Proc. Natl. Acad. Sci. USA Vol. 88, pp. 1716-1720, March 1991 Biochemistry

Effects of site-directed mutagenesis at residues cysteine-31 and cysteine-184 on lecithin-cholesterol acyltransferase activity (lipid metabolism/cholesterol/acyltransferase)

OMAR L. FRANCONE* AND CHRISTOPHER J. FIELDING Cardiovascular Research Institute and Department of Physiology, University of California Medical Center, San Francisco, CA 94143

Communicated by Richard J. Havel, November 28, 1990 (received for review September 7, 1990)

ABSTRACT Native lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein, and LCAT in which either or both of the enzyme free cysteines had been replaced with glycine residues by site-directed mutagenesis, has been expressed in cultured Chinese hamster ovary cells stably transfected with the human LCAT gene. The mass of LCAT secreted, determined by immunoassay, did not differ in the native and mutant species. LCAT specific activity was also unchanged in the mutant species. In particular, the cysteine-free double mutant, in which Cys-31 and Cys-184 had both been replaced, was fully active in the synthesis of cholesteryl esters. This result is not consistent with a catalytic role for LCAT free cysteine residues. The classical inhibitor of LCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which strongly (89%) inhibited the native enzyme, had partial (45%) inhibitory activity with mutant enzyme species containing a single -SH residue, while the double mutant was not significantly inhibited by DTNB. These data are interpreted to suggest that Cys-31 and Cys-184 are vicinal both to each other and to the "interfacial binding site" at residues 177-182, and that DTNB exerts its effect by steric inhibition.

mutagenesis have been synthesized, and their biochemical properties have been determined.

The majority of cholesteryl esters in normal blood plasma are formed by lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43). The enzyme is present as a complex with lipids and apoproteins in the high density lipoprotein fraction (1). In vivo LCAT catalyzes the transacylation of the sn-2 position fatty acid of lecithin to the 3-hydroxyl group of cholesterol, but in the absence of cholesterol LCAT effectively acts in the hydrolysis of lecithin and the transacylation of lecithin and lysolecithin (2, 3). It has usually been considered that LCAT, in a reaction analogous to that of classical serine-dependent esterases, acts via an initial deacylation of lecithin, with formation of an acylLCAT intermediate, followed by transfer of this acyl group to acceptors with a free hydroxyl function, including cholesterol and other sterols, water, or lysolecithin, with regeneration of the free LCAT protein (4). LCAT contains two free cysteine residues at positions 31 and 184 of the mature protein (5, 6). Unlike most esterases, LCAT is inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (7) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (8). Recently, a more complex mechanism has been proposed, in which transacylation to cholesterol involved an obligatory LCAT-S-acyl intermediate formed in reaction with one or both of the free cysteine residues (9, 10). To test this hypothesis, LCAT molecules modified by the removal of one or both cysteine residues by site-directed

EXPERIMENTAL PROCEDURES Isolation of LCAT cDNA. LCAT-specific cDNA was synthesized via the polymerase chain reaction from the mRNA of human hepatoblastoma (HepG2) cells. The mRNA of cells grown in 175-cm2 flasks was prepared by oligo(dT)-cellulose chromatography (K-1593-02; In Vitrogen, San Diego). The poly(A)+ mRNA was precipitated at -70'C with 0.1 vol of 2 M sodium acetate and 2 vol of 100% ethanol. A cDNA reaction mixture was prepared to contain 10 pmol of 23-mer primer with an internal Bgl II restriction site, antisense to the 3' end of LCAT mRNA (5'-A GCT AGA TCT TTA TTC AGG AGG-3') (Operon Technologies, Alameda, CA). mRNA from one flask of cells, 200 units of Moloney murine leukemia virus reverse transcriptase (BRL), 10 units of RNase inhibitor (Promega Biotec), and 0.5 mM each deoxynucleotide in 50 mM Tris HCI, pH 8.3/75 mM KCI/10 mM dithiothreitol/3 mM MgCl2. After incubation for 1 hr at 37°C, 3 ,ul of 1Ox buffer was added (100 mM Tris HCI, pH 8.3/500 mM KCI/15 mM MgCl2/0.1% gelatin) together with a further 40 pmol of the 3'-end primer and 50 pmol of a 5'-sense primer (5'-CC AAG CTT GGA ATG GGG CCG CCC-3') containing a HindlIl restriction site corresponding to the beginning ofthe coding region of LCAT cDNA and 1.25 units of Thermus aquaticus DNA polymerase (PerkinElmer/Cetus) in a final aqueous vol of 50 ,u. The polymerase chain reaction was carried out in a Perkin-Elmer/Cetus thermocycler for 40 cycles (94°C for 1 min; 55°C for 2 min; 72°C for 3 min). Synthesis of the expected full-length (1.4 kilobases) cDNA was confirmed by electrophoresis in 1.5% agarose/ethidium bromide gel. cDNA Cloning and Sequencing. The cDNA was inserted into the Sma I site of a pUC18 vector (Pharmacia LKB). The ligation mixture was transformed into DH5-a Escherichia coli and clones containing the insert subcloned into the EcoRI/BamHI sites of M13mpl8 and M13mpl9 vectors. The insert was sequenced via the dideoxynucleotide chaintermination reaction (11) using adenosine 5'-[-[35S]thio]triphosphate. The entire cDNA was then sequenced, but no differences were found from that sequence of the LCAT gene previously reported (5). Site-Directed Mutagenesis. The full-length human LCAT cDNA sequence was ligated into the EcoRP/BamHI site of pTZ18 phagemid vector (Bio-Rad). Synthetic oligonucleotides 24-30 bases long carrying one mismatched base were used to mutagenize Cys-31 to Gly (5'-CTGATTCCCCAGGCCGCCGGGCACGAG, complementary to GGC instead of TGC in the LCAT cDNA sequence) or Cys-184 to Gly

