HFA1 Encoding an Organelle-specific Acetyl-CoA Carboxylase Controls Mitochondrial Fatty Acid Synthesis in Saccharomyces cerevisiae
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m401071200
ISSN1083-351X
AutoresUrsula Hoja, Sandra Marthol, Jörg Hofmann, Sabine Stegner, Rainer Schulz, Sandra Meier, Eva Greiner, Eckhart Schweizer,
Tópico(s)Fungal and yeast genetics research
ResumoThe Saccharomyces cerevisiae gene, HFA1, encodes a >250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Δ disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to 250-kDa protein, which is required for mitochondrial function. Hfa1p exhibits 72% overall sequence similarity (54% identity) to ACC1-encoded yeast cytoplasmic acetyl-CoA carboxylase. Nevertheless, HFA1 and ACC1 functions are not overlapping because mutants of the two genes have different phenotypes and do not complement each other. Whereas ACC1 is involved in cytoplasmic fatty acid synthesis, the phenotype of hfa1Δ disruptants resembles that of mitochondrial fatty-acid synthase mutants. They fail to grow on lactate or glycerol, and the mitochondrial cofactor, lipoic acid, is reduced to 95%) of cellular fatty acids. In contrast, mitochondrial FAS is structurally similar to the non-aggregated FAS enzymes found in most bacteria where each component activity is represented by a distinct protein (type II-FAS) (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar, 3Harington A. Herbert C.J. Tung B. Getz G.S. Slonimski P.P. Mol. Microbiol. 1993; 9: 545-555Crossref PubMed Scopus (57) Google Scholar, 4Schneider R. Brors B. Burger F. Camrath S. Weiss H. Curr. Genet. 1997; 32: 384-388Crossref PubMed Scopus (66) Google Scholar). Both FAS systems are encoded by nuclear genes. Although mutational loss of cytoplasmic FAS gives rise to a fatty acid-requiring phenotype (5Schweizer E. Werkmeister K. Jain M.K. Mol. Cell. Biochem. 1978; 21: 95-107Crossref PubMed Scopus (51) Google Scholar), mutants defective in one of the mitochondrial FAS-encoding genes are fatty acid-prototrophic but fail to grow on non-fermentative media (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar, 3Harington A. Herbert C.J. Tung B. Getz G.S. Slonimski P.P. Mol. Microbiol. 1993; 9: 545-555Crossref PubMed Scopus (57) Google Scholar, 4Schneider R. Brors B. Burger F. Camrath S. Weiss H. Curr. Genet. 1997; 32: 384-388Crossref PubMed Scopus (66) Google Scholar). In accordance with these findings, Brody et al. (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar) and Wada et al. (6Wada H. Shintani D. Ohlrogge J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1591-1596Crossref PubMed Scopus (173) Google Scholar) working with either yeast or plant systems suggest that a major function of mitochondrial FAS refers to lipoic acid synthesis by providing the octanoylacyl-carrier protein precursor of this cofactor. Because the product spectra of both FAS systems are very similar in vitro (7Rössler H. Rieck C. Delong T. Hoja U. Schweizer E. Mol. Gen. Genomics. 2003; 269: 290-298Crossref PubMed Scopus (45) Google Scholar), it remains to be shown whether, apart from octanoic acid, mitochondrially produced long-chain fatty acids serve a specific function as well. Neither octanoic nor lipoic acid is capable of healing the mitochondrial FAS defect when added to the growth medium (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar, 3Harington A. Herbert C.J. Tung B. Getz G.S. Slonimski P.P. Mol. Microbiol. 1993; 9: 545-555Crossref PubMed Scopus (57) Google Scholar). This suggests that either the uptake or the activation of these acids is impossible for mitochondria or intact yeast cells. In all of the known FAS systems, chain extension depends on malonyl-CoA as a substrate. Thus, carboxylation of acetyl-CoA to malonyl-CoA is a key step of fatty acid synthesis. The respective enzyme, acetyl-CoA carboxylase (ACC), comprises three functional components, i.e. biotin carboxylase, biotincarboxyl-carrier protein, and transcarboxylase (8Lane D.M. Moss J. Polakis S.E. Cell. Regul. 