Functional Characterization and Localization of Acetyl-CoA Hydrolase, Ach1p, in Saccharomyces cerevisiae
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m213268200
ISSN1083-351X
AutoresLeh-Miauh Buu, Yee‐Chun Chen, Fang‐Jen S. Lee,
Tópico(s)Plant biochemistry and biosynthesis
ResumoAcetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, inach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast. Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, inach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast. acetyl-CoA hydrolase morpholine-ethanesulfonic acid carnitine acetyltransferase The concentration of acetyl-CoA in cells is primarily regulated by its rate of synthesis and its utilization in various metabolic pathways. In the yeast Saccharomyces cerevisiae, biosynthesis of acetyl-CoA is mainly achieved by the acetyl-CoA synthetase reaction, whereas oxidative decarboxylation by the mitochondrial pyruvate dehydrogenase complex appears to be of minor importance (reviewed in Ref. 1Pronk J.T. Yde Steensma H. Van Dijken J.P. Yeast. 1996; 12: 1607-1633Crossref PubMed Scopus (619) Google Scholar). Even under glycolytic growth conditions, S. cerevisiae converts pyruvate into acetate, catalyzed by the subsequent action of pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-CoA synthetase (2Flikweert M.T. Van Der Zanden L. Janssen W.M. Steensma H.Y. Van Dijken J.P. Pronk J.T. Yeast. 1996; 12: 247-257Crossref PubMed Scopus (201) Google Scholar). In the presence of a fermentable carbon source, acetyl-CoA may be mainly used as a precursor of fatty acid and sterol biosynthesis. On the other hand, an additional pool of acetyl-CoA is required for the glyoxylate cycle (citrate synthase and malate synthase reactions) when cells grow with a non-fermentable substrate such as ethanol or acetate. Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, was first identified in the pig heart (3Gergely J. Hele P. Ramakrishnan C.V. J. Biol. Chem. 1952; 198: 323-334Abstract Full Text PDF Google Scholar), and subsequently the enzyme has been found in many mammalian tissues (4Knowles S.E. Jarrett I.G. Filsell O.H. Ballard F.J. Biochem. J. 1974; 142: 401-411Crossref PubMed Scopus (274) Google Scholar, 5Robinson Jr., J.B. Mahan D.E. Koeppe R.E. Biochem. Biophys. Res. Commun. 1976; 71: 959-965Crossref PubMed Scopus (15) Google Scholar, 6Bernson V.S.M. Eur. J. Biochem. 1976; 67: 403-410Crossref PubMed Scopus (37) Google Scholar, 7Grigat K.-P. Koppe K. Seufert C.-D. Soling H.-D. Biochem. J. 1979; 177: 71-79Crossref PubMed Scopus (21) Google Scholar, 8Prass R.L. Isohashi F. Utter M.F. J. Biol. Chem. 1980; 255: 5215-5223Abstract Full Text PDF PubMed Google Scholar, 9Soling H.-D. Rescher C. Eur. J. Biochem. 1985; 147: 111-117Crossref PubMed Scopus (25) Google Scholar, 10Svensson L.T. Kilpelainen S.H. Hiltunen J.K. Alexson S.E. Eur. J. Biochem. 1996; 239: 526-531Crossref PubMed Scopus (11) Google Scholar). During the purification of yeast Nα-acetyltransferase, an endogenous "inhibitor" of acetyltransferase was purified and shown to be acetyl-CoA hydrolase (11Lee F.-J.S. Lin L.-W. Smith J.A. J. Biol. Chem. 1988; 263: 14948-14955Abstract Full Text PDF PubMed Google Scholar, 12Lee F.-J.S. Lin L.-W. Smith J.A. Eur. J. Biochem. 1989; 184: 21-28Crossref PubMed Scopus (16) Google Scholar). Acetyl-CoA hydrolase also inhibits purified rat brain pyruvate carboxylase (13Mahan D.E. Mushahwar I.K. Koeppe R.E. Biochem. J. 1975; 145: 25-35Crossref PubMed Scopus (11) Google Scholar) and [acyl-carrier-protein]acetyltransferase (14Lowe P.N. Rhodes S. Biochem. J. 1988; 250: 789-796Crossref PubMed Scopus (27) Google Scholar). It has been shown that the expression of acetyl-CoA hydrolase (ACH1) from S. cerevisiae is glucose-repressible (15Lee F.-J.S. Lin L.-W. Smith J.A. J. Biol. Chem. 1990; 265: 7413-7418Abstract Full Text PDF PubMed Google Scholar) and subjected to cAMP-dependent repression (16Boy-Marcotte E. Perrot M. Bussereau F. Boucherie H. Jacquet M. J. Bacteriol. 1998; 180: 1044-1052Crossref PubMed Google Scholar). The function of Ach1p1in vivo is still speculative. Previously, we have shown that the ability of ach1 mutants to grow on acetate is impaired (17Lee F.-J.S. Lin L.-W. Smith J.A. Biochim. Biophys. Acta. 1996; 1297: 105-109Crossref PubMed Scopus (20) Google Scholar).ACH1 is highly homologous to the aarC gene ofAcetobacter aceti (18Fukaya M. Takemura H. Tayama K. Okumura H. Kawamura Y. Horinouchi S. Beppu T. J. Ferment. Bioeng. 1993; 76: 270-275Crossref Scopus (45) Google Scholar) and the Neurospora crassagene acu8 (19Marathe S. Connerton I.F. Fincham J.R.S. Mol. Cell. Biol. 1990; 10: 2638-2644Crossref PubMed Scopus (24) Google Scholar, 20Connerton I.F. McCullough W. Fincham J.R.S. J. Gen. Microbiol. 