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.

Abbreviations: LCAT, lecithin-cholesterol acyltransferase; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); apoA-I, apolipoprotein A-I. *To whom reprint requests should be addressed.

1716

Biochemistry: Francone and Fielding (5 '-GAGCAAGTGTAGACCGCCGAGGCTGTGGCC, complementary to GGT in place of TGT). The double mutant LCAT-Gly-31, Gly-184 was prepared in the same way by mutagenizing LCAT-Gly-31 with the second oligonucleotide. In this protocol, first described by Kunkel (12), colonies transformed with the mutated DNA were grown to produce single-stranded pTZ18 human LCAT DNA. In each case, the predicted base sequence of the mutated LCAT cDNAs was confirmed by dideoxynucleotide sequencing as described above. In Vitro Transcription and Translation. pTZ18 plasmids containing full-length human LCAT cDNA were purified by alkaline hydrolysis (13) and via a cesium chloride gradient containing ethidium bromide; then they were linearized with BamHI and transcribed with T7 RNA polymerase (Pharmacia LKB). The transcripts were capped by addition of m7G(5')ppp(5')G in the transcription reaction. Template DNA was removed with RQ1 DNase and portions of the transcription reaction were run in 1% agarose/formaldehyde gels to verify the size and integrity of the transcripts. mRNA synthesized by T7 RNA polymerase was translated in a cell-free system containing nuclease-treated rabbit reticulocyte lysate, [35S]methionine, and RNasin in the presence or absence of canine pancreatic microsomal membranes under conditions described by the manufacturer (Promega Biotec). Primary translation products were further analyzed on a SDS/8% polyacrylamide gel. The gels were fixed, dried, and exposed to Kodak XAR-5 film at -70'C with an intensifying screen. Construction of an Expression Plasmid for Human LCAT. An expression plasma pSV2dhfr (American Type Culture Collection ATCC37146) containing the cDNA sequence of a mouse dihydrofolate reductase (Dhfr) was digested with HindIII and Bgl II to remove the Dhfr sequence. The wild-type and mutant full-length LCAT cDNA cloned in pTZ18 plasmids were digested with Bgl II and HindIII and a full-length LCAT cDNA clone extending from 8 bases before the ATG start codon to 5 bases after the TAA stop codon was then ligated to the unique HindIII/Bgl II sites of the pSV2 plasmid. Culture and Transfection of CHO Cells. CHO (Dhfr-) cells (DXB 11 line), deficient in the Dhfr gene, were grown in F-12 medium supplemented with 10% fetal calf serum and gentamycin. The expression vector pSV2hLCAT was cotransfected with pSV2dhfr in a 20:1 ratio into CHO cells by calcium phosphate-mediated transfection (14). The calcium phosphate-DNA coprecipitate was allowed to form in the tissue culture medium during prolonged incubation (15-24 hr) under controlled conditions of pH (6.96) and CO2 tension (3%) (15). Transfected cells were selected by their ability to grow in modified Eagle's (MEM) alpha medium without nucleosides, containing 10% dialyzed fetal calf serum and individual colonies were propagated for assay. Clones expressing LCAT were identified by solid-phase immunoassay and LCAT functional activity. Immunoassay of Expressed LCAT. The mass of secreted LCAT was determined by solid-phase immunoassay. Medium from CHO untransfected (control) and transfected cells was concentrated 20- to 40-fold in Millipore filters. Protein in the concentrated samples was bound to nitrocellulose screens (Sartorius, West Coast Scientific, Hayward, CA) in a Bio-Rad dot-blot apparatus. Bound medium protein, and pure antigen standard, underwent reaction as described first with site-directed antibody to the mature human LCAT (16) and then with 125I-labeled goat antibody to rabbit IgG. Screens were then assayed in a Searle 1185 y spectrometer. Assay of LCAT Activity. Wild-type and mutant clones expressing both Dhfr and human LCAT genes were grown in T25 flasks in MEM alpha medium without nucleosides supplemented with 10% dialyzed fetal calf serum and gentamycin

