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Journal of Lipid Research, Vol. 42, 1282-1291, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences

Correspondence to: Stephen L. Sturley, To whom correspondence should be addressed., sls37{at}columbia.edu (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis. Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs. Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity. A serine-to-leucine mutation in the (H/Y)SF motif (residues 338;–340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p. Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523;–529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completely abolished activity. In human ACAT1, mutagenesis of the corresponding motifs (residues 268;–270, and 403;–409, respectively) also nullified enzymatic activity.

On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes. — Guo, Z., D. Cromley, J. T. Billheimer, and S. L. Sturley. Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences. J. Lipid Res. 2001. 42: 1282;–1291.

Supplementary key words: ACAT cholesterol, steryl ester

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The conjugation of sterol with fatty acids via an acyl-CoA intermediate is mediated in mammals by the enzyme ACAT (1). The esterification reaction is critical to sterol homeostasis in all eukaryotic cells, and in mammals may contribute to several disease states including atherosclerosis (2). Human ACAT1 is a 550-amino acid, membrane-associated protein, localized primarily to the endoplasmic reticulum and expressed in a variety of tissues (3) (4) (5) (6). ACAT1 is the founding member of a gene family conserved across multiple genera for the process of neutral lipid biosynthesis (7). Database searches reveal multiple ACAT homologs between and within species as diverse as Caenorhabditis elegans, Arabidopsis thaliana, and Drosophila melanogaster (7). In humans, a second sterol-esterifying enzyme, ACAT2, exhibits 48% sequence identity with ACAT1 and, in contrast to ACAT1, is primarily expressed in hepatocytes and enterocytes (8). Murine ACAT2 likely accounts for sterol esterification in the liver and intestine as demonstrated in ACAT1- and ACAT2-induced mutant mouse models (9) (10) (11). By contrast, in humans, ACAT1 is apparently the major determinant of sterol esterification in all tissues except the intestine, where ACAT2 predominates (12). The third member of the mammalian acyltransferase gene family catalyzes the acylation of diacylglycerol, the terminal step in triglyceride biosynthesis, and has thus been termed acyl-CoA:diacylglycerol O-acyltransferase [DGAT; see refs. (8) and (13)].

In the model eukaryote Saccharomyces cerevisiae, the sterol esterification reaction is mediated by ACAT-related enzymes derived from the ARE1 and ARE2 genes (14). Disruption of both genes produces a viable yeast cell with no detectable steryl ester. The ARE2 gene product produces the majority of steryl ester mass in the cell (14) (15). However, Are1p is clearly capable of performing the esterification reaction and when overexpressed can restore sterol esterification in a double-mutant strain to wild-type levels (14). Moreover, the yeast double mutant can be complemented by heterologous expression of a human ACAT1 or ACAT2 cDNA (8) (15) (16).

In common with the other ACAT family members, yeast Are2p is predicted to have multiple transmembrane domains and phosphorylation and N-linked glycosylation sites. In general, the sequence conservation of this gene family is most pronounced toward the COOH terminus of the molecules. Previous to this report, a description of the relationship between the structure and function of these enzymes has been lacking. This prompted us to define the relevance of conserved sequence domains to the activity of these enzymes, using yeast Are2p as a model. We chose to alter regions of the ARE2-encoded protein (Are2p) and human ACAT1 that are strongly maintained across organisms. We identify two conserved motifs that are required for the enzymatic activity of Are2p and human ACAT1, and propose that they mediate substrate binding by these enzymes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General techniques
Recombinant DNA techniques were performed by conventional protocols (17) (18). DNA-modifying reagents were purchased from New England BioLabs (Beverly, MA) or Promega (Madison, WI) as indicated. DNA sequencing was performed with an Applied Biosciences (Foster City, CA) automated sequencer (Columbia University Cancer Center sequencing facility, New York, NY). Oligonucleotides ( Table 1) were synthesized by Genset (La Jolla, CA). Radioactive reagents ([¹⁴C]oleoyl-CoA, 9,10-³H(N)]oleic acid and [4-¹⁴C]cholesterol) were purchased from DuPont NEN (Boston, MA).

Yeast strains, transformations, and media
Congenic yeast strains SCY059 (MAT ade2-1 met14HpaI-SalI are1::HIS3 are2::LEU2) and SCY061 (wild-type at ARE1 and ARE2) have been described previously (16). Transformation of yeast was performed with lithium acetate and prototrophic selection (19). Yeast extract, yeast nitrogen base, Bacto-peptone, and Bacto-agar were from Difco (Detroit, MO); D-dextrose, D-galactose, and D-raffinose were from Sigma (St. Louis, MO). Complete medium (YEPD), synthetic complete medium (SC), and SC medium lacking uracil (SC-ura) were prepared as described (17).