1992; 138: 1797-1800Crossref PubMed Scopus (15) Google Scholar). An acu-8 mutant strain, characterized as acetate non-utilizing, shows strong growth inhibition by acetate but will use it as a carbon source at low concentrations (20Connerton I.F. McCullough W. Fincham J.R.S. J. Gen. Microbiol. 1992; 138: 1797-1800Crossref PubMed Scopus (15) Google Scholar). The acu-8 mutant was also shown to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate. As in Saccharomyces, the Neurosporaenzyme is acetate-inducible. The arrC-defective mutant also showed an inability to assimilate acetic acid (18Fukaya M. Takemura H. Tayama K. Okumura H. Kawamura Y. Horinouchi S. Beppu T. J. Ferment. Bioeng. 1993; 76: 270-275Crossref Scopus (45) Google Scholar). However, in all three organisms, disruption of these genes yields strains that grow normally on ethanol (17Lee F.-J.S. Lin L.-W. Smith J.A. Biochim. Biophys. Acta. 1996; 1297: 105-109Crossref PubMed Scopus (20) Google Scholar, 18Fukaya M. Takemura H. Tayama K. Okumura H. Kawamura Y. Horinouchi S. Beppu T. J. Ferment. Bioeng. 1993; 76: 270-275Crossref Scopus (45) Google Scholar, 20Connerton I.F. McCullough W. Fincham J.R.S. J. Gen. Microbiol. 1992; 138: 1797-1800Crossref PubMed Scopus (15) Google Scholar). Possibly, the acetyl-CoA balance during growth on acetate is disturbed in such mutants. Whether or not acetyl-CoA hydrolase is involved in regulating the endogenous pool(s) of acetyl-CoA remains to be established. In this study, we took an initial step to characterize the biochemical property of Ach1p in vivo and determine its subcellular localization. We demonstrate that the enzymatic activity of Ach1p is dependent upon its putative nucleotide (CoA) binding sites and show that Ach1p is a mitochondrial enzyme. In addition, we provide initial evidence that Ach1p is involved in development of pseudohyphae but not in mitochondrial biogenesis. Yeast culture media were prepared as described by Sherman et al.(21Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). YPD contained 17 Bacto-yeast extract, 27 Bacto-peptone, and 27 glucose. SD contained 0.77 Difco yeast nitrogen base (without amino acids) and 27 glucose. Nutrients essential for auxotrophic strains were supplied at specified concentrations. For comparison of Ach1p expression in different carbon sources, synthetic media containing 57 glucose, 27 galactose, 27 glycerol, and 27 potassium acetate were used. Yeast cells were transformed by the lithium acetate method (22Ito H., Y. Fukuda K. Murata K. Murata Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Plasmids were constructed by standard protocols as described by Sambrook et al. (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast strains YPH250 (MATa ade2, his3, leu2, lys2,trp1, ura3–52), YPH252 (MATα ade2,his3, leu2, lys2, trp1,ura3–52), and INVSc1 (MATα his3-1,leu2, trp1–289, ura3–52) were used in this study. For creating the yeast expression vector encoding Ach1p, the sequence encoding yeast ACH1 was amplified by PCR and inserted into the SstI-XbaI site of the yeast expression vector pVT101U, a 2-ॖ-based expression plasmid containing the ADH1 promoter (24Vernet T. Dignard D. Dignard D. Thomas D.Y. Gene (Amst.). 1987; 52: 225-233Crossref PubMed Scopus (477) Google Scholar). ACH1 constructs containing truncations of the N-terminal region (Ach1pdN, deletion of 1–64 amino acid residues), the first mutation of the putative CoA binding site (Ach1pSS, substitution of amino acids Gly-277 and Gly-279 by Ser-277 and Ser-279), and the second mutation of the putative CoA binding site (Ach1pES, substitution of amino acids Gly-393 and Gly-395 by Glu-393 and Ser-395) were made. All mutations were generated by PCR-based mutagenesis. The sequences of the resulting constructs were verified by sequencing. The open reading frame ofACH1 was obtained by PCR, by the use of primers that incorporated unique NcoI and BamHI sites at the initiating methionine and 6 bp downstream of the stop codon, respectively. For the His-tagged Ach1p, a DNA fragment containing theACH1 coding region was generated by amplifying of yeast genomic DNA with sequence-specific primers. The PCR product was purified and ligated to the expression vector pET15b (Novagen), yielding pET15bACH1. The His-tagged fusion protein was synthesized in BL21(DE3) Escherichia coli and purified on nickel-nitrilotriacetic acid resin (Qiagen, Chatsworth, CA) as described (25Huang C.-F. Buu L.-M., Yu, W.-L. Lee F.-J.S. J. Biol. Chem. 1999; 274: 3819-3827Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Denatured, purified recombinant Ach1p isolated from an SDS-PAGE gel was used as antigen for raising polyclonal antibodies in rabbits essentially as described (25Huang C.