Proc. Natl. Acad. Sci. USA 88 (1991)

1717

to =90% confluency. The cells were then extensively washed with MEM alpha medium containing gentamycin and were incubated 24-48 hr with MEM alpha medium without nucleosides supplemented with 5% Ultroser G (GIBCO). LCAT activity was determined as the rate of synthesis of 3H-labeled cholesteryl esters from [1,2-3H]cholesterol (New England Nuclear) and unlabeled egg lecithin and apolipoprotein A-I (apoA-I) (Sigma). Single walled vesicles containing cholesterol and lecithin (1:8, wt/wt; cholesterol specific activity, 1.2 x 105 dpm/,ug) were prepared with a French press (17), activated with apoA-I (18), and assayed in the presence of recrystallized human serum albumin (2.5%, wt/vol), 10 mM phosphate buffer (pH 7.5) in 0.15 M NaCI, together with protease inhibitors (19). The labeled cholesteryl ester was separated from free cholesterol by thin-layer chromatography on silica gel layers on plastic sheets (Merck) developed in hexane/diethyl ether/acetic acid (83:16:1) (vol/vol). Some incubations were done in the presence of 1.5 mM DTNB (8).

RESULTS Translation of Human LCAT mRNA. LCAT cDNA transcribed in vitro with T7 RNA polymerase generated a single 1.4-kilobase RNA with a complete absence of detectable shorter transcripts, consistent with previous findings (5, 20). The capped LCAT mRNA was then translated in vitro with a rabbit reticulocyte lysate, which (in the absence of microsomes) generates only nonglycosylated translated proteins (21). Following incorporation of [35S]methionine into the translated protein, LCAT with an apparent molecular mass of -45 kDa was obtained (Fig. 1), similar to the expected molecular mass of the protein moiety of the fulllength 416-amino acid enzyme (5, 6). When the same mRNA was translated in the presence of microsomal vesicles obtained from canine pancreas, the 45-kDa LCAT band was almost completely replaced with a band of 67 kDa apparent molecular mass, consistent with the size of mature plasma LCAT (2, 3). These data indicate the addition of substantial N-linked carbohydrate to the LCAT polypeptide catalyzed by enzymes of the endoplasmic reticulum in the early secretory pathway, to the extent of -25% of total molecular mass, consistent with direct analysis of the plasma LCAT protein

(1). Construction and Expression of Glycine Mutant LCAT Genes. Three mutant genes were constructed in which either or both cysteine codons were replaced with glycine codons. As shown in Fig. 2, LCAT-Gly-31 and LCAT-Gly-184 contained a single base mismatch at positions 183 and 622, respectively, while the double-mutant LCAT-Gly-31, Gly184 contained both modifications. MW(KDa) A 92.5 69.0_ 46.0

B

30.0_ FIG. 1. In vitro translation of wild-type LCAT mRNA. Fulllength human LCAT cDNA was cloned in a pTZ18 phagemid vector. The recombinant plasmid was digested with BamHI, and an in vitro transcription was performed. The mRNA was then translated in a reticulocyte lysate system in the absence (A) or presence (B) of canine pancreatic microsomes as a source of glycosylating enzymes.