1974; 8: 139-194Google Scholar). Depending on the organism, these components represent either three distinct proteins or they are contained as functional domains within difunctional or trifunctional ACC proteins (9Toh H. Kondo H. Tanabe T. Eur. J. Biochem. 1993; 15: 687-696Crossref Scopus (55) Google Scholar). In yeast, cytoplasmic ACC is a single trifunctional polypeptide of 2233 amino acids and with an approximate molecular mass of 250 kDa (10Mishina M. Kamiryo T. Tanaka A. Fukui S. Numa S. Eur. J. Biochem. 1976; 71: 295-300Crossref PubMed Scopus (26) Google Scholar, 11Al-Feel W. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4534-4538Crossref PubMed Scopus (82) Google Scholar, 12Hasslacher M. Ivessa A.S. Paltauf F. Kohlwein S.D. J. Biol. Chem. 1993; 268: 10946-10952Abstract Full Text PDF PubMed Google Scholar). The ACC-encoding gene has been isolated and was designated as ACC1 and FAS3, respectively (11Al-Feel W. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4534-4538Crossref PubMed Scopus (82) Google Scholar, 12Hasslacher M. Ivessa A.S. Paltauf F. Kohlwein S.D. J. Biol. Chem. 1993; 268: 10946-10952Abstract Full Text PDF PubMed Google Scholar). Unlike fatty-acid synthase mutants, which grow upon fatty acid supplementation, ACC1 disruption is lethal and cannot be compensated by external long-chain fatty acids (12Hasslacher M. Ivessa A.S. Paltauf F. Kohlwein S.D. J. Biol. Chem. 1993; 268: 10946-10952Abstract Full Text PDF PubMed Google Scholar, 13Schneiter R. Hitomi M. Ivessa A.S. Fasch E.V. Kohlwein S.D. Tartakoff A.M. Mol. Cell. Biol. 1996; 16: 7161-7172Crossref PubMed Scopus (148) Google Scholar). This characteristic is commonly attributed to the malonyl-CoA requirement of cellular very long-chain fatty acid biosynthesis. Fatty acid elongation appears to be an essential cellular function not supplementable by exogenous very long-chain fatty acid. Other than ACC1 null mutants, however, ACC1 missense mutants are potentially viable and grow upon fatty acid supplementation. The low level of ACC activity eventually retained in some of these mutants obviously fulfills the limited malonyl-CoA requirement of very long-chain fatty acid synthesis (14Mishina M. Roggenkamp R. Schweizer E. Eur. J. Biochem. 1980; 111: 79-87Crossref PubMed Scopus (32) Google Scholar). These mutants therefore provide valuable tools for biochemical studies. Although Acc1p provides the malonyl-CoA used in cytoplasmic fatty acid synthesis, the origin of this substrate in mitochondrial fatty acid synthesis is unclear. Mitochondrial membranes are unlikely to be permeable for malonyl-CoA. In higher plant mitochondria, malonic acid was suggested to be imported into the organelle with subsequent activation and transacylation to acyl carrier protein (15Gueguen V. Macherel D. Jaquinod M. Douce R. Bourgignon J. J. Biol. Chem. 2000; 275: 5016-5025Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). On the other hand, fatty acid synthesis in plant chloroplasts relies on a specific organellar ACC, which is distinctly different from its cytoplasmic counter-part (16Ohlrogge J.B. Jaworski J.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 109-136Crossref PubMed Scopus (512) Google Scholar, 17Alban C. Baldet P. Douce R. Biochem. J. 1994; 300: 557-565Crossref PubMed Scopus (104) Google Scholar, 18Konishi T. Shinohara K. Yamada K. Sasaki Y. Plant Cell Physiol. 