A Glucose-repressible Gene Encodes Acetyl-CoA Hydrolase from Saccharomyces cerevisiae”

In to the biological function and regulation of the acetyl-CoA hydro- lase, cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase. RNA blot analy- that acetyl-CoA hydrolase is encoded by a 2.5-kilobase DNA blot

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, was first identified in pig heart in 1952 (l), and subsequently the enzyme has been found in many mammalian tissues (2)(3)(4)(5)(6)(7). There have been several studies dealing with the physicochemical properties of acetyl-CoA hydrolase, although little is known about its biological function. It has been proposed that a physiological role for acetyl-CoA hydrolase may be its maintenance of the cytosolic acetyl-CoA concentration and CoASH pool for both fatty acid and cholesterol synthesis and fatty acid oxidation (6,8). The concentration of intracellular acetyl-CoA is regulated by its rate of synthesis and its rate of utilization and/or degradation.
Acetyl-CoA is primarily synthesized from pyruvate generated from the carbohydrate and from amino acids (Ala, Thr, Gly, Ser, and Cys), from acetoacetyl-CoA generated from other amino acids (Phe, Tyr, Leu, Lys, and Trp), and from P-oxidation of fatty acids, although a minor amount is also synthesized by acetyl-CoA synthetase (9). Utilization of acetyl-CoA occurs during the Krebs cycle or by its conversion to fatty acids or ketone bodies. Furthermore, the iV"-and N'acetyltransferase-catalyzed acetylation of proteins and peptides (lo- 15 This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mine) (16)(17)(18)(19), accounts for additional usage of endogenous acetyl-CoA.
During the purification of yeast N"-acetyltransferase, the presence of an endogenous "inhibitor" of acetyltransferase was detected (20), and thereafter the inhibitor was purified and shown to be acetyl-CoA hydrolase (21). In addition to its inhibition of yeast N"-acetyltransferase, acetyl-CoA hydrolase also inhibits purified rat brain pyruvate carboxylase (22), choline acetyltransferase (23), and [acyl-carrier-proteinlacetyltransferase (24). Moreover, the Klein group (25) has found that arylalkylamine N-acetyltransferase in the rat pineal gland plays a key role in generation of the circadian rhythmicity of melatonin synthesis and suggested that acetyl-CoA hydrolase may be involved in the overall regulation of acetylation. Whether or not acetyl-CoA hydrolase is involved in regulating the endogenous pool(s) of acetyl-CoA remains to be established.
The acetyl-CoA hydrolase from Saccharomyces cerevisiae was purified to apparent homogeneity (1,080-fold) and characterized as a monomeric protein, whose M, was 65,000 (21). During the purification, we noticed the enzymatic activity and yield were higher from the stationary phase or frozen cells and less from the log phase or freshly cultured cells. In order to understand the function and regulation of acetyl-CoA hydrolase and its role in controlling the acetyl-CoA and CoASH pools, we cloned the acetyl-CoA hydrolase gene and demonstrated that its expression is glucose-repressible. EXPERIMENTAL PROCEDURES'
Cloning of the Yeast Acetyl-CoA Hydrolase cDNA-After initial screening of 500,000 recombinant cDNA clones in the yeast Xgtll cDNA library, 17 clones were detected which hybridized to both oligonucleotides Al and A2. These clones, designated XAl to XA17, also hybridized with two other oligonucleotides, A3 and A4, and their cDNA inserts were analyzed by restriction enzyme digestions and DNA blot analyses. The use of four oligonucleotide probes derived from four discrete amino acid sequences allowed the unequivocal identification of the cDNA clones encoding acetyl-CoA hydrolase. XbaI digestion revealed inserts that lacked internal X&I sites and ranged from 1.8 to 2.3 kilobases. The five longest cDNA inserts were subcloned as XbaI fragments into the Bluescript plasmid, and additional restriction enzyme mapping, DNA blot analyses, and nucleotide sequence analyses were carried out. Four cDNA clones (pBA2, pBA3, pBA5, pBA17), represented as ACHS in Fig. 2B, displayed identical restriction maps, and ACH4 (derived from pBA4) overlapped them.
