©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein Requires Import into Mitochondria, Presequence Removal, and Folding (*)

(Received for publication, September 12, 1995; and in revised form, December 6, 1995)

Karen M. Robinson (§) Bernard D. Lemire (¶)

From the Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.


INTRODUCTION

Although protein import into the mitochondria has been an area of intense study, relatively little is known regarding their cofactor insertion and oligomerization into multisubunit complexes. We are using the Saccharomyces cerevisiae succinate dehydrogenase (SDH), (^1)or complex II, as a model for examining mitochondrial respiratory complex assembly. SDH is a multisubunit mitochondrial enzyme that is part of both the Krebs cycle and the electron transfer chain. Located in the inner membrane facing the matrix, SDH catalyzes the oxidation of succinate to fumarate and donates the reducing equivalents to ubiquinone. An anaerobically expressed prokaryotic enzyme, fumarate reductase, is structurally and functionally closely related to SDH. Most SDH and fumarate reductase enzymes are composed of four nonidentical subunits: a flavoprotein (Fp) of about 70 kDa, an iron-sulfur protein (Ip) of about 30 kDa, and two hydrophobic anchoring subunits of 7-17 kDa. The Fp contains the active site and the unusual cofactor, an 8alpha-N(3)-histidyl-FAD linked at a conserved histidine residue. The Ip subunit contains three different iron-sulfur clusters, a [2Fe-2S], a [3Fe-4S], and a [4Fe-4S] cluster. The hydrophobic anchoring subunits are integral membrane proteins and interact with quinone substrates. In some SDH and fumarate reductase enzymes, these subunits also contain a b-type heme (Ackrell et al., 1992; Cole et al., 1985). Together, the Fp and Ip form a catalytic dimer that is attached to the membrane by the anchoring subunits, thereby composing the holoenzyme. In yeast, the SDH Fp, Ip, and two anchoring subunits are encoded by the nuclear genes, SDH1, SDH2, SDH3, and SDH4, respectively, which have all been cloned and sequenced (Chapman et al., 1992; Robinson and Lemire, 1992; Schülke et al., 1992; Lombardo et al., 1990; Daignan-Fornier et al., 1994; Bullis and Lemire, 1994). The SDH subunits are translated in the cytoplasm, targeted to mitochondria by cleavable amino-terminal presequences, translocated across both mitochondrial membranes, and finally assembled with each other and their respective co-factors into a functional complex.

The relationships between the attachment of covalent cofactors to mitochondrial proteins and their import has been examined for several proteins. In some cases, such as the addition of pyridoxal phosphate to aspartate aminotransferase or the addition of biotin to pyruvate or propionyl-CoA carboxylases, import and processing of the precursor proteins are independent of coenzyme addition (Ahmad and Ahmad, 1991; Sharma and Gehring, 1986; Taroni and Rosenberg, 1991). In fact, the biotinylation of propionyl-CoA carboxylase can occur either before or after translocation (Taroni and Rosenberg, 1991). In contrast, heme attachment to cytochrome c or cytochrome c(1) is essential for correct localization or processing (Dumont et al., 1991; Nicholson et al., 1989). Covalent bond formation between the cofactor and the apoprotein is usually a catalyzed process (Gross and Wood, 1984; Nargang et al., 1988; Nicholson et al., 1989; Schmidt et al., 1969). However, a notable exception is the Arthrobacter oxidans flavoprotein 6-hydroxy-D-nicotine oxidase, which has the same 8alpha-N(3)-histidyl-FAD linkage as the SDH Fp. Covalent FAD attachment to the purified 6-hydroxy-D-nicotine oxidase apoprotein is suggested to be autocatalytic (Brandsch and Bichler, 1991).