-F. Buu L.-M., Yu, W.-L. Lee F.-J.S. J. Biol. Chem. 1999; 274: 3819-3827Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Crude yeast lysates were prepared, and acetyl-CoA hydrolase activity was determined by radioactive assay, as described previously (15Lee F.-J.S. Lin L.-W. Smith J.A. J. Biol. Chem. 1990; 265: 7413-7418Abstract Full Text PDF PubMed Google Scholar). One unit of activity is defined as the amount of enzyme that hydrolyzes 1 nmol of [1-14C]acetyl-CoA in 1 min. Yeast total proteins were prepared and subjected to Western blot analysis as described previously (26Lee F.-J.S. Huang C.-F., Yu, W.-L. Buu L.-M. Lin C.-Y. Huang M.-C. Moss J. Vaughan M. J. Biol. Chem. 1997; 272: 30998-31005Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cells were prepared for immunofluorescence staining as described by Huang et al.(25Huang C.-F. Buu L.-M., Yu, W.-L. Lee F.-J.S. J. Biol. Chem. 1999; 274: 3819-3827Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Alexa 594- or Alexa 488-conjugated anti-IgG antibodies (Molecular Probes, Eugene, OR) were used as secondary antibodies. H33258 was diluted in mounting solution for nucleic acid staining. Fluorescence microscopy was performed with a Nikon Microphot SA microscope. The monoclonal anti-yeast mitochondria porin antibodies were purchased from Molecular Probes. The monoclonal anti-yeast ribosomal protein Rpl3p antibody (also called anti-TCM1 antibody) was a gift of Dr. J. Warner (Albert Einstein College of Medicine, Bronx, NY), and the polyclonal anti-Kar2p antibody was a gift from Dr. M. Rose (Princeton University, Princeton, NJ). Polyclonal anti-porin antibody was diluted 1:20,000 for Western blot analysis and 1:5000 for immunofluorescence staining. The polyclonal antibodies against yeast Arf1p were generated by the use of recombinant proteins from our laboratory (26Lee F.-J.S. Huang C.-F., Yu, W.-L. Buu L.-M. Lin C.-Y. Huang M.-C. Moss J. Vaughan M. J. Biol. Chem. 1997; 272: 30998-31005Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Yeasts grown in selective minimal medium or YPD medium were harvested by centrifugation and washed once with 10 mm NaN3, before Lyticase digestion of cell walls in a solution of 1.2 m sorbitol and 100 mm potassium phosphate, pH 6.5. Spheroplasts were suspended in buffer containing 0.1 m sorbitol, 20 mmHEPES (pH 7.4), 50 mm potassium acetate, and 1 mm EDTA with protease inhibitors and disrupted on ice with 20 strokes in a Dounce homogenizer. The lysate was centrifuged (450 × g) to remove debris and unbroken cells. Cleared lysate (0.8 ml) was loaded on top of a manually generated six-step sucrose gradient (0.7 ml each of 60, 50, 40, 30, 20, and 107 sucrose in lysis buffer), which was then centrifuged at 170,000 ×g for 3 h in a Beckman SW55 rotor at 4 °C. Proteins in samples (100 ॖl) of fractions, collected manually from the top, were precipitated with 107 trichloroacetic acid, separated by SDS-PAGE, and analyzed by immunoblotting. Diluted antibodies against mitochondrial porin (1:500) (Molecular Probes), Kar2 (1:1000), Emp47 (1:5000), and Arf1p (1:5000) (26Lee F.-J.S. Huang C.-F., Yu, W.-L. Buu L.-M. Lin C.-Y. Huang M.-C. Moss J. Vaughan M. J. Biol. Chem. 1997; 272: 30998-31005Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) were used to identify organelles. The medium formula, cell treatments, and fractionated centrifugation were performed as described (27Elgersma Y. van Roermund C.W.T. Wanders R.J.A. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (164) Google Scholar, 28van Roermund C.W.T. Elgersma Y. Singh N. Wanders R.J.A. Tabak H.F. EMBO J. 1995; 14: 3480-3486Crossref PubMed Scopus (261) Google Scholar, 29Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (307) Google Scholar, 30Wilusz C.J. Gao M. Jones C.L. Wilusz J. Peltz S.W. RNA (N. Y.). 2001; 7: 1416-1424PubMed Google Scholar) and with some modifications. Wild-type yeast cells were cultured in 50 ml of synthetic medium containing 27 glucose at 30 °C with shaking overnight. Then the overnight culture was harvested and transferred into 100 ml of oleic acid-containing medium (0.37 yeast extract, 0.37 peptone, 0.17 oleic acid, 0.27 Tween 40, and 0.57 potassium phosphate) to induce the formation of peroxisomes. After 24 h, the yeast cells were harvested and washed once with 0.1 mpotassium phosphate buffer (pH 6.3). Then cells were suspended in 3 ml of spheroplast solution (1.2 m sorbitol, 0.1 mpotassium phosphate, pH 6.3), 150 ॖl of औ-mercaptoethanol was added, and the cells were kept at room temperature for 15 min with gentle rocking. After spinning down and resuspending in 3 ml