Isolation and nucleotide sequence of the Saccharomyces cerevisiae gene for the succinate dehydrogenase flavoprotein subunit.

Succinate dehydrogenase (EC 1.3.99.1) of the mitochondrial inner membrane is a four-subunit membrane-bound enzyme that catalyzes the oxidation of succinate to fumarate and the transfer of electrons into the electron transport chain to oxygen. The catalytic domain of the enzyme is composed of a flavoprotein subunit which contains a covalently attached FAD cofactor and an iron-sulfur subunit with three nonidentical iron-sulfur clusters. We have isolated a complete genomic clone for the flavoprotein subunit of the succinate dehydrogenase from Saccharomyces cerevisiae and determined its nucleotide sequence. The sequence predicts a protein of 70,185 Da (640 amino acids) that shows more similarity to the Escherichia coli succinate dehydrogenase flavoprotein subunit than it does to the only other mitochondrial homologue, the human flavoprotein subunit. The yeast flavoprotein subunit precursor was synthesized in a cell-free translation system and shown to possess a mitochondrial targeting sequence that directs its import into isolated, energized mitochondria where it is processed by the matrix-localized protease. The genes for the flavoprotein and the iron-sulfur subunits reside on different chromosomes and hence form different transcriptional units.

membrane protein complexes. All four subunits are nuclearencoded, cytosolically synthesized, and transported into the mitochondrion for assembly. The succinate dehydrogenase flavoprotein subunit will provide an opportunity to investigate the mechanism and temporal sequence of cofactor attachment with respect to transmembrane transport. It has been suggested that mammalian mitochondria may contain an enzyme responsible for the modification of several covalent flavoproteins (Lang et al., 1991). Alternatively, the covalent modification of a mitochondrial precursor in the cytosol has been shown not to inhibit import (Taroni and Rosenberg, 1991).
Recently, the role of covalent flavin attachment has been investigated by the isolation of site-directed mutants of His-44 of the Escherichia coli fumarate reductase (Blaut et al., 1989). The covalent linkage of the FAD considerably raises its redox potential with respect to free FAD. Fumarate reductase containing noncovalently bound FAD loses the ability to act as a succinate dehydrogenase while retaining considerable fumarate reductase activity. The covalent interaction between the enzyme and its cofactor appears to be necessary to modulate the redox properties of the FAD and enable the enzyme to act as a succinate dehydrogenase.
In this paper, we describe the isolation of a complete genomic clone for the flavoprotein subunit gene, SDHA, by screening a plasmid library and by complementation of a mutant containing a targeted gene disruption of this gene. This is the first complete nucleotide sequence of a eucaryotic SDHA gene to be determined; it predicts a hydrophilic protein of 70,185 Da bearing strong resemblance to the flavoprotein subunits of other succinate dehydrogenases and fumarate reductases. The in vitro expressed protein is synthesized as a precursor that can be imported into isolated, energized mitochondria. Gene mapping indicates that at least two of the succinate dehydrogenase subunits are located on different chromosomes.

EXPERIMENTAL PROCEDURES
Strains, Vectors, and Media-The Saccharomyces cerevisiae and E, coli strains, as well as media, have previously been described (Robinson et al., 1991) except D273-10B (MATa; ATCC 25657). The vectors pRS416 and pBluescriptl1 were obtained from Stratagene (La Jolla, CA).
Cloning and Sequencing-A 0.95-kb' polymerase chain reaction product (Robinson et al., 1991) was labeled with digoxigenin-dUTP as described by the manufacturer (Boehringer Mannheim), and used as a probe to screen 4000 colonies of a yeast genomic library in the vector YCp50 (Rose et al., 1987). DNA sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using both single-and double-stranded template DNA (Mierendorf and Pfeffer, 1987). Exonuclease digestions for the production of nested deletions were performed according to the supplier (Stratagene). All The abbreviations used are: kb, kilobase; SDS, sodium dodecyl sulfate. sequences were determined using information obtained from both strands of the DNA.