Isolation and expression of the ARE2 gene
A 4,280-bp BamHI-XbaI fragment encompassing the entire ARE2 gene was isolated from cosmid 14-21 [a gift of T. Pohl (14)] and introduced into the BamHI and XbaI sites of pAlter1 (Promega) to produce pAlter1-ARE2₄₂₈₀. To remove a flanking, extraneous open reading frame (ORF) (YLR020c) at the 3' end of ARE2, an 826-bp XhoI-SalI fragment was deleted by religation of the 2,836-bp SacII-XhoI and 6,276-bp SacII-SalI fragments from pAlter1-ARE2₄₂₈₀ to produce pAlter1-ARE2₃₄₅₀.

To manipulate Are2p levels, the ARE2 gene was subcloned from pAlter1-ARE2₃₄₅₀ into three yeast vectors: YEp352, YCp50, and pS5. The 3,460-bp BamHI-SphI fragment from pAlter1-ARE2₃₄₅₀ was subcloned to the same sites in YEp352 or YCp50 to produce YEpARE2 and YCpARE2, respectively. The ARE2 gene in these two constructs is under the control of its own promoter. To express ARE2 from the ADH1 promoter in pS5ARE2, the 2,642-bp NcoI (filled-in with DNA polymerase I, Klenow fragment)-PstI fragment of pAlter1-ARE2₃₄₅₀ was subcloned into the EcoRI (Klenow treated) and PstI sites of pS5 (20).

Truncation and deletion mutagenesis of ARE2
Three carboxyl-terminal-truncated are2 mutants were generated by insertion of a 12-bp translation-terminating XbaI linker that contains stop codons in all three ORF. YCpARE2 was digested with AflII, filled in with Klenow, and then ligated with the above-mentioned XbaI linker. This are2 mutant was named are2-C68 because it lacks the COOH-terminal 68 amino acids. Similarly, YCpARE2 was digested with SnaBI or Ecl136II and ligated with the XbaI linker to generate the are2-C208 and are2-C253 alleles, respectively. The three mutants were further subcloned into YEp352 (for overexpression) at the BamHI and SphI sites to produce YEpare2-C68 (amino acids 1;–574), YEpare2-C208 (amino acids 1;–434), and YEpare2-C253 (amino acids 1;–389), respectively.

Two ARE2 deletions of 46 and 139 amino acids (are2-46 and are2-139, representing in-frame deletions of residues 389;–434 and 435;–573, respectively) were produced by digestion of YCpARE2 with SacI (blunt ended with T4 DNA polymerase) and SnaBI, or with SnaBI and AflII (filled in with Klenow), respectively. In each case, the large fragment was gel purified and self-ligated. Both mutants were further subcloned into YEp352 at BamHI and SphI sites for overexpression.

ARE2 was also truncated from the NH₂ terminus. A 12-bp NcoI linker was first ligated into YEpARE2 at SalI (Klenow). After NcoI digestion, the 547-bp band was removed, and the remaining two fragments were religated. This mutant, are2-N178, retains the initiator methionine and the 5' untranslated region but lacks amino acids 2;–178. To create a mutant lacking the NH₂-terminal 31 residues (are2-N31), a 583-bp fragment of ARE2 encoding an NH₂-terminal fragment starting at residue 32 was amplified by PCR with oligonucleotides ARE2-SalI (SalI arises due to one mismatched nucleotide) and ZM6. The PCR product was digested with SalI and ligated into YEpare2-N178 at the SalI site. Both mutants were further subcloned into YCp50 at the BamHI and SphI sites.

Missense mutagenesis of ARE2
Tyr₅₂₄ was mutated to alanine to produce are2-Y₅₂₄A in the vector pAlter1 by in vitro site-directed mutagenesis (Promega), using the mutagenic oligonucleotide ARE2-tyr. Similarly, Ser₃₃₉ was mutated to leucine to produce are2-S₃₃₉L with the mutagenic oligonucleotide ARE2-HSF. The mutated sequences were used to replace the wild-type sequences in YCpARE2 and YEpARE2, using BstEII and AflII sites. The paired tryptophan residues at positions 527 and 528 were replaced by alamine-serine in are2-WW_{527, 528}AS, using overlapping PCR. A PCR fragment corresponding to nucleotides 1582;–1961 of ARE2 was amplified with oligonucleotides WW-C (NheI site included) and SS1, and then digested with NheI and AflII to give a 140-bp fragment. Another PCR fragment corresponding to nucleotides 960 to 1581 was amplified with oligonucleotides ZM4 and WW-N (NheI site included), and then digested with NheI and SnaBI to give a 279-bp fragment. The 140-bp NheI-AflII fragment and the 279-bp NheI-SnaBI fragment were ligated with the 10,810-bp AflII-SnaBI from YCpARE2. The are2-WW_527,528AS allele was further subcloned into YEp352 at BamHI and SphI sites.