of fresh spheroplast solution containing 30 ॖl of औ-mercaptoethanol, 1,000 units of lyticase were added. The cells were mixed gently and incubated at 30 °C for generation of spheroplasts. The spheroplasts were spun down, washed once in ice-cold spheroplast solution, and finally suspended in lysis buffer (0.6 m sorbitol, 5 mmMES, pH 6.0, 1 mm KCl, and 0.5 mm EDTA). The spheroplasts were lysed by passage through a 26-gauge needle 20 times and incubated on ice for 30 min. Unbroken cells and nuclei were removed by centrifugation at 1,500 × g for 5 min. The supernatant was centrifuged at 15,000 rpm for 15 min. The crude organelle pellet, consisting mainly of mitochondria and peroxisomes, was gently resuspended in lysis buffer and centrifuged at low speed (600 × g) to remove larger aggregates. The organelle suspension was loaded on a 14–367 Nycodenz gradient to further fractionate mitochondria and peroxisomes. The gradient was centrifuged at 32,500 rpm, 4 °C, for 3.5 h. After centrifugation, the sample was divided into 14 fractions from 14 to 367 Nycodenz gradients; then proteins were precipitated by 107 trichloroacetic acid and analyzed by Western blotting. Yeast was grown in YPGal to early stationary phase. Mitochondria were isolated as described previously (31Huang C.-F. Chen C.-C. Tung L. Buu L.-M. Lee F.-J.S. J. Cell Sci. 2002; 115: 275-282PubMed Google Scholar). Mitochondria were resuspended in lysis buffer to give an approximate final concentration of 10 mg of protein/ml. To isolate the mitochondrial intermembrane space, a suspension of mitochondria (10–20 mg of protein/ml in 0.6m sorbitol, 10 mm Tris, pH 7.4) was diluted with 5 volumes of 10 mm Tris, pH 7.4, to a final sorbitol concentration of 0.1 m. The suspension was incubated at 4 °C with gentle rocking for 20 min. The "shocked" mitochondria were sedimented at 20,000 rpm in a Beckman SW55 Ti rotor for 20 min. The supernatant contains the contents of the intermembrane space; then proteins were precipitated by 107 trichloroacetic acid and analyzed by Western blotting. To isolate mitochondrial membrane and matrix, shocked mitochondria were resuspended in 10 mm Tris, pH 7.4, to a protein concentration of about 2 mg/ml with five strokes in a Dounce homogenizer and left on ice to allow further swelling of the mitochondrial matrix space. After 5 min, "shrinking buffer" (one-third of the suspension volume) containing 1.8 msucrose, 8 mm ATP, 8 mm MgC12, adjusted to pH 7.4 with KOH, was added. The suspension was mixed carefully by three strokes in the Dounce homogenizer and left on ice. After 5 min, the suspension was exposed to ultrasonic irradiation for 3 × 5 s on ice. Total mitochondrial membranes were sedimented for 60 min at 35,000 rpm in a Beckman SW55 Ti rotor at 4 °C. The supernatant represents the matrix fraction; then proteins were precipitated by 107 trichloroacetic acid and analyzed by Western blotting. To characterize theACH1 gene product, we prepared a rabbit antiserum against anE. coli synthesized recombinant full-length His-tagged Ach1p fusion protein. Among total cellular proteins, antibodies prepared against Ach1p reacted only with a protein of ∼64 kDa, the expected size for Ach1p (Fig. 1). This protein was not detected in an ach1 mutant (Fig. 1) or by the preimmune serum (not shown). Immunoblotting with this antiserum detected nanogram amounts of Ach1p (data not shown) as well as various mutant forms of Ach1p (Fig. 1A). As in previous RNA blot analysis (15Lee F.-J.S. Lin L.-W. Smith J.A. J. Biol. Chem. 1990; 265: 7413-7418Abstract Full Text PDF PubMed Google Scholar), Ach1p was subjected to glucose-dependent repression (Fig.1B). A data base search showed significant homologies among Ach1p and other CoA-transferases, including Schizosaccharomyces pombe ACH1 (SpACH1; 647 identity), N. crassa Acu8 (NcAcu8; 577 identity), E. coli ACH1-like (EcCat1, GenBankTM accession U28377; 387 identity),A. aceti AarC (AarC; 397 identity), Clostridium kluyveri CAT1 (CkCat1, succinyl-CoA:coenzyme A transferase; 377 identity), and C. kluyveri CAT2 (CkCat2, butyryl-CoA-acetate Coenzyme A; 207 identity). The amino acid sequence of the CoA (ADP) binding site (GXGXX(G/A)) was reported from an analysis of the known three-dimensional structures of ADP binding औ-α-औ-folds (32Wierenga R.K. Terpstra P. Hol W.G. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (1019) Google Scholar). Fig. 2shows that the conserved CoA (ADP) binding site (GXGXX(G/A)) from heterodimeric CoA transferases are present in the amino acid sequences of Ach1p and homologous CoA-transferases (33Parales R.E. Harwood C.S. J. Bacteriol. 1992; 174: 4657-4666Crossref PubMed Google Scholar, 34Sohling B Gottschalk G. J. Bacteriol. 