We report here the results of investigations carried out both in vivo and in vitro on the cofactor attachment to the yeast SDH Fp. In previous work, we constructed a mutant SDH1 gene that encodes an apoprotein unable to undergo cofactor addition because the conserved histidine, which is normally modified, had been converted to a serine (Robinson et al., 1994). The resulting His-90 Ser Fp is imported into mitochondria, binds FAD noncovalently, and is assembled into a nonfunctional SDH holoenzyme, demonstrating that covalent FAD attachment is necessary for enzyme activity but is dispensable for both import and assembly. Thus, the mutant Fp serves as a useful control for examining the flavinylation of the wild-type Fp. In this study, flavinylation is assayed by immunoprecipitation with an anti-FAD serum that recognizes the holo-Fp but neither the apo-Fp nor the His-90 Ser Fp (Robinson and Lemire, 1995). We show that FAD attachment in vivo occurs after import and proteolytic processing of the apo-Fp and that the rate of FAD attachment varies markedly with the carbon source upon which the cells are grown. Interestingly, modification of the Fp is stimulated by the Ip subunit. Carboxyl-terminal truncations of the apo-Fp completely eliminate modification by FAD, while Krebs cycle intermediates function as activators. Our results are consistent with flavinylation being a post-translocational process that occurs during or after mature Fp folding and prior to its assembly.


MATERIALS AND METHODS

Strains, Media, and Plasmids

Yeast strains used are described in Table 1. Yeast media, the Escherichia coli strains, UT580 and DH5alpha, and the plasmids, pSDH1 and pS1H90S, have been described previously (Robinson et al., 1991; Robinson and Lemire, 1992; Robinson et al., 1994). The plasmid, pSfRHAC, which encodes the SDH1 gene without any 5`-untranslated sequence, was created in two steps. pSDH1 was cut with SfaN1 and the ends were blunted with the Klenow fragment of DNA polymerase and digested with EcoRI (see Fig. 5). The SfaN1/EcoRI fragment encoding the Fp amino terminus was cloned into EcoRV- and EcoRI-digested pBluescript II SK- (Stratagene, La Jolla, CA), placing the partial SDH1 coding sequence under the expression of the T7 promoter and creating pSfR1. To reconstruct the entire SDH1 gene, pSDH1 was digested with NdeI, the ends were blunted, and it was cut with EcoRI, and the NdeI/EcoRI fragment encoding the Fp carboxyl terminus was cloned into EcoRI- and SmaI-digested pSfR1. The SDH1 gene from pSfRHAC, was inserted into the multicopy vector, YEplac195 (Gietz and Sugino, 1988) and placed under the control of the copper metallothionein promoter (CUP1) obtained from the vector, Yep96 (Ellison and Hochstrasser, 1991), to create the plasmid, pCuSDH1. To place SDH2 under CUP1 control, the SDH2 open reading frame was amplified by a polymerase chain reaction and cloned into the vector, pTCu, which is YEplac112 (Gietz and Sugino, 1988) bearing the CUP1 promoter, to create pCuB. The integrity of the SDH2 gene was established by complementation of an SDH2 disruption mutant. To select for the SDH2 gene in the yeast strain Sdh1Ad1, the LYS2 gene (Fleig et al., 1986) was inserted into pCuB, making the plasmid pKCuSDH2.




Figure 5: Restriction map of the yeast SDH1 gene. The SDH1 open reading frame is depicted as a box, while flanking regions are depicted as lines. The hatched box represents sequence encoding the mitochondrial targeting sequence; the stippled boxes represent the AMP binding domains; and the filled box represents the active site. The arrow indicates the His-90 codon, which encodes the histidine to which the FAD is covalently attached. The origins and sizes of the Fp proteins expressed in this work are indicated; preFp, precursor form of the Fp; Pvu, Fp translated from an mRNA truncated at the PvuI1 site; Eco, Fp translated from an mRNA truncated at the EcoRI site.



