Purification, Functional Reconstitution, and Characterization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p

Jessica L Anderson,Hilary Frase,Susan Michaelis,Christine A Hrycyna

doi:10.1074/jbc.m410292200

Abstract

Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and alpha-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-beta-d-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure.

Highlights

Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and ␣-carboxyl methylesterification of the isoprenylated cysteine
This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes
His-tagged Ste14p expressed from a strong constitutive promoter is overexpressed to levels 20-fold higher than described previously, suggesting that geranylgeranyl-L-cysteine; DDM, n-dodecyl-␤-D-maltopyranoside; SAM, S-adenosyl-L-methionine; 14C-SAM, S-adenosyl-L-[14C-methyl]methionine; PGK, 3Ј-phosphoglycerate kinase; AEBSF, aminoethylbenzenesulfonyl fluoride hydrochloride; HRP, horseradish peroxidase; MES, 2-morpholinoethanesulfonic acid; OP, o-phenanthroline; Zincon, 2-carboxy-2Ј-hydroxy-5Ј-sulfoformazylbenzene; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; PI, phosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ␣, anti; FTase, farnesyltransferase

Summary

EXPERIMENTAL PROCEDURES

Materials—All oligonucleotides were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). A 734-bp EagI-SacII fragment containing the STE14 gene was amplified from pSM187 and cloned into the EagI and SacII sites of pCHH10m3N and pCHH10m6N to produce pCHH10m3N-STE14 and pCHH10m6NSTE14, respectively These plasmids encode Ste14p with a 10ϫ histidine tag followed by a 3 or 6 myc epitope repeat at the N terminus. A 724-bp XmaI fragment containing STE14 was amplified from pSM187 and cloned into the XmaI site of pCHm3H10C and pCHm6H10C to produce pCHm3H10C-STE14 and pCHm6H10CSTE14, respectively These plasmids encode Ste14p with either a 3 or 6 myc epitope repeat followed by a 10ϫ histidine tag at the C terminus. A 975-bp EagI-SacII fragment containing the RAS2 gene was amplified from pSM1326 and cloned into the EagI and SacII sites of pCHH10m3N to produce pCHH10m3N-RAS2 This plasmid encodes Ras2p with a 10ϫ histidine tag followed by a 3 myc epitope repeat at the N terminus expressed under the control of the PGK promoter. A clear zone, or halo, indicates the secretion of mature a-factor by the MATa cells which in turn inhibits the growth of the lawn, which is supersensitive to mature a-factor

RESULTS

Fold enrichment

DISCUSSION