Mitochondrial Protein Import-Mitochondria were isolated and import experiments performed as described (Gasser et al., 1982). Coupled in vitro transcription and translation in rabbit reticulocyte lysate was performed as described by the supplier (Promega Corp., Madison, WI) using Tran"'S-Label (ICN Biomedicals, St. Laurent, Quebec). Published procedures were used to assay inaccessibility to externally added proteinase K (Hurt et al., 1985) and for polyacrylamide gel electrophoresis and fluorography (van Loon et al., 1986).
Other Methods-Enzyme assays, Southern and Western blot analyses, and the determination of covalently bound flavin, transformations, and recombinant DNA methods have been described (Robinson rt al., 1991).

RESULTS AND DISCUSSION
Isolation and Subcloning of the SDHA Gene-A yeast genomic library was screened by colony hybridization with a previously isolated partial gene fragment that had been labeled with digoxigenin-dUTP in a polymerase chain reaction (Robinson et al., 1991). Of approximately 4000 colonies, three demonstrated strong hybridization. Plasmid DNA from each of the positives was isolated and further examined by restriction mapping; the three plasmids were identical. The SDHA gene was localized to a 3.5-kb BamHI fragment by Southern analysis. This fragment was cloned into the vector, pRS416, a n autonomously replicating, single-copy yeast-E. coli shuttle vector carrying the yeast-selectable marker, URA3, to produce the plasmid, pSDHA. In the following experiments, pRS416 and pSDHA were found to be retained by a t least 70% of the cells in each culture.
Complementation of a n SDHA Mutant-To determine whether pSDHA encoded a functional gene, sdhAGL, a mutant constructed by targeted gene disruption of the SDHA gene, was transformed with the plasmid. sdhA6L grows very slowly on glycerol; however, sdhA6L transformed with pSDHA displayed growth similar to MH125, the parental wild type strain (data not shown).
Western Blot Analysis-Mitochondria from sdhA6L transformed with either pSDHA or the pRS416 vector were isolated and analyzed by Western blot analysis. The SDHA polypeptide was not detectable in the mutant transformed with pRS416, but was clearly evident as a 67-kDa protein in the cells bearingpSDHA ( Mitochondria from sdhA6L transformed with the plasmids pRS416 (-) or with pSDHA (+) were isolated as described (Daum et al., 1982). Mitochondrial protein (50 pg per lane) was separated by SDS-gel electrophoresis, transferred to nitrocellulose membranes, and analyzed by Western blot analysis. ance of the iron-sulfur subunit (Fig. 1B).
pSDHA Restores Succinate Dehydrogenase Activity to sdhAGL-Membranes were isolated from the parental wildtype strain, MH125, and from sdhA6L transformed with either pRS416 or pSDHA. Succinate-dichlorophenol indophenol reductase activity is not detectable in membranes from the mutant plus vector, but is comparable in wild type membranes and membranes from the mutant plus pSDHA (Table  I).
pSDHA Restores Trichloroacetic Acid-precipitable Flavin Levels in sdhAGL-The only yeast protein with a covalently attached flavin is the succinate dehydrogenase flavoprotein subunit (Singer et al., 1965). Therefore, the amount of trichloroacetic acid-precipitable flavin is a direct measure of succinate dehydrogenase content. While membranes from the mutant plus vector have no trichloroacetic acid-precipitable flavin above background levels, membranes from the mutant plus pSDHA contain levels comparable to those of wild type membranes ( Table I).
The genomic DNA insert in pSDHA contains a functional copy of the succinate dehydrogenase flavoprotein subunit gene; the plasmid hybridizes to a partial clone we had previously isolated (Robinson et al., 1991), is able to complement an SDHA mutant, and restores succinate dehydrogenase activity, polypeptides recognized by antisera to the SDHA and SDHB subunits, and the levels of covalent FAD detected in submitochondrial membranes.