1996; 37: 117-122Crossref PubMed Scopus (227) Google Scholar). Similarly, identification of the unassigned yeast reading frame, HFA1, encoding a putative protein with striking similarity in its length and amino acid sequence to cytoplasmic ACC suggested the existence of a second and functionally differentiated acetyl-CoA carboxylase being present in this organism as well (19Kearsey S.E. DNA Seq. 1993; 4: 69-70Crossref PubMed Scopus (8) Google Scholar). For many years, however, the biochemical function of HFA1 remained elusive because hfa1Δ disruptants were, other than acc1 mutants, fatty acid-prototrophic and exhibited no obvious ACC deficiency. Accordingly, the fatty acid-requiring phenotype of ACC1 mutants was not compensated by functional HFA1 DNA (12Hasslacher M. Ivessa A.S. Paltauf F. Kohlwein S.D. J. Biol. Chem. 1993; 268: 10946-10952Abstract Full Text PDF PubMed Google Scholar, 13Schneiter R. Hitomi M. Ivessa A.S. Fasch E.V. Kohlwein S.D. Tartakoff A.M. Mol. Cell. Biol. 1996; 16: 7161-7172Crossref PubMed Scopus (148) Google Scholar). A first clue to the physiological function of HFA1 came from an observation in our laboratory that HFA1 mutants failed to grow on non-fermentable carbon sources. As will be demonstrated in the present study, the HFA1 gene product is located in the mitochondria and the phenotype of HFA1 mutants conforms to that of yeast mitochondrial FAS mutants. For complementation of hfa1 null mutants, the putative mitochondrial targeting sequence of HFA1 proved indispensable. On the other hand, signal-free HFA1 DNA restored cytoplasmic ACC activity in acc1 missense mutants. From these findings, it is concluded that HFA1 encodes a mitochondrial acetyl-CoA carboxylase providing malonyl-CoA for organellar fatty acid biosynthesis. Yeast Strains, Plasmids, and Media—The Saccharomyces cerevisiae strain X2180-1A (R. Mortimer, Yeast Genetic Stock Center, Berkeley, CA) served as a haploid wild type reference. The ACC1-missense mutant acc1-318/45 was from our own collection (14Mishina M. Roggenkamp R. Schweizer E. Eur. J. Biochem. 1980; 111: 79-87Crossref PubMed Scopus (32) Google Scholar) The ACC1/acc1Δ heterozygous diploid, Y25391, was obtained from Euroscarf (Frankfurt, Germany). The diploid JS95.3 (MATa/α his3/his3 ura3/URA3 leu2/LEU2) was from H.-J. Schüller (Greifswald, Germany) and served for chromosomal disruption of HFA1. Plasmid pSE303 containing the N-terminal portion of HFA1 together with its 5′-flanking chromosomal DNA was obtained from S. E. Kearsey (20Kearsey S.E. Edwards J. Mol. Gen. Genet. 1987; 210: 509-517Crossref PubMed Scopus (18) Google Scholar). Plasmid YEp6 (21Botstein D. Falco S.C. Stewart S.E. Brennan M. Scherer S. Stinchcomb D.T. Struhl K. Davis R.W. Gene (Amst.). 1979; 8: 17-24Crossref PubMed Scopus (548) Google Scholar) was used for PCR amplification of S. cerevisiae HIS3-DNA. Plasmid p48.8.3 containing intact ACC1-DNA was isolated from a YEp24-based yeast gene bank by acc1-mutant complementation (Dr. Lilian Schweizer). For HFA1 disruption, the 2684-bp XbaI fragment of pSE303 was subcloned into pUC19 giving plasmid pRSHFA1. In pRSHFA2, the 386-bp EcoRI/SstI fragment was replaced by S. cerevisiae HIS3 DNA, which was prepared by PCR amplification from YEp6 (cf. Fig. 1). From the resulting construct, the inserted HIS3 gene together with its flanking HFA1 sequences was isolated as NcoI/SpeI fragment. Using the one-step gene disruption procedure of Rothstein (22Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2014) Google Scholar), the NcoI/SpeI fragment served for disruption of chromosomal HFA1 DNA in the diploid JS95.3 (Fig. 1). Thus, the heterozygous diploid SC1343 (MATa/α, hfa1Δ::HIS3/HFA1 his3/his3 ura3/URA3 leu2/LEU2) was obtained. The haploid HFA1 null mutant SC 1517 (MATα Δhfa1::HIS3 his3) was a meiotic segregant of SC1343. The multicopy yeast expression vector, pVT100-U, of Vernet et al. (23Vernet T. Dignard D. Thomas D.Y. Gene (Amst.). 