Proc. Natl. Acad. Sci. USA 88 (1991)

Biochemistry: Francone and Fielding

1718

9@(c\ G

A T C G A T

XC*

c

A

c~Gy3

\c

uan

z"

.:I. .

tCcy G

ATG

Cys-31

Cys-1 84

163

622

idtp

A leu

wild type

C/g

/c

MYG/l Gly 31 mutant

AATAAA 1328

Ax C )cys G/ Gx

-M.

XG sG leu gty

Bglll

Hind III

A

\1

fn an

LCAr

B A leu

leu A

\G \G A T C G A T /C*

gly( _

Gx C )gly

-w

gly c

G/ -

_

A

_:

C )CyS _a" A-

S§

G\ A )leu T/

leu gA \T his

/G / Ply 184 mutant,

(T

wild type

I

\)

XG

FIG. 2. Comparison of the wild-type and mutant LCAT cDNA species. The sequences of wild-type and either Gly-31 (A) or Gly-184 (B) mutant LCAT cDNAs were determined by the dideoxynucleotide chain-termination method. The double-mutant Gly-31, Gly-184 was obtained by mutagenizing the Gly-31 single mutant as described for the preparation of Gly-184. The gel shows the complementary base sequence of the codons indicated.

The structure of the expression vector used in this study is shown in Fig. 3. As described in Experimental Procedures, cells were cotransfected with this vector together with the vector pSV2dhfr, which includes the Dhfr gene, permitting transfected colonies to grow in nucleoside-free medium. Of the colonies surviving in nucleoside-free medium, 30-40%o expressed both LCAT and Dhfr genes as judged by the ability of these colonies both to grow in the absence of nucleotides and to secrete LCAT protein into the culture medium. Of these colonies, three of the wild type and three of each mutant were selected for enzymatic analysis. Enzymatic Properties of Wild-Type and Mutant LCAT Species. LCAT activity in the medium of cells transfected with wild-type LCAT was 9.3 2.1 ng of cholesterol esterified per hr in the complete assay medium described in Experimental Procedures. Nontransfected cells secreted no detectable LCAT activity under the same conditions. Very similar rates of secretion were obtained for each single ±

AmpR

FIG. 3. Human LCAT expression vector. The pSV2dhfr plasmid HindlII/Bgl II to remove the Dhfr sequence and of cloned human LCAT cDNA was inserted at the unique HindIII/Bgl lI site in the vector. pSV2hLCAT was cotransfected with pSV2dhfr as described. The expression of human LCAT and mouse Dhfr genes was driven by the simian virus 40 (SV40) early promoter. Cysteine residues selected for site-specific mutagenesis are shown beneath the LCAT cDNA. AmpR, ampicillin resistance.

was digested with a 1336-base-pair fragment

mutant and the double-mutant species (Table 1). In the case of both wild-type and mutant enzymes, the production of cholesteryl esters was linear over at least 6 hr at 37°C. The secretion of LCAT protein by cells transfected with wild-type and mutant LCAT DNA is shown in Fig. 4. Rates of secretion of LCAT protein by cells transfected with either wild-type or mutant genes were similar (Table 1). This finding indicates that the secretion of LCAT was also unaffected by the Cys-to-Gly substitutions in the mutant enzyme species. As a result, the specific activity of LCAT was similar in each case, whether or not the protein contained free cysteine residues. The activity of LCAT is characteristically stimulated in the presence of apoA-I. Wild-type and mutant enzymes were assayed in the presence or absence of apoA-I in the assay medium. There was no significant difference in the -fold activation obtained with the two LCAT species. The wild-type enzyme was strongly inhibited in the presence of 1.5 mM DTNB (Table 1). This inhibition is similar to

Table 1. Catalytic rate of wild-type and mutant LCAT species ApoA-I Activity, ng of Inhibition by activation, Specific cholesteryl -fold DTNB, % activity Mass, ng ester per hr ± 3.2 ± ± ± ± 7.3 2 89 0.1 11.2 0.3 34.5 9.3 2.1 Wild type 46 ± 4 0.2 + 0.1 39.1 ± 3.1 7.5 ± 0.7 Cys-31 to Gly 45 ± 14 0.3 ± 0.1 31.6 + 14.1 10.1 ± 1.9 Cys-184 to Gly 5.8 ± 2.0 8± 3 0.3 ± 0.1 30.7 ± 7.0 8.9 ± 2.0 Cys-31, Cys-184 to Gly (5 with [3H]cholesterol hr, 370C) incubation (6 by was determined Enzyme activity Aug/ml; 1.2 x 105 (5 ,tg/ml) as described. Specific activity cpm/gg) and egg lecithin (40 ,jg/ml) in the presence of apoA-IInhibition was determined from the ratio is expressed as ng of cholesterol esterified per ng of protein. of LCAT activities obtained from assays carried out in the presence or absence of 1.5 mM DTNB, under the same assay conditions. In studies carried out at the same time with LCAT protein isolated from human plasma, cholesteryl ester synthesis was inhibited 92% ± 1% by 1.5 mM DTNB. Values shown are means ± 1 SEM for five to seven different experiments. Activity and mass measurements are expressed per T25 flask. The -fold activation by apoA-I is expressed as the ratio between LCAT rates with lecithin-cholesterol vesicles in the presence and absence of apoA-I (1.0 tkg/8 ug of lecithin).

