DAVID S. KINGt, AND JASPER RINE*1 *Department of Molecular and Cell Biology, Division of Genetics, and tHoward Hughes Medical Institute, University of California, Berkeley, CA 94720
Communicated by Robert Tjian, January 9, 1992
ABSTRACT Numerous eukaryotic proteins containing a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) require a three-step posttranslational processing for localization and function. The a mating factor of Saccharomyces cerevisiae is one such protein, requiring cysteine farnesylation, proteolysis of the terminal three amino acids, and carboxyl methylation for biological activity. We have used farnesylated a-factor peptides to examine the proteolytic step in the maturation of CAAX-containing proteins. Three distinct carboxyl-terminal protease activities were found in yeast cell extracts that could remove the terminal three residues of a-factor. Two of the proteolytic activities were in cytosolic fractions. One of these activities was a PEP4dependentcarboxypeptidasethatwassensitivetophenylmethylsulfonyl fluoride. The other cytosolic activity was PEP4independent, sensitive to 1,10-phenanthroline, and effectively inhibited by an unfarnesylated a-factor peptide. In contrast, a protease activity in membrane fractions was unaffected by phenylmethylsulfonyl fluoride, 1,10-phenanthroline, or unfarnesylated a-factor peptide. Incubation of membrane preparations from either yeast or rat liver with a radiolabeled farnesylated a-factor peptide released the terminal three amino acids intact as a tripeptide, indicating that this reaction occurred by an endoproteolytic mechanism and that the enzyme most likely possesses a broad substrate specificity. The yeast endoprotease was not significantly affected by a panel of protease inhibitors, suggesting that the enzyme is novel. Zinc ion was shown to inhibit the endoprotease (K; < 100 pM). The specific activities of the a-factor carboxyl-terminal membrane endoprotease and methyltransferase clearly indicated that the proteolytic reaction was not rate-limiting in these processing reactions in vitro.
(21-24). The yeast a mating factor has the carboxyl-terminal sequence CVIA and has been shown to be farnesylated (4). The isolation of yeast a-specific sterile mutants has identified several of the genes required for posttranslational processing of a-factor. DPRI/RAMI (also known as STE16) and RAM2 have been shown to encode distinct subunits of the protein farnesyltransferase (25-27). Substrates of this protein farnesyltransferase include a-factor, RAS2, and the 'y subunit of a trimeric GTP-binding protein (10). STEJ4 encodes a protein methyltransferase that mediates carboxyl methylation of both a-factor and RAS2 in yeast (18, 28). Secretion of mature a-factor requires the STE6 gene product (29). In contrast, no mutants defective in proteolytic maturation of RAS2 or a-factor have been isolated. Studies on the maturation of a-factor and RAS peptides in vitro have shown that cytosolic fractions from yeast and mammalian cells contain protein farnesyltransferase activity (23, 25, 30, 31). Carboxyl-terminal methyltransferase activity is present in plasma membrane preparations (28). Removal of the terminal three amino acids of farnesylated p21Ki-Ras(B) translated in vitro will take place when incubated with membrane fractions from COS cells (15). We have begun to characterize the carboxyl-terminal protease involved in the maturation of a-factor and RAS prenylated precursors. We have identified comparable proteolytic activities in membrane preparations from both yeast and rat liver that will correctly process a farnesylated a-factor peptide. We have learned that this processing activity is an endoprotease that depends upon a prenylated substrate and may define a class of protease.