Missense mutagenesis of human ACAT1
The human ACAT1 cDNA was subcloned into pAlter1 (Promega) at SacI and KpnI sites from pRS424-ACAT (16). To generate the equivalent of the yeast are2-Y₅₂₄A mutant, Tyr₄₀₄ was mutated to alanine in human ACAT1, using the mutagenic oligonucleotide ACAT1-tyr, to produce ACAT1-Y₄₀₄A. Similarly, human ACAT1-S₂₆₉L was created with the mutagenic oligonucleotide ACAT1-HSF. The NotI-SmaI insert from pAlter1-ACAT1_Y404A or pAlter1-ACAT1_S269L was then ligated at NotI and Ecl136II sites of pRS426GP for expression in yeast.

In vivo assays of sterol esterification
Yeast strains containing wild-type ARE2, are2 mutants, or vector controls, were grown in 5 ml of SC-ura-2% glucose medium to a density of 10⁷ cells/ml (14) (16). [9,10-³H(N)]oleic acid, 1 µCi/ml in tyloxapol;–ethanol 1:1 (v/v), was added and cells were pulsed for a further 4 h. Total lipids were prepared and analyzed as described (14). Incorporation of label into steryl ester was determined after scintillation counting and normalization to a [4-¹⁴C]cholesterol internal standard and the dry weight of the cells.

In vitro microsomal sterol esterification assay
Yeast cells containing the constructs of interest or vector control were grown under conditions similar to those of the in vivo assays. The cultures were diluted to 500 ml in SC-ura medium containing 2% glucose or 2% galactose-1% raffinose and grown overnight. Yeast microsomes were prepared as described (16) and frozen at -70°C. Enzyme activity was determined, in vitro, by the rate of incorporation of [¹⁴C]oleoyl-CoA into steryl ester as described previously (16) (21). The standard assay (200 µl)containing 1 mM glutathione and 0.1 M potassium phosphate buffer, pH 7.4, 200 µg of microsomal protein, 1 mg of BSA, 20 nmol of oleoyl-CoA, and, where appropriate, 20 µg of cholesterol suspended in Triton WR-1339 (22), was performed for 2.5 min and terminated by the addition of chloroform;–methanol 2:1 (v/v). In control assays, labeled oleoyl-CoA was added after the reaction had been stopped. Phase separation was induced by the addition of water, and [³H]cholesteryl oleate and 15 µg of cholesteryl oleate were added as the internal standard and carrier, respectively. In some experiments, the final concentration of oleoyl-CoA was varied. Lipids were separated and quantified as described previously (16).

Production of Are2p-glutathione S-transferase (GST) fusion proteins and generation of antibody
To generate antibodies specific to the unique NH₂-terminal portion of Are2p, residues 16;–179 of this enzyme were fused with glutathione S-transferase (GST). A 1,921-bp ARE2 fragment was PCR amplified with oligonucleotides ARE2-N (BamHI site included) and SS1, and then digested with BamHI and SalI (filled in with Klenow). The resulting 498-bp fragment (ARE2 nucleotides 45 to 542) was gel purified and ligated into pGEX-3X (Pharmacia Biotech, Piscataway, NJ) at the BamHI and SmaI sites. The construct was sequenced to confirm the in-frame fusion of the ARE2 and GST sequences. Expression of the GST-Are2p fusion protein was induced in Escherichia coli with isopropyl-1-thio-ß-D-galactopyranoside. The resultant fusion protein was purified directly from bacterial lysates, using glutathione-Sepharose 4B (Pharmacia Biotech). The fusion protein was eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, and dialyzed against PBS. About 200 µg of the purified protein was used to generate chicken egg yolk immunoglobulin (IgY) antibodies. The antigen, in 0.2% SDS, was injected into two chickens. Eggs were collected daily, and the IgY were purified by differential precipitations with an EGGstract IgY purification system (Promega). The purified IgY was filter sterilized and stored at 4°C in buffer containing 0.01 M K₂HPO₄ (pH 7.4), 0.1 M NaCl, and gentamicin sulfate (50 mg/ml).