Biochemistry: Francone and Fielding

Proc. Natl. Acad. Sci. USA 88 (1991)

1719

FIG. 4. Immunoblots of cell culture medium from untransfected (control) and pSV2hLCAT wild-type and mutant transfected cells. Medium from CHO Dhfr- cells (untransfected) and transfected cells was concentrated and the samples were applied in duplicate to nitrocellulose membranes as described. The LCAT protein was detected by reaction with a rabbit site-directed antibody to the mature LCAT protein and then with 1251-labeled goat antibody to rabbit IgG.

that obtained with the human plasma protein (8). The mutant LCAT species carrying a single -SH residue were only partially (45-46%) inhibited by DTNB under the same conditions. DTNB did not significantly inhibit the double-mutant LCAT, which contained no free sulfhydryl residues. These results indicate that substitution of glycine for cysteine residues in LCAT had little or no effect on secretion or catalytic rates, or activation by apoA-I, but reduced the dependence of activity on free -SH residues.

DISCUSSION The data in this study provide strong evidence that the free sulfhydryl residues of LCAT are not directly involved in the catalytic mechanism leading to the synthesis of cholesteryl esters. In particular, the double mutant containing no free cysteine residues has full catalytic activity in the esterification of cholesterol. For this reason, the earlier proposal (9, 10) that an S-acyl covalent intermediate was part of the catalytic mechanism of LCAT appears to be incorrect. Recent studies of several other enzyme reactions, such as those catalyzed by RNase and hydroxymethylglutaryl CoA reductase (21, 22) (in which an essential thiol function had been previously proposed) have also shown, by mutagenesis, that the cysteine-free enzyme retained its activity. It will be important to modify the free cysteines in other lipases related to LCAT that have reported functional sulfbydryl residues (e.g., gastric lipase) (23) to determine whether in these, as in LCAT, the free cysteine residue has only a steric effect. As it is clear that sulfhydryl reagents such as DTNB strongly inhibit native LCAT activity, some other explanation must be sought for its effect, and for other evidence interpreted to support the S-acyl intermediate hypothesis. DTNB may act to sterically hinder one or more of the triad of active site residues implicated in the activity of many serine esterases (24). It is of interest that one of the two cysteine residues of LCAT (at position 184) is located close to the serine residue at position 181, which has been proposed as part of an interfacial substrate binding site (1). The same residue is also one of two serines (the other at position 216), which in different studies were modified by the antiesterase diisopropyl fluorophosphate (25, 26). The reactivity of LCAT with the bifunctional reagent aminophenylarsene dichloride clearly indicated that Cys-31 and Cys-184 are closely apposed in LCAT (10). For this reason, substitution by DTNB at either Cys-31 or Cys-184 would be likely to inhibit sterically a reaction mechanism involving this region of the protein. This concept is quite consistent with the additive effect of DTNB (Table 1) as it reacts with one or two -SH groups in LCAT.