Fungal mating pheromones were the first proteins shown to undergo carboxyl-terminal posttranslational processing involving modification with an isoprenoid compound (1-3). Since then a large number of eukaryotic proteins have been shown to undergo similar modifications. Such proteins include a-factor (4), the p21 family of Ras proteins (5-7), some subunits of trimeric GTP-binding proteins (8-10), and nuclear lamins (11, 12). A key determinant for proteins destined for this posttranslational processing is the carboxyl-terminal tetrapeptide sequence CAAX (C, cysteine; A, aliphatic; X, any amino acid). Processing includes prenylation of the cysteine residue with either a farnesyl or a geranylgeranyl moiety (1-12), proteolytic removal of the terminal three amino acids (13-15), and, finally, methylesterification of the newly exposed carboxylate group of the prenylated cysteine residue (16-20). The terminal (X) amino acid of the motif may be a determinant for distinguishing among protein prenyltransferases, since CAAX-containing proteins terminating in leucine are prenylated by a geranylgeranyl diterpenoid, whereas proteins terminating in cysteine, serine, alanine, or methionine are prenylated with a farnesyl sesquiterpenoid
MATERIALS AND METHODS Materials. Saccharomyces cerevisiae strain JRY2594 (MATa ade2-101 his3-200 lys2-801 met ura3-52) was the yeast strain used. S-adenosyl-L-[methyl-14C]methionine (47 mCi/ mmol; 1 Ci = 37 GBq) was from ICN. L-[4,5-3H]isoleucine (100 Ci/mmol) was from Amersham. The peptides dansylWDPAC(S-t,t-farnesyl) and dansyl-WDPAC(S-t,t-farnesyl)VIA were a generous gift from S. Rosenberg and C. C. Yang (Protos, Emeryville, CA). Peptide Synthesis. The radiolabeled peptide KWDPAC(St,t-farnesyl)V[4,5-3H]IA was synthesized onp-hydroxymethylphenoxy-derivatized polystyrene/divinylbenzene resin by using 2-(lH-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumactivated 9-fluorenylmethoxycarbonyl (Fmoc) amino acids in the presence of 1-hydroxybenzotriazole and diisopropylethylamine in an Applied Biosystems 431A peptide synthesizer employing user-derived cycles. Protecting groups were tbutoxycarbonyl [K(Boc)], t-butyl [D(t-Bu)], and trityl [C(Trt)]. Fmoc-[3H]Ile was synthesized from L-[4,53H]isoleucine by using Fmoc N-succinimide ester in ethylene glycol dimethyl ether with carbonate as catalyst. Labeled peptide was cleaved and deprotected with reagent K (32) for
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.
Abbreviation: PMSF, phenylmethylsulfonyl fluoride. tTo whom reprint requests should be addressed.
4613
4614
Biochemistry: Ashby et al.
Natl.
Proc.
Acad. Sci. USA 89 (1992)
3 hr (room temperature), precipitated in t-butyl methyl ether, purified by reversed-phase HPLC with an acetonitrile gradient in 0.1% trifluoroacetic acid, andIyophilized. The peptide thiolate anion was generated with carbonate and alkylated with farnesyl bromide (85-90%o yield). The radiolabeled farnesylated peptide was purified by reversed-phase HPLC to yield material of purity >96% (24.8 mCi/mmol). Structures were confirmed by derivative UV spectrometry and by electron spray ionization MS. The peptide was then coupled to N-hydroxysuccinimideactivated CH-Sepharose 4B (Pharmacia) as instructed by the manufacturer. The lysine at the amino terminus of the peptide was utilized in place of the naturally occurring phenylalanine at this position to ameliorate the coupling reaction. Peptide coupling efficiency was =94%. Crude Membrane and Cytosol Preparations. Yeast cultures
Reactions were initiated by addition of 1 Ag of cell protein and incubated at 370C for 15 min. For quantitative determinations the reaction mixtures were centrifuged at 10,000x g for 2 min. Aliquots of the supernatant were taken and the soluble radioactivity was determined by liquid scintillation counting. Under these conditions the release of soluble [3H]isoleucine was linear with respect to time and cell protein concentration. The concentration of the Sepharose-coupled peptide was in excess (data not shown). For qualitative analyses by HPLC, supernatants from five individual reactions were pooled and centrifuged through a Centricon-30 (Amicon) filter. Material passing through the filter (