1987; 52: 225-233Crossref PubMed Scopus (461) Google Scholar) served as a recipient of the HFA1 coding sequence in pHFA1. The latter plasmid was restituted from three different PCR fragments representing nucleotides –216 to 1974 (fragment 1), 1975–3885 (fragment 2), and 3886–6423 (fragment 3) relative to the first ATG codon in the HFA1 reading frame. In pHFA1, an in-frame ATG start codon in combination with the ADH1 promoter was contributed by the vector (cf. Fig. 2). Fragments 1–3 were flanked by HindIII/SpeI (fragment 1), SpeI/XhoI (fragment 2), and XhoI/NheI (fragment 3) restriction sites, respectively, allowing their appropriate ligation and integration into the vector. A second HFA1 expression plasmid, pMA1, was prepared accordingly but using a C-terminally hexahistidine-tagged fragment 3 (Fig. 2). Another HFA1 expression plasmid, pMA3, contained the complete HFA1 reading frame in combination with its genuine promoter sequence and a C-terminal hexahistidine-encoding nucleotide sequence. According to Fig. 2, pMA3 was obtained by inserting the XbaI fragment of pMA1 into the unique XbaI site of pMA2. In pMA2, a 589-bp PCR fragment of the HFA1 upstream region had been cloned into the yeast expression vector Yep 352 (24Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1078) Google Scholar). According to Fig. 3, the HFA1 expression plasmid pUH 33.3 was constructed from pMA3 by replacing the 5′-terminal 3439-1 bp HindIII/SphI fragment of the inserted HFA1 DNA by a shorter 2779-bp PCR-generated HindIII/SphI fragment from the same region. The resulting construct, pUH32, was used for inserting the 846-bp HindIII/HindIII PCR fragment of the ACC1 promoter DNA into the unique HindIII site of pUH32 (cf. Fig. 3). The inserted DNA comprises the first 11 bp of the ACC1 reading frame together with 835 bp of its 5′-flanking upstream region.Fig. 2HFA1 constructs used for expression studies. Details of the plasmid construction protocols are described under "Experimental Procedures." HFA1 coding sequences are indicated in white. Non-coding HFA1, ADH1, or ACC1 upstream DNA sequences are shaded and marked appropriately. Numbering refers to the first ATG codon in the HFA1 reading frame (black asterisk). ATG initiation codons provided by the ADH1 or ACC1 promoter sequences are indicated as white asterisks. The C-terminal hexahistidine tag in pMA3, pMA1, and pUH33.3 is indicated in black.View Large Image Figure ViewerDownload (PPT)Fig. 3pUH33.3 construction scheme. The HindIII/SphI and HindIII fragments incorporated into pMA3 and pUH32, respectively, were generated by PCR amplification from appropriate plasmid DNAs. HFA1 coding sequences are white. Indication of non-coding HFA1 and ACC1 DNA regions as well as the numbering conforms to Fig. 2.View Large Image Figure ViewerDownload (PPT) Yeast cells were routinely grown on complex YPD medium containing 1% yeast extract (Invitrogen), 2% peptone (Invitrogen), and 2% dextrose. Respiratory competence was examined on complex medium containing, alternatively, 3% glycerol (YPG), 3% lactate (YPL), or 3% ethanol (YPE) instead of dextrose. The fatty requirement of yeast mutants was analyzed by replica plating from fatty acid containing YPDFA plates (YPD supplemented with 0.5% Tween 40 and 0.015% each of 14:0 and 16:0 fatty acids) onto YPD agar. The uracil-free (SCD/-U) or histidine-free (SCD/-H) synthetic complete medium used for selecting and identifying pHFA1 transformants and hfa1Δ disruptants, respectively, was prepared as described previously (25Harrer R. Schwank S. Schüller H.J. Schweizer E. Curr. Genet. 1993; 24: 136-140Crossref PubMed Scopus (14) Google Scholar). RT-PCR Amplification of HFA1-mRNA—Total RNA from yeast strain X2180-1A grown in YPD medium was extracted with TRIzol (Invitrogen) as described by the manufacturer. 