1996; 178: 871-880Crossref PubMed Google Scholar). To determine whether these putative nucleotide (CoA) binding sites are required for Ach1p activity, we generated two mutants, Ach1pSS and Ach1pES, by site-directed mutagenesis (as described under "Materials and Methods"). Wild-type Ach1p, Ach1pSS, and Ach1pES were expressed in the ach1mutant, and their expression was confirmed by Western blotting with anti-Ach1p antibody (Fig. 1A). Enzyme assays of protein extracts from these strains confirmed that Ach1pSS and Ach1pES expressed in the ach1 mutant contained little (<57) detectable acetyl-CoA hydrolyzing activity, whereas the untransformed wild type had normal enzyme activity (Fig. 1A). In addition, in comparison with wild-type yeast, overexpressing Ach1p in theach1 mutant had ∼3.8-fold of activity. Because of the low solubility of E. coli producing Ach1p recombinant proteins, we failed to isolate recombinant Ach1p and its mutant constructs to assess their enzymatic activity. Previously, we have shown that the ability of ach1 mutants to grow on acetate is impaired (17Lee F.-J.S. Lin L.-W. Smith J.A. Biochim. Biophys. Acta. 1996; 1297: 105-109Crossref PubMed Scopus (20) Google Scholar). We next tested whether overexpressed Ach1p and its mutant constructs have biological function in ach1 mutant yeast. Specific growth rates of tested strains (wild-type strain, ach1strain, ach1 strains overexpressing Ach1p, Ach1pES, and Ach1pSS) were obtained by the growth of cells in the synthetic medium containing acetate, and A600values were determined at specific time intervals. Overexpressing Ach1p in ach1 mutant can grow on acetate medium; however, the ability of the ach1 mutant and strains overexpressing Ach1pES and Ach1pSS to grow on acetate is impaired (data not shown). These data suggest that Ach1pES and Ach1pSS lose their biological activity in vivo. To study the subcellular distribution of Ach1p, the total yeast spheroplast-homogenized lysate was fractionated by 30–607 discontinuous sucrose gradient centrifugation. As shown in Fig.3A, distribution of most of Ach1p was similar to that of the mitochondrial protein porin, although little Ach1p was found in cytoplasmic fractions. To determine further whether Ach1p may also be present in peroxisomes, a homogenate of oleate-grown cells was first subjected to differential centrifugation to obtain an organellar pellet. This material was further fractionated by density gradient centrifugation on Nycodenz. Fig. 3Bshows good resolution between mitochondria (porin marker) and peroxisomes (thiolase marker), and the distribution of Ach1p was similar to that of the mitochondrial protein porin. By immunofluorescence microscopy, endogenous Ach1p, similar to porin, appeared to be localized to mitochondria (Fig. 3C). Yeast mitochondria can form branched networks distributed evenly around the circumference of the cell in the peripheral cytoplasm. Abnormal mitochondrial morphology was not seen in ach1 mutant yeast. To further localize the Ach1p within the purified mitochondria, we analyzed the intermembrane space, matrix, and membrane fractions (Fig.4). Ach1p cofractionated with Mge1p but not with cytochrome oxidase subunit IV and porin, indicating that it is localized to the mitochondrial matrix.Figure 4Ach1p is localized to mitochondrial matrix. The mitochondria of yeast were subfractionated as described under "Materials and Methods." Protein samples of submitochondrial fractions were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with specific antisera to detect the following proteins: membrane markers (Porin and cytochrome oxidase IV), matrix marker (Mge1p), and Ach1p protein. Mem, mitochondrial membrane proteins; IMS, mitochondrial intermembrane space;Matx, mitochondrial matrix; MW, molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Most proteins targeted to the mitochondrial matrix contain a cleavable N-terminal presequence with basic and hydroxylated amino acids interspersed throughout their length (35Schatz G. J. Biol. Chem. 1996; 271: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 36Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (985) Google Scholar). Although the N terminus of Ach1p contains no typical matrix-targeting sequence, we suspected that deletion of the N-terminal domain from Ach1p might interfere with its mitochondrial localization. After expression in the ach1mutant, Ach1pdN, lacking 64 amino acids at the N terminus, was recovered in the least dense fractions of the lysate (data not shown). In cells, most of the Ach1pdN mutant was in the cytoplasmic region with a punctate distribution (Fig. 5), and the mitochondrial morphology was similar to that