Biochemistry
Endoproteolytic processing of a farnesylated peptide in vitro (CAAX motifs/prenylation/pttranslational procsing/carboxyl-terminal protease)
MATTHEW N. ASHBY*, DAVID S. KINGt, AND JASPER RINE*1 *Department of Molecular and Cell Biology, Division of Genetics, and tHoward Hughes Medical Institute, University of California, Berkeley, CA 94720
Communicated by Robert Tjian, January 9, 1992
ABSTRACT Numerous eukaryotic proteins containing a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) require a three-step posttranslational processing for localization and function. The a mating factor of Saccharomyces cerevisiae is one such protein, requiring cysteine farnesylation, proteolysis of the terminal three amino acids, and carboxyl methylation for biological activity. We have used farnesylated a-factor peptides to examine the proteolytic step in the maturation of CAAX-containing proteins. Three distinct carboxyl-terminal protease activities were found in yeast cell extracts that could remove the terminal three residues of a-factor. Two of the proteolytic activities were in cytosolic fractions. One of these activities was a PEP4dependentcarboxypeptidasethatwassensitivetophenylmethylsulfonyl fluoride. The other cytosolic activity was PEP4independent, sensitive to 1,10-phenanthroline, and effectively inhibited by an unfarnesylated a-factor peptide. In contrast, a protease activity in membrane fractions was unaffected by phenylmethylsulfonyl fluoride, 1,10-phenanthroline, or unfarnesylated a-factor peptide. Incubation of membrane preparations from either yeast or rat liver with a radiolabeled farnesylated a-factor peptide released the terminal three amino acids intact as a tripeptide, indicating that this reaction occurred by an endoproteolytic mechanism and that the enzyme most likely possesses a broad substrate specificity. The yeast endoprotease was not significantly affected by a panel of protease inhibitors, suggesting that the enzyme is novel. Zinc ion was shown to inhibit the endoprotease (K; < 100 pM). The specific activities of the a-factor carboxyl-terminal membrane endoprotease and methyltransferase clearly indicated that the proteolytic reaction was not rate-limiting in these processing reactions in vitro.
(21-24). The yeast a mating factor has the carboxyl-terminal sequence CVIA and has been shown to be farnesylated (4). The isolation of yeast a-specific sterile mutants has identified several of the genes required for posttranslational processing of a-factor. DPRI/RAMI (also known as STE16) and RAM2 have been shown to encode distinct subunits of the protein farnesyltransferase (25-27). Substrates of this protein farnesyltransferase include a-factor, RAS2, and the 'y subunit of a trimeric GTP-binding protein (10). STEJ4 encodes a protein methyltransferase that mediates carboxyl methylation of both a-factor and RAS2 in yeast (18, 28). Secretion of mature a-factor requires the STE6 gene product (29). In contrast, no mutants defective in proteolytic maturation of RAS2 or a-factor have been isolated. Studies on the maturation of a-factor and RAS peptides in vitro have shown that cytosolic fractions from yeast and mammalian cells contain protein farnesyltransferase activity (23, 25, 30, 31). Carboxyl-terminal methyltransferase activity is present in plasma membrane preparations (28). Removal of the terminal three amino acids of farnesylated p21Ki-Ras(B) translated in vitro will take place when incubated with membrane fractions from COS cells (15). We have begun to characterize the carboxyl-terminal protease involved in the maturation of a-factor and RAS prenylated precursors. We have identified comparable proteolytic activities in membrane preparations from both yeast and rat liver that will correctly process a farnesylated a-factor peptide. We have learned that this processing activity is an endoprotease that depends upon a prenylated substrate and may define a class of protease.