10-μg RNA fractions were incubated with 1 unit of RNase-free RQ1-DNase (Promega) for various time intervals ranging from 0 to 35 min. 0.5 μg of DNase-treated RNA was reverse-transcribed for 50 min at 42 °C with 1 unit of Superscript II reverse transcriptase (Invitrogen) and 0.5 μmHFA1-specific primer I (cf.Fig. 4). The reaction mixture contained in a volume of 20 μl, 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 0.5 mm dNTP, and 1 mm dithiothreitol. 2 μl of the cDNA synthesis mixture were subsequently applied to PCR amplification using in a total volume of 50 μl the Expand High Fidelity PCR system (Roche Applied Sciences) and 200 nm of primers. 30 cycles of 94 °C denaturing (1.5 min), 52 °C annealing (2 min), and 72 °C extension (2 min) were run in an automated thermal cycler (Biometra, Göttingen, Germany). The following primers were used: primer A, 5′-atcgaggcaatattcataaac-3′; primer B, 5′-aaagggaagacaattacacac-3′; primer C, 5′-ACGCGTCGACttaagggtaactggg-3′; primer D, 5′-ACGCGTCGACagtaataatagcatcattcatc-3′; primer E, 5′-ACGCGTCGACctatcgctagctttccac-3′; primer F, 5′-ACGCGTCGACgcattctgaaagtgagatag-3′; primer G, 5′-ATCAGTCGACgcgacagccaaacgaggccc-3′; primer H, 5′-ATCAGAATTCcaccgcagcaataccattg-3′; primer I, 5′-agttcgtcttgttatcgata-3′; and primer J, 5′-tattttcctttgtgaactagc-3′. Capitals letters indicate non-HFA1 sequences, HFA1 sequences are indicated in small letters, and restriction sites are underlined. 5 μl of each reaction mixture were applied to 1% agarose gel electrophoresis. The synthetic oligonucleotides used in this study were supplied by MWG-Biotech (Ebersberg, Germany). Acetyl-CoA Carboxylase Assay—Cells were grown in 500 ml of uracil-free SCD/-U liquid medium containing 0.5% Tween 40 and 0.15% of each of the 14:0 and 16:0 fatty acids. After harvesting at early stationary phase, the wet cells (∼2 g) were resuspended in 1 volume of extraction buffer (0.3 m sorbitol, 0.1 m NaCl, 5 mm MgCl2, 10 mm Tris-HCl, pH 7.4) containing the protease inhibitors (Sigma) chymostatin (10 ng/ml), aprotinin (200 ng/ml), pepstatin A (100 ng/ml), leupeptin (50 ng/ml), antipain (250 ng/ml), p-benzamidine (50 ng/ml), and phenylmethanesulfonyl fluoride (20 ng/ml). After cell breakage with glass beads, cell debris were removed by 5-min centrifugation at 5000 rpm. Acetyl-CoA carboxylase was precipitated from the supernatant by 30% saturation with ammonium sulfate. The protein pellet was dissolved in 0.1 m potassium phosphate buffer (pH 7.5), and ACC activity was assayed spectrophotometrically in combination with purified fatty-acid synthase essentially as described by Matsuhashi (26Matsuhashi M. Methods Enzymol. 1969; 14: 3-8Crossref Scopus (16) Google Scholar). In detail, the procedure was as follows. 1 ml of assay mixture contained 50 μmol of cysteine, 0.3 mg of bovine serum albumin, 2 μmol of ATP, 8 μmol of MgCl2, 0.4 μmol of NADPH, 50 μg of purified yeast fatty-acid synthase, and 10–30 μl (20–40 μg) of the ACC preparation. The reaction was started by adding 20 μmol of acetyl-CoA. Subsequently, the decrease of absorbance at 334 nm was followed. Yeast FAS was purified as described by Lynen (27Lynen F. Methods Enzymol. 1969; 14: 17-33Crossref Scopus (147) Google Scholar). Demonstrating HFA1 Transcription and Mapping the Transcriptional Start Site—Using total RNA from S. cerevisiae wild type cells as a template, HFA1 transcription was verified by RT-PCR amplification of the HFA1 transcript (Fig. 4). Using appropriate controls, it was shown that formation of the PCR product was strictly dependent on the addition of reverse transcriptase. From this result and from the fact that product formation proved to be insensitive to DNase pretreatment, it is evident that RNA, rather than traces of residual DNA, served as a template (Fig. 4A). Using