In Vivo Labeling

Labeling of cellular proteins was based on a published procedure (Brandt, 1991). Cells were grown overnight on semisynthetic media containing 0.15% yeast extract as well as the necessary auxotrophic markers and a carbon source of either 2% glucose, galactose, or lactate. MH125 and related strains were supplemented with 0.1% glucose when grown on galactose. Strains carrying plasmids with the CUP1 promoter were labeled without induction. When the A of the culture was between 0.8 and 1.0, it was quickly harvested and resuspended in prewarmed labeling buffer containing auxotrophic markers and 2% carbon source. Radiolabeled methionine was added (TranS-label, 81 µCi/ml; 1037 Ci/mmol; ICN Biomedicals, Mississauga, Ontario), and the cells were labeled for 3 min at 30 °C with vigorous shaking. Cycloheximide and cold methionine were added to 100 µg/ml and 2 mM, respectively, and the chase continued at 30 °C with vigorous shaking. At the indicated times, 0.5-ml aliquots were taken, lysed with NaOH and 2-mercaptoethanol, and precipitated with trichloroacetic acid (Yaffe and Schatz, 1984). Lysates were pelleted (14,000 times g for 10 min), resuspended in 1 ml of 2% SDS and 1% trichloroacetic acid. 0.1 ml of 55% trichloroacetic acid was added to reprecipitate proteins, and the sample was incubated at room temperature for 10 min and then on ice for an additional 10 min. We found it necessary to wash the precipitates in SDS and to reprecipitate with trichloroacetic acid for the anti-FAD serum to efficiently immunoprecipitate holo-Fp. Lysates were pelleted, washed with 1 ml of ice-cold acetone, resuspended in 115 µl of 2% SDS, 100 mM Tris-HCl, pH 7.5, and heated to between 65 and 90 °C for 10 min. Insoluble material was removed by centrifugation at 14,000 times g for 10 min, and the supernatant was used for immunoprecipitations. The efficiencies of the labeling and the chase were monitored as described previously (Brandt, 1991).

In Vitro Transcription, Translation, and Import

Template mRNA was produced from the plasmids, pSDH1, pS1H90S, or pSfRHAC using T7 RNA polymerase and translated in rabbit reticulocyte lysate as described by the supplier (Promega Corp.). Carboxyl-terminal truncated Fps were produced from the plasmid, pSfRHAC, that had been digested with the restriction enzymes, EcoRI or PvuI1, before transcription and translation. Import reactions containing 400 µg of isolated mitochondria or mitoplasts in a volume of 320 µl were performed essentially as described using 10 µl of lysate/import reaction (Gasser et al., 1982; Robinson et al., 1994). Import reactions were supplemented with 20 mM succinate, 10 mM malate, and 50 µM FAD unless stated otherwise. Import was allowed to proceed for 1 h at 30 °C. After proteinase K treatments (50 µg for 15 min at 0 °C), re-isolated mitochondria or mitoplasts were resuspended in 1 ml of 2% SDS, 0.1 M Tris-HCl, pH 7.5, and prepared for immunoprecipitation as described above. 90% of the sample was used for immunoprecipitation, and 4.5% was separated by denaturing gel electrophoresis to determine protein import.