Sequence Analysis of the cDNA Clones-The complete nucleotide sequence, both orientations, of pBA4 and pBA5 were determined using exonuclease III deletions and the doublestranded dideoxy chain termination method. The nucleotide sequence of the yeast acetyl-CoA hydrolase cDNA is shown in Fig. 2C. Compared with pBA5, pBA4 lacks 266 base pairs from the 3' end and extends 537 base pairs further toward the 5' end. Together both cDNAs contain a long open reading frame extending from nucleotides 1 to 1578 which encodes all 16 tryptic peptides. The location of the initiator methionine was assigned based on the fact that there are no intervening methionine residues between this residue and the first identified tryptic peptide and that there is an in-frame termination codon (nucleotides -93 to -91) preceding this proposed site of initiation.
DNA blot analysis of restriction enzyme-digested yeast genomic DNA (Fig. 4, Miniprint) with the same probes revealed a fragment pattern consistent with a single-copy gene encoding the sequences present in the cDNA. Chromosomal analysis with these same probes indicated that the yeast acetyl-CoA hydrolase gene (ACHl ) is located on chromosome II (Fig. 5, Miniprint).
Hydrophobicity Profile for Acetyl-CoA Hydrolase-The hydrophobicity profile was determined using the algorithm of Kyte and Doolittle (42) with a window size of 9 (Fig. 6,  Miniprint).
Comparison of cDNA and Protein Sequence Data for Yeast Acetyl-CoA Hydrolase with DNA and Protein Sequence Data Bases-A computer search of the GenBank data base was conducted using the FASTA program, described by Pearson and Lipman (43). Comparisons between the protein sequence of yeast acetyl-CoA hydrolase and other protein sequences in the Swiss Protein data base were also conducted. There were no significant similarities (~30%) revealed by either comparison.
Yeast Acetyl-CoA Hydrolase Is a Mannose-containing Glycoprotein-The calculated molecular weight for acetyl-CoA hydrolase derived from cDNA sequence (58,369) is smaller than the M, determined from sodium dodecyl sulfate-poly-a&amide gel electrophoresis (65,000 + 2,000) (21). To determine whether this disparity could be attributed to acetyl-CoA hydrolase being a glycoprotein, the presence of a carbohydrate moiety on acetyl-CoA hydrolase was assessed using a lectin-binding method. Concanavalin A (binds to a-mannose), Sophora japonica agglutinin (binds to galactose and N-acetylgalactosamine), and Pisum sativum agglutinin (binds to amannose and N-acetylchitobiose linked to cy-fucose) were used as the lectins. Concanavalin A, but not S. juponica agglutinin and P. sativum agglutinin, bound to yeast acetyl-CoA hydrolase, indicating that the enzyme contains Ly-mannose and is an intracellular glycoprotein. Glucose Repression of ACHl Gene Expression--In an attempt to understand the regulation of ACHZ gene expression, we measured the levels of ACHZ mRNA and acetyl-CoA hydrolase activity in different growth phases and examined the effects of various carbon sources on the levels of ACHZ mRNA expression.
Total RNA, isolated from the early log, late log, and stationary phase yeast cultures, was run on formaldehydelagarose gel, transferred onto GeneScreen Plus membrane, and hybridized with 32P-labeled pBA4 cDNA insert. The blot membrane was also hybridized with yeast tubulin as an internal control. The ACHl mRNA and enzyme activity were increased in stationary phase but were decreased in early and late log phases (Fig. 7A).
An additional RNA blot analysis of total RNA prepared from mid-log yeast cultures grown in either glucose, glycerol, galactose, or acetate-containing medium was also performed. ACHZ mRNA level of yeast grown in glucose was barely detectable and was much less than the mRNA levels of yeasts grown in media containing glycerol, galactose, or acetate (Fig.  7B). In addition, acetyl-CoA hydrolase activity is much higher when yeast are grown in media containing glycerol, galactose, or acetate as compared with glucose. Furthermore, the addition of glucose to galactose or glycerol medium caused a rapid reduction in ACHl mRNA level within 2 h after addition of glucose, and the ACHl mRNA level was reduced to that of yeast grown in glucose medium (data not shown). Furthermore, the addition of glycerol or galactose into glucose medium did not increase the level of ACHl mRNA (data not shown). DISCUSSION In order to study the biological function and regulation of the acetyl-CoA hydrolase and eventually its role in controlling the intracellular level of acetyl-CoA, we cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase. This is the first report of the cloning and sequencing of an acetyl-CoA hydrolase gene from any species, and we have utilized this gene to establish that the acetyl-CoA hydrolase is glucose-repressible.