SDS-PAGE and immunodetection
Microsomes from yeast strains containing wild-type ARE2, are2 mutants, or vector controls, were prepared as described (16). Denaturing gel electrophoresis (5 µg of total protein per lane) was performed with 10% polyacrylamide for the resolving gel in the presence of 0.1% SDS (23). After SDS-PAGE separation, the proteins were electroblotted to nitrocellulose. The membrane was blocked in 5% nonfat milk in 20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20 (TBST) and probed with chicken IgY antibody against Are2p (3.4 µg/ml in TBST-1% nonfat milk) for 1 h. Detection of the immune complexes was attained with horseradish peroxidase-conjugated secondary anti-chicken IgY antibody (Promega) and ECL Western blotting detection reagent (Amersham, Arlington Heights, IL). Strains expressing human ACAT1 were similarly processed and analyzed with an ACAT1-specific monoclonal antibody (DM10, generously provided by T-Y. Chang, Department of Biochemistry, Dartmouth Medical School, Hanover, NH).

RNA analysis
Total RNA was prepared from yeast by using a hot acidic phenol extraction method (17). Equal amounts of total RNA (15 µg) were resolved in 1.2% formaldehyde agarose gels and transferred to nylon membranes by conventional procedures (17). A 471-bp probe (nucleotides 538;–1009 of ARE2) was radiolabeled with [³²P]dCTP (Prime-it; Stratagene, La Jolla, CA) and used in hybridizations at 65°C in ExpressHyb buffer (Clontech, Palo Alto, CA). The membrane was washed in 2x SSC (150 mM NaCl, 15 mM sodium citrate)-0.1% SDS (at room temperature), and in 0.1% SSC-0.1% SDS (at 60°C).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sequence conservation in the O-acyltransferase superfamily
The sequences of members of the O-acyltransferase superfamily that in an oleoyl-CoA-dependent manner esterify sterols (an ACAT-like reaction) or diacylglycerols (a DGAT reaction), were aligned with BLASTP (24) ( Fig 1a). An FYxDWWN heptapeptide forms the invariant core of a 28-residue motif (consensus 1; Fig 1a). In addition to some flanking residues, the core tyrosine and tryptophans are conserved in all database entries for this gene family (consensus 2; Fig 1a). The only exception to this consensus is a C. elegans entry CAA99773, where the first tryptophan is conservatively replaced by phenylalanine. The FYxDWWN sequence is thus a hallmark of all characterized O-acyltransferases. By contrast, the H(Y)SF motif (His₂₆₈ in human ACAT1, Tyr₂₄₄ in human ACAT2) is conserved in the sterol-esterifying ACAT enzymes but absent from the DGATs [see Fig 1b and ref. (8)].

View larger version (68K): [in this window] [in a new window]   Figure 1. Sequence motifs in the ACAT superfamily of O-acyltransferases. A: Conservation in the ACAT superfamily of O-acyltransferases. Phenylalanine-tyrosine-x-aspartic acid-tryptophan-tryptophan-asparagine (FYxDWWN, residues 403;–409 in human ACAT1) is invariant in enzymes that esterify sterol or diacylglycerol. Consensus 1 is derived from six mammalian ACAT1 sequences (human, mouse, rat, hamster, African green monkey, and cynomolgus monkey), three mammalian ACAT2 sequences (human, murine, and simian), human and murine DGAT, three plant DGAT sequences, and the yeast Are enzymes. The second consensus includes eight predicted uncharacterized proteins (organisms and GenBank accession numbers as indicated). Upper case letters indicate identity with mammalian ACAT1 and x indicates any amino acid. B: Conserved motifs and mutations in yeast Are2p and human ACAT1. Both sequences predict the retention of motifs common to the ACAT gene family; the FYxDWWN heptapeptide, and a histidine (or tyrosine)-serine-phenylalanine (H/YSF) tripeptide (8). Mutations were created and confirmed at the nucleotide level as described in text. They comprise NH2-terminal truncations (N), COOH-terminal truncations (C), in-frame deletions (, missing sequence indicated by the filled boxes), and missense variants (indicated by the residue changed).

Expression of yeast ARE2
As a first step toward determining the role of conserved sequence motifs in the esterification reaction, we isolated the gene encoding Are2p, the major sterol esterification isoform in yeast, and varied its expression by altering transcription or gene copy number. A 3.45-kb BamHI-XhoI fragment of yeast chromosome XIV, comprising the ARE2 ORF with flanking regions, was subcloned into single-copy and multicopy vectors (YCp50 and YEp352, respectively). The ARE2 ORF was also expressed from the alcohol dehydrogenase (ADH1) promoter in the low copy vector pS5. These ARE2 constructs were transformed individually into strain SCY059, which lacks the endogenous ARE genes (14) and were assayed for the incorporation of [³H]oleate into steryl ester. ARE2 expressed from the high copy number vector (YEpARE2) conferred the highest activity (1.8-fold higher than YCpARE2 or pS5ARE2; Fig 2a).