Immunoprecipitations

Immunoprecipitations were performed as described previously (Brandt, 1991) with the following modifications. 45 µl of in vivo cell lysate was added to 1 ml of BTNTE (2 mg/ml bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, and 0.02% NaN(3)) containing 5 µl of preimmune serum and 1 mM phenylmethylsulfonyl fluoride. Samples were incubated 1 h at room temperature with rotation and centrifuged at 14,000 times g for 10 min. 8 µl of protein A-Sepharose beads (Sigma; binding capacity of 20 mg human IgG/ml) were added to the supernatants incubated as above. The beads were removed by centrifugation, and 10 µl of either anti-FAD serum or anti-Fp serum (Robinson et al., 1991) were added. The anti-FAD serum immunoprecipitates only the modified or holo-Fp; the anti-Fp serum immunoprecipitates both the modified and the apo-Fp. Samples were incubated overnight with rocking at 4 °C. Aggregated protein was removed by centrifugation at 14,000 times g for 10 min, and 16 µl of protein A-Sepharose beads were incubated with the supernatants for 1 h at room temperature. The beads were pelleted with a brief centrifugation, the supernatant was removed, and the beads were washed twice with NaTNTE (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% Triton X-100, 5 mM EDTA), twice with TNTE (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA), and finally twice with NTE (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA). The beads were resuspended in 20 µl of IPLB (0.1 M Tris-HCl, pH 6.8, 5% SDS, 5 mM EDTA, 0.005% bromphenol blue, 25% glycerol, 5% 2-mercaptoethanol), heated for 10 min at 65-90 °C, and spun briefly, and the supernatant was resolved by SDS-polyacrylamide gel electrophoresis. The gels were treated for fluorography, exposed to x-ray film, and, when desired, the bands were quantified with a model BAS1000 phosphoimager (Fuji Photo Film Co., Ltd.). To determine the extent of Fp modification, the amount of Fp immunoprecipitated by the anti-FAD serum was divided by the amount of Fp immunoprecipitated by the anti-Fp serum. For immunoprecipitations from in vitro translation experiments, 90 µl of lysate was added to 1 ml of BTNTE containing 1 mM phenylmethylsulfonyl fluoride and 10 µl of protein A-Sepharose beads. Samples were treated as above except that 20 µl of anti-FAD serum and 30 µl of protein A-Sepharose beads were used.


RESULTS

In Vivo Flavinylation of the Yeast Fp

We have previously shown that our anti-FAD serum only recognizes flavinylated protein and that this recognition can be competed for with free FAD, FMN, or riboflavin (Robinson and Lemire, 1995). The anti-Fp serum immunoprecipitates both apo- and holo-Fp forms of the protein. Therefore, the amount of FAD-modified Fp can be expressed as the amount of Fp immunoprecipitated by the anti-FAD serum divided by the amount of Fp immunoprecipitated by the anti-Fp serum. Quantitation is performed with a phosphoimager. The in vivo rate of FAD attachment to the Fp was examined with a pulse-chase experiment in the wild-type strain, D273-10B (Table 1). Aliquots taken during the chase period were analyzed by immunoprecipitation with the anti-FAD and the anti-Fp sera. Only the 67-kDa mature Fp is detected in this experiment since all of the Fp molecules rapidly reach the mitochondrial matrix where they are proteolytically processed (Brandt, 1991). The amount of labeled Fp is constant throughout the chase period, indicating that the chase has been effective and that the mature Fp is stable over this time period (Fig. 1, alpha-Fp row). In contrast, the amount of flavinylated Fp immunoprecipitated with the anti-FAD serum is initially undetectable and increases rapidly with time (Fig. 1, alpha-FAD row), demonstrating that the Fp is modified post-translationally after its presequence has been removed. Although it did not exceed 15%, flavinylation occurred with a half-time of about 5 min. The low efficiency is in part due to incomplete immunoprecipitation by the anti-FAD serum; larger volumes of serum do not significantly increase the amount of Fp precipitated. However, reprecipitation of the supernatant of a first round of immunoprecipitation indicates significant quantities of FAD-modified Fp remain (KMR, unpublished observations).


Figure 1: The in vivo flavinylation of the Fp. D273-10B was grown in sulfate-free medium with lactate as the carbon source to an A of approximately 1.0. Cells were labeled and chased as described under ``Materials and Methods.'' Aliquots were taken during the 20-min chase at the indicated times, and immunoprecipitated with the anti-Fp serum (alpha-Fp row) or the anti-FAD serum (alpha-FAD row) as described under ``Materials and Methods.''



We investigated whether the presence of the other SDH subunits could influence FAD attachment. A positive result would suggest that modification is a late step in the assembly of the SDH complex. The wild-type parent strain, MH125, and the derived SDH2, SDH3, and SDH4 disruption mutants (Table 1) were labeled and subjected to immunoprecipitations as described above. As a control, sdh1L6, an SDH1 disruption mutant transformed with the plasmid, pS1H90S, encoding the flavinylation-incompetent His-90 Ser mutant Fp was also analyzed. We compared the amount of FAD-modified Fp in each mutant strain with the amount detected in the wild-type strain, which was set to 100% (Fig. 2). As expected, the His-90 Ser Fp was not immunoprecipitated by the anti-FAD serum. Flavin attachment to the Fp was consistently reduced 2-3-fold in the SDH2 mutant, was less affected in an SDH4 mutant and was not affected in an SDH3 mutant. Thus, FAD attachment to the Fp can proceed in the absence of any one of the other subunits, although the Ip and possibly the SDH4 subunits enhance the process.