As part of the S-acyl hypothesis (9), it has also been reported that the phospholipase activity of LCAT was not DTNB dependent; it was proposed that the S-acyl intermediate was required for cholesteryl ester synthesis but not the release of unesterified fatty acid from lecithin. Several other observations in the literature are not consistent with this, however. In comparative studies with lecithin and lecithincholesterol vesicles, Aron et al. (2) found that both phospholipase and transacylase activities of LCAT were similarly inhibited by DTNB. Swaney et al. (27) found LCATmediated phospholipase and transacylase activities with rat high density lipoprotein to be similarly inhibited by DTNB. Subbhaiah et al. (3) found the lysolecithin-lecithin exchange reaction of LCAT to be as much inhibited by sulfhydryl reagents as was the generation of cholesteryl esters. These data, like those obtained in the present research, argue against a unique catalytic role for LCAT -SH residues in cholesterol esterification. Finally, Jauhiainen and Dolphin (9) described the interaction of LCAT sulfhydryl groups with long-chain acyl CoA. However, the extent of reaction (1 mol of acyl CoA per reactive residue) provides no evidence for catalytic turnover of acyl CoA at these sites. It seems more likely that the reaction of acyl CoA is simply a derivatization of LCAT protein driven by the high-energy acyl-S-CoA bond. In summary, the present study indicates that the free cysteine residues of LCAT are not required for cholesteryl ester synthesis. The inhibition of LCAT by sulfhydryl inhibitors may best be explained by the proximity of both residues to a functionally important region centered around Ser-181. The general mechanism of LCAT would then be similar to that of other lipases (including pancreatic, lipoprotein, and hepatic triglyceride lipases) with comparable structure in this region (1). The expert technical assistance of Lolita Evangelista is acknowledged. O.L.F. is an American Heart Association, California Affiliate, Research Fellow. This work was done during the tenure of a research fellowship from the American Heart Association, California Affiliate, and with funds contributed by the Alameda County chapter. It was also supported by the National Institutes of Health through Arteriosclerosis Grant SCOR HL 14237.

1. Fielding, C. J. (1990) in Advances in Cholesterol Research, eds. Esfahani, M. & Swaney, J. B. (Telford, Telford, NJ), pp. 271-314. 2. Aron, L., Jones, S. & Fielding, C. J. (1978) J. Biol. Chem. 253, 7220-7226. 3. Subbaiah, P. V., Albers, J. J., Chen, C. H. & Bagdade, J. D. (1980) J. Biol. Chem. 255, 9275-9280. 4. Piran, U. & Nishida, T. (1979) Lipids 14, 478-482. 5. McLean, J., Fielding, C. J., Drayna, D., Dieplinger, H., Baer,

1720

6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16.

Biochemistry: Francone and Fielding B., Kohr, W., Henzel, W. & Lawn, R. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339. Yang, C. Y., Manoogian, D., Pao, Q., Lee, F. S., Knapp, R. D., Gotto, A. M. & Pownall, H. J. (1987) J. Biol. Chem. 262, 3086-3091. Glomset, J. A. (1968) J. Lipid Res. 9, 155-168. Stokke, K. T. & Norum, K. R. (1971) Scand. J. Clin. Lab. Invest. 27, 21-27. Jauhiainen, M. & Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7043. Jauhiainen, M., Stevenson, K. J. & Dolphin, P. J. (1988) J. Biol. Chem. 263, 6525-6533. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. Bimboim, H. C. (1983) Methods Enzymol. 100, 243-255. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456-467. Chen, C. & Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752. Francone, 0. L., Gurakar, A. & Fielding, C. J. (1989) J. Biol. Chem. 264, 7066-7072.

Proc. Nati. Acad. Sci. USA 88 (1991) 17. Hamilton, R. L., Goerke, J., Guo, L. S. S., Williams, M. C. & Havel, R. J. (1980) J. Lipid Res. 21, 981-992. 18. Fielding, C. J., Shore, V. G. & Fielding, P. E. (1972) Biochem. Biophys. Res. Commun. 46, 1493-1498. 19. Castro, G. R. & Fielding, C. J. (1988) Biochemistry 27, 25-29. 20. McLean, J., Wion, K., Drayna, D., Fielding, C. & Lawn, R. (1986) Nucleic Acids Res. 14, 9397-9406. 21. Kanaya, S., Kimura, S., Katsuda, C. & Ikehara, M. (1990) Biochem. J. 271, 59-66. 22. Jordan-Starck, T. C. & Rodwell, V. W. (1989) J. Biol. Chem. 264, 17919-17923. 23. Gargouri, Y., Moreau, H., Pieroni, G. & Verger, R. (1988) J. Biol. Chem. 263, 2159-2162. 24. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358. 25. Farooqui, J. Z., Wohl, R. C., Kezdy, F. J. & Scanu, A. M. (1988) Arch. Biochem. Biophys. 261, 330-335. 26. Park, Y. B., Yuksel, K. U., Gracy, R. W. & Lacko, A. G. (1987) Biochem. Biophys. Res. Commun. 143, 360-363. 27. Swaney, J. B., Orishimo, M. W. & Girard, A. (1987) J. Lipid Res. 28, 982-992.