View larger version (89K): [in this window] [in a new window]   Figure 2. In vivo esterification and expression analysis of yeast cells expressing wild-type ARE2. A: Sterol esterification activity. Yeast strains (are1 are2) expressing wild-type ARE2 and vector controls were grown in selective media to retain plasmids and were labeled with [3H]oleate for 4 h. The data (incorporation into steryl ester per milligram dry weight of cells) are expressed as means of triplicate assays over at least two experiments with the corresponding standard deviations. The asterisk (*) indicates a statistically significant (paired t test, P < 0.01) difference from wild-type controls. DPM/mg dry weight, disintegrations per minute, per milligram of dry weight of cells. B: Immunoblot of microsomal protein preparations from strains with altered expression of ARE2. Microsomes were prepared from SCY059 (are1 are2) strains expressing ARE2 or vector controls grown in selective media and analyzed with an Are2p-specific antibody as described in text. The arrow indicates the predicted intact monomeric form of the enzyme. Molecular weight determination was performed relative to indicated Bio-Rad (Hercules, CA) markers.

To confirm the expression of the Are2p protein, we used isoform-specific antibodies generated against residues 16;–179 of the Are2 protein. Microsomes were prepared from strain SCY059 containing YEpARE2 and YCpARE2 and analyzed by immunoblotting. Several bands were detected at levels commensurate with the observed enzyme activity (Fig 2b). The predominant species had a mobility indicating a mass of approximately 78 kDa, with a minor but distinct degradation product of approximately 52 kDa. Are2p is predicted to contain 642 amino acid residues, with a calculated molecular mass of 74 kDa. Purified microsomes from strain SCY059 transformed with a vector control did not exhibit immunoreactive species with these mobilities, indicating the specificity of the antibody. The YEpARE2 construct was used as the recipient for the subsequent mutagenesis experiments.

Truncation and deletion mutagenesis of ARE2
To determine the functional significance of sequence conservation in the O-acyltransferases, we first performed a deletion analysis of the Are2p molecule (Fig 1b). COOH-terminal truncation mutants of 68 residues (are2-C68) or greater (are2-C208 and are2-C253, not shown) and two internal deletions (are2-46 and are2-139) that encompass regions of sequence similarity between the O-acyltransferases were created. In addition, two NH₂-terminal truncation mutants, are2-N31 and are2-N178, were constructed to express 31- and 178-residue deletions, respectively, from the NH₂ terminus of the Are2p molecule. The ARE2 mutants were transformed into strain SCY059 and assayed in vivo for sterol esterification. Truncations or deletions from the COOH terminus consistently resulted in a complete loss of enzymatic activity ( Fig 3a; and data not shown for are2-C208, are2-C253). By contrast, the are2-N31 allele produced significantly higher sterol esterification activity than wild-type ARE2, indicating that the NH₂-terminal 31 amino acids are certainly dispensable and may in fact inhibit enzymatic function. The are2-N31 allele in a single-copy YCp50 vector also produced higher activity than the corresponding YCpARE2 wild-type control (not shown). Deletion of 178 amino acids from the NH₂ terminus of Are2p (in are2-N178) produced an enzyme with residual activity that was significantly higher than that of the vector control (approximately 5% of wild-type Are2p).

View larger version (141K): [in this window] [in a new window]   Figure 3. In vivo esterification analysis and expression of mutant alleles of ARE2. A: Yeast strains (wild-type or are1 are2) expressing wild-type ARE2, are2 mutants (see Fig 1), or vector controls metabolically labeled with [3H]oleate were analyzed as described previously for sterol esterification. The data (incorporation into steryl ester per milligram dry weight of cells) are expressed as means of triplicate assays over at least two experiments with the corresponding standard deviations (which if absent were too small to be visible on this scale). The single asterisk (*) and double asterisks (**) indicate statistically significant (paired t test, P < 0.01) differences from wild-type and are1 are2 controls, respectively. DPM/mg dry weight, disintegrations per minute, per milligram of dry weight of cells. B: Immunoblot of microsomal protein from strains expressing Are2p variants. Microsomes were prepared from SCY059 (are1 are2) strains expressing wild-type ARE2, are2 mutants, or vector controls as described above. All protein samples were prepared and analyzed with an Are2p-specific antibody as described. The arrow indicates the predicted intact monomeric form of the normal enzyme. Molecular weight determination was performed relative to indicated Bio-Rad markers. C: RNA blot hybridization of strains expressing mutant alleles. Samples were prepared, resolved, and probed with a 471-bp ARE2-specific radiolabeled probe as described. Arrow indicates the predicted full-length transcription product. Molecular weight determination was performed relative to the indicated markers (Clontech).