Yeast acetyl-CoA hydrolase is encoded by an open reading frame of 1,578 bases and consists of 526 amino acids. The molecular weight calculated from the amino acid composition is 58,369, which is smaller than the previously determined M, of 65,000 f 2,000 (21). We have demonstrated that yeast acetyl-CoA is a mannose-containing glycoprotein, and we propose that the presence of such a carbohydrate moiety accounts for the difference between the theoretical and ob- served molecular masses. Although acetyl-CoA hydrolase contains three putative N-glycosylation sites (i.e. Asn-X-Ser (or Thr) sequences) at residues 314-316, 381-383, and 386-388, the position(s) of N-glycosylation remains to be determined. Previous protein sequence analysis of the native protein revealed it to be N-terminally blocked (21), and we assume (but have not yet established) that the N-terminal residue of acetyl-CoA hydrolase is a N"-acetylated threonyl residue. A comparison of the protein sequences of acetyl-CoA hydrolase to other protein sequences in the data bank failed to reveal any appreciable percent similarity with any protein in the Swiss Protein data base.
The RNA, DNA, and chromosomal blot hybridizations indicate that there is a single 2.5-kilobase mRNA and a single gene (ACHI) encoding acetyl-CoA hydrolase located on chromosome II. However, it remains to be established whether or not yeast contains other acetyl-CoA hydrolases.
The mRNA level and enzyme activity of acetyl-CoA hydrolase are markedly reduced in glucose-containing medium and increased in glucose-free medium indicating that the expression of ACHl gene is repressed by glucose. Since the acetyl-CoA hydrolase is highly expressed in the stationary phase and in media containing nonfermentable carbon sources (glycerol, galactose, and acetate), we suspect that under conditions of limited nutrition that a large amount of acetyl-CoA is likely generated from fatty acids or amino acids and that it is not effectively incorporated into the tricarboxylic acid cycle. Such an excess of acetyl-CoA could lead to autoacetylation of proteins, as well as to the generation of toxic ketone bodies and other noxious metabolites.
It is possible, albeit not yet established, that the intracellular level of acetyl-CoA could be regulated at a "safe" level by hydrolysis of excessive acetyl-CoA by acetyl-CoA hydrolase.
The concentration of acetyl-CoA in cells is primarily regulated by its rate of synthesis and its utilization in various metabolic pathways. Contemporary knowledge of the structure and function of acetyl-CoA hydrolases (i.e. cytosolic (6) and mitochondrial (3)) is incomplete.
The role of such enzymes catalyzing the scission of acetyl-CoA's high energy thioester bond with no apparent metabolic advantage represents a biochemical conundrum.
Several functions for acetyl-coA hydrolases from higher eukaryotes have been proposed. First, the cytoplasmic form of the enzyme may be involved in the maintenance of the cytosolic acetyl-CoA concentration and CoASH pool for both fatty acid and cholesterol synthesis and fatty acid oxidation (6,8). Second, the mitochondrial form of the enzyme may be involved in the metabolism of brown fat in hibernating animals (4). Third, the enzyme may be involved in the regulation of acetylation during melatonin synthesis in the pineal gland (25). However, in none of these cases has a specific biological function for acetyl-CoA hydrolase been firmly established.
Furthermore, although it is known that iV-acetylation reduces the rate of protein turnover, it is unclear how the intracellular concentration of acetyl-CoA relates to the rate of N"-acetylation.
Appropriate manipulation of the ACHl gene may shed light on this important question. We are investigating the role of acetyl-CoA hydrolase in regulating the acetyl-CoA pool utilized for N"-acetylation of proteins. In addition, we are studying the regulatory pathways of the glucose repression of ACHl gene and are comparing it with other glucose-repressible genes.