in wild-type cells (Fig.5). In addition, fusion between the N terminus (64 amino acids) of Ach1p and GFP protein failed to be imported into mitochondria (data not shown). We further tested whether overexpression of Ach1p or its mutant constructs (Ach1pES and Ach1pSS) in yeast might cause dominant-negative effects on mitochondrial morphology. By immunofluorescence microscopy, Ach1pES and Ach1pSS, like overexpressed Ach1p, were present in some tubular or spherical structures that also stained with anti-porin antibody (Fig. 5). Thus, N terminus, but not putative Co-A binding sites of Ach1p, was required for Ach1p mitochondrial localization. Because most of Ach1p is localized in the mitochondria, we next examined whether Ach1p can affect mitochondrial function. Yeast, when cultured in glycerol medium, requires mature active mitochondria for oxidative metabolism and growth. Yeast grown in glucose initially does not need active, mature mitochondria, and the mitochondria are not well developed before being switched from anaerobic glucose fermentation to aerobic ethanol oxidation. The wild-type strain, ach1strain, and ach1 strains overexpressing Ach1p and its deleted or mutated constructs (Ach1pES, Ach1pSS, and Ach1pdN) were cultured overnight in synthetic medium containing glucose. Cells were harvested, suspended in double distilled H2O, subjected to serial dilution, and dropped onto glucose, acetate, glycerol, succinate, and ethanol media plates. Fig.6 shows that all strains grew on the glucose, glycerol, succinate, and ethanol media plates; however, theach1 mutant and the Ach1pES, Ach1pSS, and Ach1pdN expression strains did not grow in acetate medium plates. Moreover, the Ach1p-overexpressing cells grew as well as the wild-type yeast on acetate plates. These results suggested that Ach1p is not involved in cellular events in mitochondrial biogenesis, which is required for cells to grow in non-fermentable carbon sources. Diploid cells of the yeast S. cerevisiae undergo pseudohyphal differentiation in response to nutrient limitation (37Gimeno C.J. Ljungdahl P.O. Styles C.A. Fink G.R. Cell. 1992; 68: 1077-1090Abstract Full Text PDF PubMed Scopus (1004) Google Scholar). We have characterized the connection between acetyl-CoA changes and pseudohyphal growth. Wild-type, ach1/ach1 mutant, and ach1/ach1-overexpressing Ach1p yeast cells were grown in low ammonium sulfate (SLAD; 50 ॖm) medium to characterize their pseudohyphal differentiation. Importantly, we found that cells lacking the Ach1p were completely defective in pseudohyphal differentiation, whereas ach1/ach1-overexpressing Ach1p restores pseudohyphal growth (Fig.7). However, expression of Ach1p deleted or mutated constructs (Ach1pES, Ach1pSS, and Ach1pdN) failed to restore pseudohyphal growth (data not shown). In this study, we show that putative conserved nucleotide (CoA) binding sites and N terminus of Ach1p require its enzyme activity. Our data also show that Ach1p is localized to the mitochondria, and the N terminus of Ach1p is required for its localization. Finally, we show that Ach1p is not involved in mitochondrial biogenesis but may be involved in pseudohyphal differentiation. The amino acid sequences of Ach1p and homologous CoA-transferases contain two conserved CoA (ADP) binding sites (GXGXX(G/A)) from heterodimeric CoA transferases (Fig. 2) (9Soling H.-D. Rescher C. Eur. J. Biochem. 1985; 147: 111-117Crossref PubMed Scopus (25) Google Scholar, 33Parales R.E. Harwood C.S. J. Bacteriol. 1992; 174: 4657-4666Crossref PubMed Google Scholar). We determined whether these putative nucleotide (CoA) binding sites are required for Ach1p activity. Enzyme assays confirmed that Ach1pSS and Ach1pES expressed in the ach1 mutant contained little detectable acetyl-CoA hydrolyzing activity. We also showed that utilization of acetate as carbon source by theach1 mutant is impaired, and overexpression of Ach1pES, or Ach1pSS, cannot restore this activity. Our data indicate that one or more putative nucleotide (CoA) binding sites are required for Ach1p enzymatic activity. The subcellular localization of a protein is an important characteristic with functional implications. Our data show that most of Ach1p is localized to the mitochondria, although a little Ach1p was also found in small punctate form distributed in the cytoplasm. The majority of mitochondrial matrix-targeting signals are cleaved upon import into the mitochondria (36Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (985) Google Scholar). The matrix-located MTF1 protein in yeast, which is a transcription-stimulating factor (38Sanyal A. Getz G.S. J. Biol. Chem. 1995; 270: 11970-11976Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), is