Nucleotide Sequence Determination-The nucleotide sequence of the pSDHA insert was determined by sequencing cloned restriction fragments and nested deletions constructed by exonuclease digestion. Sequence determination was performed with M13 universal primers and on one occasion, with a specifically designed oligonucleotide primer. Sequence was determined from both strands for the entire open reading   The reading frame identified as SDHA starts with a methionine initiation codon at nucleotide +1 and terminates with an ochre codon at nucleotide +1921. Only the sequence of the sense strand is shown. The amino acid sequence of the succinate dehydrogenase flavoprotein subunit is shown below the nucleotide sequence. Shown in bold type are two potential TATAA boxes and underlined is a possible polyadenylation signal.

T T T A T T T A T T I I C T T T C I U T~~G C A T T T C C T T~~~T T C T C C A A T T T T~T K T C A T T A G C C A G A T G T G~~T T T K T G G C C C K A~T
GAGCAGGAGARAGTCATATGGCGAACGTAMTATGTIUCT~TTAAGATGGGCAGACATTTATCATTTTGCTTATGACTAMCCGC~TTGCTGTA CAAGGGTGCTGTCATGGTCAGCTAMCCAR~TTTAT~GAA   (Brutlag et al., 1990). The values represent percentage identities (matches/matches + mismatches + gaps). The predicted size of the SDHA polypeptide precursor is 70,185 Da, and as expected, it displays considerable sequence similarity with other flavoprotein subunits of this family (Table 11). The yeast SDHA subunit is most closely related to its E. coli homologue. Surprisingly, it is least similar to the only other mitochondrially derived flavoprotein subunit, a partial sequence from a human cDNA library (Malcovati et al., 1991). This is in marked contrast to the case for the ironsulfur subunits of yeast and human where extensive sequence identity (68.7%) occurs (Gould et al., 1989). However, the human sequence does not contain the amino terminus of the polypeptide with the highly conserved regions involved in flavin attachment and binding. Despite the low similarity of the yeast sequence to its human counterpart, it is clear that pSDHA encodes the S. cerevisiae SDHA gene.
The alignment of the yeast SDHA polypeptide sequence with related flavoprotein subunits is displayed in Fig. 4. The FAD cofactor of the bovine succinate dehydrogenase is covalently bound via the &-methyl group of the isoalloxazine ring to the N(3) of a histidine (Singer and Edmondson, 1974). The sequence of a 23-residue flavopeptide from this enzyme was determined (Singer et al., 1973), and comparison with the yeast sequence reveals that 17 of 23 positions are identical; this identifies His-90 as the probable site of flavin attachment in the yeast protein. In contrast to the other flavoprotein subunits listed in Fig. 4, this histidyl residue is not the first in the yeast sequence, but is preceded by His-47 and His-51. This observation is inconsistent with the notion that FAD is attached cotranslationally to the first histidine residue en-countered, but rather suggests that some minimal tertiary structure is necessary to accommodate the cofactor (Hamm and Decker, 1978) or that the process is posttranslational (Cecchini et al., 1985). The role of the presequence in flavin attachment to a mitochondrially localized protein is under investigation.
From comparisons with glutathione reductase, a noncovalent flavoprotein for which three-dimensional structural data exist, the succinate dehydrogenase and fumarate reductase flavoprotein subunits are believed to interact noncovalently with the AMP moiety of the FAD in two regions (Schulz et al., 1982;Wierenga et al., 1983;Cole et al., 1985). One such region contains a Rossman nucleotide binding fold which contacts the bottom of the AMP and is located near the amino terminus. The second region appears in the center of the polypeptide and forms the top of the AMP binding domain (Fig. 4). The residues in these regions, along with those near the site of FAD attachment, are among the most conserved.