Fungal mating pheromones were the first proteins shown to undergo carboxyl-terminal posttranslational processing involving modification with an isoprenoid compound (1-3). Since then a large number of eukaryotic proteins have been shown to undergo similar modifications. Such proteins include a-factor (4), the p21 family of Ras proteins (5-7), some subunits of trimeric GTP-binding proteins (8-10), and nuclear lamins (11, 12). A key determinant for proteins destined for this posttranslational processing is the carboxyl-terminal tetrapeptide sequence CAAX (C, cysteine; A, aliphatic; X, any amino acid). Processing includes prenylation of the cysteine residue with either a farnesyl or a geranylgeranyl moiety (1-12), proteolytic removal of the terminal three amino acids (13-15), and, finally, methylesterification of the newly exposed carboxylate group of the prenylated cysteine residue (16-20). The terminal (X) amino acid of the motif may be a determinant for distinguishing among protein prenyltransferases, since CAAX-containing proteins terminating in leucine are prenylated by a geranylgeranyl diterpenoid, whereas proteins terminating in cysteine, serine, alanine, or methionine are prenylated with a farnesyl sesquiterpenoid
MATERIALS AND METHODS Materials. Saccharomyces cerevisiae strain JRY2594 (MATa ade2-101 his3-200 lys2-801 met ura3-52) was the yeast strain used. S-adenosyl-L-[methyl-14C]methionine (47 mCi/ mmol; 1 Ci = 37 GBq) was from ICN. L-[4,5-3H]isoleucine (100 Ci/mmol) was from Amersham. The peptides dansylWDPAC(S-t,t-farnesyl) and dansyl-WDPAC(S-t,t-farnesyl)VIA were a generous gift from S. Rosenberg and C. C. Yang (Protos, Emeryville, CA). Peptide Synthesis. The radiolabeled peptide KWDPAC(St,t-farnesyl)V[4,5-3H]IA was synthesized onp-hydroxymethylphenoxy-derivatized polystyrene/divinylbenzene resin by using 2-(lH-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumactivated 9-fluorenylmethoxycarbonyl (Fmoc) amino acids in the presence of 1-hydroxybenzotriazole and diisopropylethylamine in an Applied Biosystems 431A peptide synthesizer employing user-derived cycles. Protecting groups were tbutoxycarbonyl [K(Boc)], t-butyl [D(t-Bu)], and trityl [C(Trt)]. Fmoc-[3H]Ile was synthesized from L-[4,53H]isoleucine by using Fmoc N-succinimide ester in ethylene glycol dimethyl ether with carbonate as catalyst. Labeled peptide was cleaved and deprotected with reagent K (32) for
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.
Abbreviation: PMSF, phenylmethylsulfonyl fluoride. tTo whom reprint requests should be addressed.
4613
4614
Biochemistry: Ashby et al.
Natl.
Proc.
Acad. Sci. USA 89 (1992)
3 hr (room temperature), precipitated in t-butyl methyl ether, purified by reversed-phase HPLC with an acetonitrile gradient in 0.1% trifluoroacetic acid, andIyophilized. The peptide thiolate anion was generated with carbonate and alkylated with farnesyl bromide (85-90%o yield). The radiolabeled farnesylated peptide was purified by reversed-phase HPLC to yield material of purity >96% (24.8 mCi/mmol). Structures were confirmed by derivative UV spectrometry and by electron spray ionization MS. The peptide was then coupled to N-hydroxysuccinimideactivated CH-Sepharose 4B (Pharmacia) as instructed by the manufacturer. The lysine at the amino terminus of the peptide was utilized in place of the naturally occurring phenylalanine at this position to ameliorate the coupling reaction. Peptide coupling efficiency was =94%. Crude Membrane and Cytosol Preparations. Yeast cultures
Reactions were initiated by addition of 1 Ag of cell protein and incubated at 370C for 15 min. For quantitative determinations the reaction mixtures were centrifuged at 10,000x g for 2 min. Aliquots of the supernatant were taken and the soluble radioactivity was determined by liquid scintillation counting. Under these conditions the release of soluble [3H]isoleucine was linear with respect to time and cell protein concentration. The concentration of the Sepharose-coupled peptide was in excess (data not shown). For qualitative analyses by HPLC, supernatants from five individual reactions were pooled and centrifuged through a Centricon-30 (Amicon) filter. Material passing through the filter (