an exceptional case. This protein lacks a recognizable matrix-targeting sequence, and its import is reported to be independent of outer membrane receptors. How specificity of targeting is achieved in this case and whether there is an entirely separate pathway for importing this protein remain to be clarified. We demonstrate that N-terminal but not putative CoA binding sites of Ach1p are required for localization to mitochondria. Thus, translocation of Ach1p from the cytoplasm to the mitochondrial matrix may, like that of MTF1, require that the N-terminal sequence lacks a recognizable matrix-targeting signal. A recent report described that Ach1p has two different protein spots by two-dimensional gel electrophoresis (39Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Mol. Cell. Biol. 1999; 19: 1720-1730Crossref PubMed Scopus (3221) Google Scholar). These two Ach1ps have the same relative molecular weight but differ in their pI, suggesting that Ach1p might be modified post-translationally. However, we attempted, but failed, to confirm that there are two different protein spots of Ach1p by two-dimensional gel electrophoresis. 2L.-M. Buu, Y.-C. Chen, and F.-J. S. Lee, unpublished data. Carnitine acetyltransferase (CAT) is known to be present in mitochondria and peroxisomes of oleate-grown S. cerevisiae, and both proteins are encoded by the same gene, YCAT (27Elgersma Y. van Roermund C.W.T. Wanders R.J.A. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (164) Google Scholar). We also speculated whether Ach1p in oleate-grown cells might have a different subcellular localization. Our data showed that the majority of Ach1p is present in mitochondria but not in peroxisomes. Thus, we concluded that Ach1p is a mitochondrial enzyme and may execute its physiologic function in the matrix space. Contemporary knowledge of the structure and function of acetyl-CoA hydrolases (i.e. cytosolic (8Prass R.L. Isohashi F. Utter M.F. J. Biol. Chem. 1980; 255: 5215-5223Abstract Full Text PDF PubMed Google Scholar, 40Suematsu N. Okamoto K. Shibata K. Nakanishi Y. Isohashi F. Kraus T. Uttamsingh V. Anders M.W. Wolf S. Eur. J. Biochem. 2001; 268: 2700-2909Crossref PubMed Scopus (25) Google Scholar) and mitochondrial (5Robinson Jr., J.B. Mahan D.E. Koeppe R.E. Biochem. Biophys. Res. Commun. 1976; 71: 959-965Crossref PubMed Scopus (15) Google Scholar)) is incomplete. Ach1p resembles the rat mitochondrial acetyl-CoA hydrolase, is not affected by ADP or ATP, and is inhibited by औNADH (9Soling H.-D. Rescher C. Eur. J. Biochem. 1985; 147: 111-117Crossref PubMed Scopus (25) Google Scholar, 12Lee F.-J.S. Lin L.-W. Smith J.A. Eur. J. Biochem. 1989; 184: 21-28Crossref PubMed Scopus (16) Google Scholar). The Km of the Ach1p is similar to the mitochondrial acetyl-CoA hydrolase from hamster brown fat (6Bernson V.S.M. Eur. J. Biochem. 1976; 67: 403-410Crossref PubMed Scopus (37) Google Scholar,12Lee F.-J.S. Lin L.-W. Smith J.A. Eur. J. Biochem. 1989; 184: 21-28Crossref PubMed Scopus (16) Google Scholar). In addition, the pH optima for Ach1p and rat brain mitochondrial acetyl-CoA hydrolase are identical (pH ∼8) (5Robinson Jr., J.B. Mahan D.E. Koeppe R.E. Biochem. Biophys. Res. Commun. 1976; 71: 959-965Crossref PubMed Scopus (15) Google Scholar). Our data demonstrate that Ach1p is localized to mitochondria, consistent with the previous finding that the biochemical properties of Ach1p is similar to those of mitochondrial acetyl-CoA hydrolases. Diploid cells of the yeast S. cerevisiae undergo pseudohyphal differentiation in response to nutrient limitation (37Gimeno C.J. Ljungdahl P.O. Styles C.A. Fink G.R. Cell. 1992; 68: 1077-1090Abstract Full Text PDF PubMed Scopus (1004) Google Scholar). Studies on the way in which nitrogen and carbon starvation induce invasive and filamentous growth suggest multiple regulatory points in each pathway and cross-talk between the pathways (41Palecek S.P. Parikh A.S. Kron S.J. Microbiology (Oxf.). 2002; 148: 893-907Google Scholar). Changing any single component may rearrange metabolic fluxes in a manner that is difficult to predict. New approaches to metabolic system modeling and design are likely to contribute to identifying the particular components that signal invasion and filamentation and offer predictions for how to regulate the intracellular activities of those components. Our study showed that Ach1p was required for pseudohyphal formation, suggesting a physiologic connection between acetyl-CoA changes and pseudohyphal growth. In recent studies, sequence alignment of genes with similar regulation patterns revealed a putative regulatory promoter element (CCWTTSRNCCG) for the glyoxylate cycle (42van den Berg M.A. de Jong-Gubbels P. Steensma H.Y. Yeast. 