It is believed that a histidine residue plays an important role in the function of the fumarate-succinate oxidoreductases. This residue is conserved in the sequence His-Pro-Thr present in all flavoprotein subunits except the yeast subunit where this sequence is His-Pro-Ser (residues 287-289). Near this sequence are the putative active site cysteine residues of the E. coli SDHA (Cys-257) and E.  subunits (Wood et al., 1984). The analogous cysteine is conserved in the Proteus vulgaris FRDA (Cys-248; Cole, 1987) and the Wollinella succinogenes FRDA (Cys-272;Lauterbach et al., 1990), but not in the Bacillus subtilis or the human SDHAs where alanines are located; it is for this reason that the B. subtilis enzyme is believed to be insensitive to thiolreactive reagents (Phillips et al., 1987). In the yeast sequence, the analogous residue is Ala-302; however, there is a cysteine residue nearby (Cys-296) that may account for the enzyme's sensitivity to p-chloromercuribenzoate (Singer et al., 1957).
Import into Isolated Mitochondria-The SDHA gene was transcribed in vitro with T7 RNA polymerase and the mRNA translated in a cell-free reticulocyte lysate in the presence of 35S-labeled methionine. As expected, the SDHA polypeptide is able to bind to mitochondria (Fig. 5, lanes 2 and 5). Import into mitochondria is membrane potential-dependent (lanes 5 and 6); only in the presence of energized mitochondria is a faster migrating species seen (lanes 2 and 3 ) . This species is resistant to externally added proteinase (lane 3) except when mitochondrial integrity is disrupted with detergent ( l a n e 4 ) .
As is commonly observed with in vitro synthesized precursor proteins, a fraction is present in an insoluble form (lane 7). This fraction is however, totally sensitive to proteinase (lune 8).
The amino terminus of the SDHA precursor contains the consensus sequence for cleavage upon import by two separate proteases: RX(F)XX(S) where R is arginine; X is any amino acid; (F) is phenylalanine or other hydrophobic residues; and (S) is serine, threonine, or glycine (Hendrick et al., 1989). In the yeast SDHA subunit, this motif is composed of Arg-19, Phe-21, and Ser-24 and would predict that the yeast SDHA precursor is first cleaved between Thr-20 and Phe-21 and that a subsequent cleavage by the matrix processing protease to a  Table I1 for a description of the sequences used.
mature species occurs between Arg-28 and Gln-29. The purpose of such a dual cleavage event has not been elucidated.
The latter cleavage can be inhibited in vitro by the addition of o-phenanthroline, a metal chelating agent (data not shown). We have not detected an intermediate in our in vitro import reactions, and are currently determining the mature amino terminus of the imported precursor.
SDHA gene was mapped by probing a nylon membrane to which had been transferred S. cereuisiue chromosomal DNA separated by contour-clamped homogeneous electric-field electrophoresis. As controls, two known genes, the succinate dehydrogenase iron-sulfur subunit ( S D H B ) gene on chromosome VI1 and the URA3 gene on chromosome V were also mapped (Lombardo et al., 1990). For the SDHA gene, a strong signal corresponding to chromosome number XI was observed ( Fig. 6). A weaker signal corresponding to chromosome XIV may indicate the presence of a related gene in the genome.
The SDHA and SDHB genes are located on different chromosomes in the S. cereuisiue genome. In all procaryotic organisms in which fumarate reductases or succinate dehydrogenases have been examined, the enzymes are expressed from operons encoding all of the subunits. Therefore, mitochondrially localized, multisubunit enzymes likely have evolved from the translation of polycistronic messages to the expression of individual subunits from different transcriptional units within the nucleus. This evolution requires that each subunit gene must not only acquire transcriptional signals but also an in-frame mitochondrial targeting sequence. It is improbable that each of the succinate dehydrogenase subunit genes would simultaneously undergo these changes. Gene transfer as an evolutionary process is a basic tenet of the endosymbiotic theory which proposes that mitochondria and chloroplasts were once free-living organisms (Gray and Doolittle, 1982). Regardless of whether the nuclear, precursor-encoding succinate dehydrogenase genes originated from the mitochondrial progenitor or not, it is probable that a t some time during evolution, the enzyme was composed of both nuclear and mitochondrially encoded subunits in a situation similar to those of complexes I, 111, IV, and V in most modern eucaryotes (Attardi and Schatz, 1988).