Missense mutagenesis of the conserved motifs FYxDWWN and H/YSF
The deletion mutagenesis described above suggests that a conserved carboxyl domain of 184 amino acids (residues 389;–573, as defined by are2-46 and are2-139) is critical to the enzymatic activity of Are2p. The inactive COOH-terminal deletion encoded by are2-139 encompasses the 28-residue consensus identified in Fig 1a. Within this region, the invariant tyrosine of the core FYxDWWN motif is a predicted phosphorylation target for tyrosine kinases (PROSITE PD00007) (25), the consensus for which is retained by many members of this superfamily. Furthermore, paired tryptophans are a relatively rare conjunction that has been shown to participate in cholesterol binding in thiol-activated cytolysins such as perfringolysin O (26) (27). Thus, we mutated the invariant Tyr₅₂₄ to alanine in are2-Y₅₂₄A and the paired tryptophan residues at positions 527 and 528 to alanine-serine in are2-WW_527,528AS (Fig 1b). The missense mutations were subcloned into a YEp vector, transformed into strain SCY059, and assayed in vivo for their ability to esterify sterols. As shown in Fig 3a, the enzymatic activity conferred by are2-Y₅₂₄A was reduced 4-fold compared with wild-type ARE2. The are2-WW_527,528AS allele lost completely the ability to confer enzymatic activity. These data indicate that the conserved motif, FYxDWWN, is critical to the sterol esterification reaction.

A histidine-serine-phenylalanine motif is invariant in ACAT1, Are1, and Are2p, conserved in ACAT2, and is critical to the sterol esterification activity of hamster and simian ACAT1 and simian ACAT2 (28) (29). To confirm a role for the HSF motif in the activity of the yeast enzyme, we mutated the corresponding Ser₃₃₉ to leucine in Are2p. The are2-S₃₃₉L variant did not catalyze esterification of sterols in yeast (Fig 3a).

Expression of mutant variants of Are2p
We tested expression of the variant Are2p proteins in microsomes from strain SCY059 transformed with the mutant alleles by immunoblot with the Are2-specific IgY (Fig 3b). The are2-N31-, are2-46-, and are2-139-encoded proteins were observed at the predicted reduced molecular weights. The missense mutants are2-Y₅₂₄A and are2-WW_527,528AS were expressed at levels and electrophoretic mobility comparable to that of wild-type ARE2. By contrast, the are2-S₃₃₉L-encoded variant was expressed at significantly lower levels than wild-type Are2p. This observation is consistent with the phenotype of the analogous ACAT1 mutation in the Chinese hamster ovary (CHO) cell line SRD4, which also produces an unstable variant of ACAT1. The Are2p variants were detected in microsomes, suggesting that the mutations do not significantly alter membrane localization.

The expression of the are2-N178-encoded protein could not be examined with this antibody because this allele lacks the NH₂-terminal epitope used to generate the antibody. In addition, the proteins encoded by the are2-C68 alleles were not detected in whole-cell lysates or microsomes, suggesting that the mutations affect the stability of the proteins. To investigate the expression of these variants at the transcriptional level we performed Northern blots (Fig 3c). We did not detect any differences in ARE2 mRNA abundance between the variants, suggesting that a posttranscriptional mechanism may account for the differences in enzyme activities.

Confirmation of the mutant phenotypes in microsomal assays
Microsomes from strain SCY059, transformed with expression vectors harboring no insert, wild-type ARE2 or the are2 mutants, were assayed in vitro for the incorporation of [¹⁴C]oleate into steryl ester. As shown in Table 2, the in vitro assays confirm our in vivo results. Wild-type ARE2 on the YEp vector formed ergosterol ester at a rate of 5,150 pmol/min per mg microsomal protein (1,000-fold over background). Microsomes from strains expressing the are2-N31 allele demonstrated about 30% higher activity than wild-type ARE2 (P < 0.01). The activity conferred by are2-N178 was decreased with respect to wild type, but still 16-fold higher than background (P < 0.01). The activity expressed from the are2-Y₅₂₄A allele was about 24% of the activity of wild-type ARE2. The are2-S₃₃₉L, are2-WW_527,528AS, and are2-C68 alleles did not express detectable in vitro sterol esterification activity.