two different HFA1-specific upstream primers (A and B) in combination with the same downstream primer (J), the size difference of the resulting products was according to expectation (Fig. 4A). The cDNA thus prepared from the 5′ end of HFA1-mRNA subsequently served as a template for a series of PCR experiments. These "primer walking" experiments were designed to map the 5′ end of HFA1-mRNA. When the same downstream primer (H) was used in combination with six differently located upstream primers (B–G), only the combinations containing upstream primers B–E generated PCR products of the expected successively increasing lengths (Fig. 4, B and C). In contrast, the two most distal primers, G and F, were ineffective in the PCR assay (Fig. 4B). Therefore, the transcriptional initiation site of HFA1 is suggested to be located at a position between 607 (primer E) and 633 nucleotides (primer F) upstream of the first ATG in the HFA1 coding sequence (cf. Figs. 2 and 4C). This corresponds to a distance of 152–183 nucleotides upstream of the last stop codon in front of the HFA1 reading frame (Figs. 2 and 4C). HFA1 Disruptants Are Respiratory Defective and Fail to Synthesize Lipoic Acid—One of the two HFA1 alleles in the diploid strain, JS95.3, was disrupted by inserting the S. cerevisiae HIS3 gene according to the scheme depicted in Fig. 1. After sporulation of the resulting heterozygous diploid, SC1343, HFA1 disruptants were isolated by tetrad analysis and subsequently characterized both biochemically and by their growth requirements. The mutants proved to be viable on glucose-containing medium and segregated according to their histidine-prototrophic character in a regular Mendelian fashion (Fig. 5, A–C). Other than mutants of the sequentially related ACC1 gene, growth of HFA1 disruptants was independent of fatty acid supplementation (Fig. 5A). However, when cultivated on non-fermentable carbon sources such as lactate, glycerol, or ethanol, HFA1 disruptants exhibited the characteristics of respiratory-defective "petite" (Fig. 5, B, D, and E). Thus, HFA1 appears to be necessary for maintaining functional mitochondria in yeast. When different non-fermentative carbon sources were compared, growth of HFA1 mutants on lactate was strictly negative, whereas growth on ethanol was retarded although not completely abolished (Fig. 5, D and E). In this respect, HFA1 mutants differ from yeast mutants defective in one of the respiratory chain or ATP synthase functions. Instead, they rather resemble the recently described mutants of mitochondrial fatty acid synthesis lacking the mitochondrial cofactor, lipoic acid (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar). To confirm the similarity between HFA1-defective and mitochondrial FAS-defective mutants further, several meiotic segregants of the hfa1Δ/HFA1 heterozygous diploid, SC1343, were analyzed for their lipoic acid content. As is indicated in Table I, cellular lipoic acid was indeed reduced drastically in hfa1Δ disruptants. This finding compares well to the lipoic acid deficiency observed in mitochondrial FAS-defective acp1Δ or ppt1Δ mutants (2Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (130) Google Scholar, 28Stuible H.P. Meier S. Wagner C. Hannappel E. Schweizer E. J. Biol. Chem. 1998; 273: 22334-22339Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To definitely correlate these characteristics to the loss of HFA1 function, hfa1Δ disruptants were transformed with the multicopy HFA1 expression plasmids, pHFA1 and pMA3, respectively. For transformation, both the heterozygous diploid, SC1343, and the haploid hfa1Δ-disruptant, SC1517, were used as recipients. It turned out that the extrachromosomal HFA1 DNA of either plasmid complemented the lactate-negative phenotype of the HFA1 disruption