1998; 14: 1089-1104Crossref PubMed Scopus (26) Google Scholar). This specific element was present in seven genes, including CIT2 (citrate synthase in peroxisomal matrix), ICL1 (isocitrate lyase), MLS1 (malate synthase in peroxisomal matrix), MDH2 (malate dehydrogenase in peroxisomal matrix), CAT2 (carnitine acetyltransferase in peroxisomal matrix and mitochondria), ACR1(succinate-fumarate transporter in mitochondrial inner membrane), andACH1, which were derepressed on ethanol or acetate. Consistent with this observation, three glyoxylate cycle genes,ICL1, MLS1, and MDH2, showed the same regulation pattern as ACH1 (42van den Berg M.A. de Jong-Gubbels P. Steensma H.Y. Yeast. 1998; 14: 1089-1104Crossref PubMed Scopus (26) Google Scholar). In addition,acu-8 mycelium exhibited no significant flux through the glyoxylate cycle 10 h after transfer to acetate. Thus, it is reasonable to speculate that Ach1p may be involved in the glyoxylate cycle. Recently, Lorenz and Fink (43Lorenz M.C. Fink G.R. Nature. 2001; 412: 83-86Crossref PubMed Scopus (590) Google Scholar) showed that live S. cerevisiae cells isolated from the phagolysosome are induced for genes of the glyoxylate cycle. These findings in fungi, in conjunction with reports that isocitrate lyase is both up-regulated and required for the virulence of Mycobacterium tuberculosis, demonstrate the wide ranging significance of the glyoxylate cycle in microbial pathogenesis. It will be interesting to learn whether Ach1p, similar to genes involved in the glyoxylate cycle, can be induced in phagolysosomes. The role of acetyl-CoA hydrolases catalyzing the scission of the high energy thioester bond acetyl-CoA with no apparent metabolic advantage represents a biochemical conundrum. Because acetyl-CoA hydrolase is highly expressed in yeast when media contain acetate, we suspect that, under conditions when acetate is used as the main carbon source, a large amount of acetyl-CoA is generated but not effectively incorporated into the trichloroacetic acid or glyoxylate cycle. Such an excess of acetyl-CoA could lead to autoacetylation of proteins, as well as to the generation of toxic ketone bodies or 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. Another interpretation is that the ACH1 enzyme, in vivo, is regulated by its associated factor, which can alter the Ach1p enzyme to be an acetyltransferase. It has been shown that the distinction between acyltransferases and thioesterases is quite narrow (44Witkowski A. Witkowska H.E. Smith S. J. Biol. Chem. 1994; 269: 379-383Abstract Full Text PDF PubMed Google Scholar). Moreover, acetyltransferases, in the absence of the acetyl acceptor, can transfer the acetyl group from acetyl-CoA to water and act as hydrolasesin vitro. Interestingly, a recent proteomic analysis showed that exposure of S. cerevisiae to sorbic acid in YEPD medium, pH 4.5, resulted in the up-regulation of 10 proteins, including Ach1p (45Nobel D.D. Lawrie L. Brul S. Klis F. Davis M. Alloush H. Coote P. Yeast. 2001; 18: 1413-1428Crossref PubMed Scopus (101) Google Scholar), suggesting that the induction of Ach1p may confer resistance to the inhibitory effects of sorbic acid. Furthermore, Ach1p was indicated to have high homologies (427 identity and 627 similarity) to C. kluyveri CAT1 (CkCat1, succinyl-CoA:coenzyme A transferase) and was suggested to be a succinyl-CoA:CoA transferase. However, the specific enzymatic activity of C. kluyveri CAT1 in recombinant E. coli clones was very low (9Soling H.-D. Rescher C. Eur. J. Biochem. 1985; 147: 111-117Crossref PubMed Scopus (25) Google Scholar). We attempted, but failed, to prove that Ach1p has such enzymatic activity.2 In conclusion, this study has confirmed that putative conserved nucleotide (CoA) binding sites of Ach1p are required for its enzyme activity in vivo. We also demonstrated that Ach1p is a mitochondrial enzyme, although its potential function in the glyoxylate cycle needs to be investigated further. We also showed that Ach1p is not involved in mitochondrial biogenesis, and our data suggest that the metabolism of acetyl-CoA by Ach1p is involved indirectly in pseudohyphal differentiation. Although yeast can use acetate or ethanol as carbon source by converting them to acetyl-CoA in the metabolic pathway, it will be interesting to know how ach1 mutants could impair acetate but not ethanol utilization. The exact physiologic role of this mitochondrial Ach1p needs to be investigated further. We thank Drs. T. Langer and M. Rose for providing us with antibodies. We thank Chih-Hsin Chen for preparing the anti-Ach1p antibody.
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