Oleoyl-CoA affinity in the are2-Y₅₂₄A variant
To assess whether the are2-Y₅₂₄A mutation conferred altered affinity for substrates, we performed an in vitro substrate saturation analysis. Because of its lability and hydrophilicity, endogenous acyl-CoA is undetectable in our microsomal preparations. By contrast, the concentrations of endogenous sterol in these membranes are high but invariant between cells expressing different are2 alleles (data not shown). We thus performed in vitro sterol esterification assays with increasing concentrations of oleoyl-CoA and constant microsomal input. The major difference between the normal Are2 and Are2-Y₅₂₄A enzymes lies in their affinity for oleoyl-CoA ( Fig 4). A Lineweaver-Burk plot of the data suggests saturable, noncooperative kinetics as observed for human ACAT1 with regard to oleoyl-CoA binding (30). Nonlinear least-squares regression of the primary data indicated the microsomal esterification reaction conferred by wild-type protein had a 6-fold lower apparent K_m than the Are2-Y₅₂₄A enzyme (35.6 vs. 229.0 µM, respectively) and a 2-fold higher apparent V_max. We performed the same experiment with microsomes prepared from strains expressing the are2-N31 allele. By contrast to the Are2-Y₅₂₄A enzyme, the apparent V_max of the reaction mediated by the are2-N31 allele was significantly higher than wild type although the apparent K_m was unchanged (Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 4. Oleoyl-CoA substrate saturation of wild-type and mutant Are2 enzymes. Microsomes were prepared from SCY059 expressing wild-type ARE2 and are2-Y524A or are2-N31 alleles and assayed in vitro for sterol esterification in the presence of increasing concentrations of oleoyl-CoA. Because of the presence of multiple acyl-CoA acceptors (such as membranes and albumin), this approach estimates an "apparent" Km and Vmax, which can then be used to compare the reactions mediated by the different are2 alleles. The mean of two representative experiments is shown, and standard errors at each point did not exceed 8%. Nonlinear least-squares regression (Sigmaplot) of these data gave the apparent Km (µM) and Vmax (pmol/min/mg).

Missense mutagenesis of human ACAT1
To assess the role of the FYxDWWN and (H/Y)SF motifs in another member of the ACAT gene family, we mutated the corresponding residues of human ACAT1 (Fig 1b) and expressed the variant cDNAs in yeast strain SCY059, using the GAL1/10 promoter (16). Assays of sterol esterification were performed in vitro in the presence of cholesterol (ACAT1 does not utilize ergosterol) with microsomes prepared from yeast expressing the normal and mutant ACAT1 alleles. Human ACAT1-Y₄₀₄A and ACAT1-S₂₆₉L were enzymatically inactive ( Table 3), confirming the importance of these motifs in enzymatic activity. We verified the expression of the ACAT1 variants by immunoblots of microsomal preparations ( Fig 5). The ACAT1-Y₄₀₄A variant was expressed at levels comparable to those of normal ACAT1; however, the ACAT1-S₂₆₉L protein was detected at significantly lower levels, although not to a level proportional to the complete loss in activity.

View larger version (47K): [in this window] [in a new window]   Figure 5. Expression of human ACAT1 variants. Microsomes were prepared from SCY059 (are1 are2) strains expressing human ACAT1 or variants thereof, grown in selective induction media to retain plasmids and express the cDNAs from the GAL1/10 promoter. Samples were analyzed with an ACAT1-specific antibody (DM10) as described. Molecular weight markers were from Bio-Rad. The arrow indicates the mobility of the monomeric form of ACAT1. Higher mobility forms represent multimers resistant to denaturation as described previously (5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The acyl-CoA O-acyltransferase gene family currently includes 16 sequence homologs with demonstrated esterification activity against substrates such as cholesterol and diacylglycerol (7) (8) (10) (13) (14) (31) (32) (33). In this study, we have identified critical regions of these enzymes on the basis of sequence conservation and mutagenesis of the S. cerevisiae ARE2 and human ACAT1 genes. Our data demonstrate two phylogenetically conserved carboxyl-terminal motifs to be required for sterol esterification. Conversely, the nonconserved, amino-terminal region appears to exert negative regulatory control of this reaction.