both in haploid transformants (Fig. 8) and in the meiotic segregants of the transformed diploid (data not shown). From these findings, it is evident first that the mutant phenotype is indeed related to the loss of HFA1 function. Secondly, HFA1 was functionally expressed both with its genuine promoter and with the heterologous ADH1 promoter when the latter was fused in pHFA1 to an artificial ATG codon 220 nucleotides upstream of the first ATG codon of the HFA1 reading frame (Fig. 8). Thirdly, the respiratory-defective hfa1Δ-mutant (SC1517) used for these transformations obviously had not undergone a secondary Rho– mutation, otherwise complementation by intact HFA1 DNA would not have been observed.Table ILipoic acid content of wild type and HFA1-disrupted yeast cellsStrainCellular lipoic acid contentmg/g cellsSC1343-derived spore tetradSpore A0.44 ± 0.02Spore B0.10 ± 0Spore C0.12 ± 0.01Spore D0.55 ± 0.17X2180-1A0.50 ± 0.09 Open table in a new tab Fig. 8Complementation of hfa1Δ disruptants by intact but not by N-terminally truncated HFA1 DNA. SC 1517 cells were transformed with the indicated plasmids. Transformants were selected on uracil-free SCD/-U medium and subsequently grown for 4 days at 30 °C on either glucose (YPD) or lactate-containing (YPL) complex medium. Plasmids were as indicated under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT) In Vivo Expression of HFA1 and Mitochondrial Localization of Its Gene Product—The respiratory-defective phenotype of hfa1Δ mutants suggested that HFA1 controls mitochondrial function and, hence, encodes a mitochondrial protein. Inspection of the amino acid sequence immediately following the first ATG codon in the HFA1 reading frame revealed ∼90% of identical or equivalent positions compared with cytoplasmic acetyl-CoA carboxylase. No obvious mitochondrial import signal is contained in this region. However, if the additional 150 codons of the 5′-flanking and stop codon-free portion of HFA1 upstream of the first ATG triplet were included in this analysis, the characteristics of a typical yeast mitochondrial import signal became apparent within this sequence (cf. Fig. 6) (29Hendrick J.P. Hodges P.E. Rosenberg L.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4056-4060Crossref PubMed Scopus (262) Google Scholar). Besides many hydrophobic and hydroxy amino acids, this extended N-terminal part of Hfa1p comprises 27 basic and only 3 acidic amino acids. Furthermore, a putative signal peptide cleavage site is as predicted by the algorithm of Claros and Vincens (30Claros M.G. Vincens P. Eur. J. Biochem. 1996; 241: 779-786Crossref PubMed Scopus (1357) Google Scholar) maps between the HFA1-specific leader and the subsequent ACC1-homologous Hfa1p sequence (cf. Fig. 6). To confirm the possible mitochondrial function of Hfa1p, we tested both the expression of HFA1 and the intracellular localization of its product biochemically. For this experiment, yeast cells were transformed with pMA3 encoding hexahistidine-tagged Hfa1p. After growth to mid-log phase, cells were broken and the homogenate was fractionated into cytoplasm and purified mitochondria. Subsequently, both cell fractions were analyzed by Western blotting for the presence of Hfa1p. As is evident from Fig. 7B, anti-pentahistidine antibodies elicited a strong signal with the mitochondrial fraction but not with the cytoplasm. The gel position of the signal conformed to a protein of a molecular mass of >250 kDa. A second signal corresponding to approximately half of this size probably represents a defined degradation product of Hfa1p. A comparable signal was not obtained with cells that had been transformed with the empty vector (Fig. 7B, lane 3). A minor band of <75 kDa observed with
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