The regulation of the sterol esterification reaction in mammalian cells is achieved primarily by the allosteric activation of the ACAT enzymes by sterols and oxysterols (34). The molecular mechanisms or cis-acting sequences that result in this regulation are unknown. By contrast to the phenotypes created by mutating conserved regions of these molecules, deletion of 31 residues from the poorly conserved NH₂ terminus of Are2p (are2-N31) consistently increased enzymatic activity with no effect on protein expression. The in vitro activity conferred by the are2-N31 allele was also increased, primarily because of an elevation in the apparent V_max of this reaction. Even deletion of 28% of the molecule (in are2-N178) from the NH₂ terminus retained significant enzymatic activity. The 31-amino acid NH₂-terminal truncation of ARE2 is similar to the ald1 allele in AKR mutant mice (35). This allele of murine ACAT1 comprises two conservative amino acid changes and a 33-amino acid NH₂-terminal deletion (35) and catalyzes in vitro cholesterol esterification at levels equivalent to or greater than that of the wild-type enzyme. Paradoxically, male ald1 mice display adrenal depletion of neutral lipids and defective in vivo sterol esterification, suggesting altered regulation of this protein in an androgen-dependent manner. We propose that the NH₂-terminal domains of Are2p, and of ACATs in general, may act on enzymatic activity in a negative fashion because of interaction with unidentified regulatory factors.

The O-acyltransferase reaction is yet to be defined at the mechanistic level. The FYxDWWN motif identified here is invariant within the conserved COOH-terminal region of ACAT and DGAT enzymes from humans, rodents, simians, plants, and yeast (7) (8) (10) (13) (15) (36) and is also conserved in several uncharacterized sequence homologs (Fig 1). Although other regions of local homology are evident, this motif is the hallmark of all currently available sequence entries for this gene family. Consistent with this conservation, the alteration of the paired tryptophan residues in the FYxDWWN motif to alanine-serine (the are2-WW_527,528AS allele) resulted in total loss of enzymatic activity. In contrast to the carboxyl-terminal truncations, these substitutions had no effect on microsomal localization or levels of the mutant protein, suggesting that the changes were relatively tolerated with regard to protein folding. We initially speculated that the paired aromatic residues could participate in sterol binding as they do in thiol-activated cytolysins (26) (27). The total ablation of activity in the are2-WW_527,528AS allele is consistent with this hypothesis. The tyrosine residue in the FYxDWWN consensus is a putative target for a phosphorylation reaction, although we have been unable to detect phosphorylation of Are2p (Z. Guo and S. L. Sturley, unpublished data). However, it is clearly a critical residue; the alteration of tyrosine to alanine in yeast Are2p (are2-Y₅₂₄A) and human ACAT1 (ACAT1-Y₄₀₄A) resulted in a significant reduction in enzymatic activity, with no effect on protein levels or microsomal association. This mutation significantly reduced the apparent oleoyl-CoA affinity of this reaction in vitro, indicating its participation in binding this substrate.

The definitive identification of the active sites of this class of enzymes will result from investigation of a crystal structure. In the meantime, it is interesting to note that the protein catalyzing the reaction opposing that of ACAT (i.e., cholesteryl ester hydrolysis by serine esterases) possesses serine, histidine, and aspartic acid as a catalytic triad (37). These residues are present in the ACAT motifs characterized here. For example, the invariant serine residue of the HSF motif encoded by the yeast ARE2, and present in human ACAT1 and CHO cell ACAT1 (28), is essential for sterol esterification enzyme activity and the stability of the enzyme. Because this sequence is absent from DGAT enzymes, which esterify diacylglycerol, we speculate that the (H/Y)SF motif plays a role in sterol binding by the ACAT enzymes. By contrast, because of its retention in all acyltransferases, we hypothesize that the FYxDWWN domain primarily mediates binding of acyl-CoA, the common substrate for these reactions.

	FOOTNOTES

Abbreviations: ARE1, ACAT-related enzyme 1; ARE2, ACAT-related enzyme 2; DGAT, acyl-coenzyme A:diacylglycerol O-acyltransferase.
¹ Present address: Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.

	ACKNOWLEDGMENTS

We are indebted to Andre Bensadoun and Jim Young of the Cornell University animal facility for assisting in the production of the chicken antisera, to Alan Attie and Rajasekhar Ramakrishnan for advice on enzyme kinetics, and to Jessica Rich and Peter Oelkers for editorial assistance. This work was supported in part by a Grant-in-Aid/Investigatorship from the American Heart Association (NYC Affiliate), the Hirschl/Weil-Caulier Trust, and the Ara Parseghian Medical Research Foundation (all to S.L.S.). S.L.S. is an Established Investigator of the American Heart Association.

Manuscript received October 6, 2000; and in revised form January 29, 2001; and in revised form April 9, 2001

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