Figure 2: Roles of the SDH2, SDH3, and SDH4 subunits in Fp flavinylation. MH125, the wild-type strain (Wt), sdh1L6 carrying the plasmid, pS1H90S (H90S), sdh2L1 (sdh2), sdh3W22 (sdh3), and sdh4W2 (sdh4) were grown in sulfate-free medium with galactose as the carbon source to an A of approximately 1.0. Cells were labeled and chased as described under ``Materials and Methods.'' Aliquots were removed after chasing for 20 min and immunoprecipitated with the anti-Fp or the anti-FAD sera. The extent of Fp modification was determined by calculating the ratio of holo-Fp to total Fp present in each strain. The level of flavinylated Fp in the wild-type is set to 100%. The values presented are from a single experiment, but comparable data were obtained in two replicates.



The expression levels of many mitochondrial proteins are quite different between glucose repressed or nonrepressed cells. To investigate the possible involvement of additional factors in Fp modification, we examined the flavinylation of the Fp under respiratory (lactate as the carbon source) and nonrespiratory (glucose as the carbon source) conditions. We transformed the SDH1 disruption strain Sdh1Ad1 with the plasmid, pCuSDH1, which contains the SDH1 gene under the control of the CUP1 promoter. Uninduced levels of expression from this promoter are not significantly affected by the carbon source (Hottiger et al., 1994). Our observations indicate that Fp expression on glucose is about 2-fold higher than on lactate (not shown). When grown on lactate, the Fp will be expressed from the CUP1 promoter, while the other subunits will be expressed from chromosomally encoded genes; when grown on glucose, only the Fp will be present, as expression of the SDH2, SDH3, and SDH4 genes will be repressed (Daignan-Fornier et al., 1994; Lombardo et al., 1992). Since the level of FAD attachment was reduced in the absence of Ip subunit (Fig. 2), Sdh1Ad1 was also transformed with both pCuSDH1 and pKCuSDH2; the latter plasmid has the SDH2 gene under control the CUP1 promoter. In this case, the Fp and Ip subunits will both be expressed regardless of carbon source. We labeled the cellular proteins of cells grown on either glucose or lactate with radioactive methionine and removed aliquots for immunoprecipitation with the anti-Fp or the anti-FAD sera. These data are presented as the percentage of counts precipitated by the anti-FAD serum as compared with the counts precipitated by the anti-Fp serum (Fig. 3). Both the rate and the extent of FAD attachment are severely reduced when cells are grown on glucose rather than lactate. Although expressed, the Ip subunit did not change the kinetics of cofactor attachment when the cells were grown on glucose. This result is surprising since the absence of the Ip subunit did substantially affect Fp modification when the cells were grown on galactose (see Fig. 2). The difference in FAD attachment with carbon source may reflect a requirement for an additional protein whose expression is repressed by glucose or a need for a metabolite such as FAD that is less abundant when the cells are grown on glucose.


Figure 3: Effect of carbon source on FAD attachment. Sdh1Ad1 carrying the plasmid pCuSDH1, or both plasmids, pCuSDH1 and pKCuSDH2, was grown in sulfate-free medium with either lactate or glucose as the carbon source, labeled, chased, and analyzed as described under ``Materials and Methods.'' The expression of the SDH1 gene is under control of the CUP1 promoter in all cases. With pKCuSDH2, the expression of the SDH2 gene is also under control of the CUP1 promoter. Otherwise, the SDH2, SDH3, and SDH4 genes are chromosomally encoded. The symbols represent Sdh1Ad1 carrying pCuSDH1 grown on lactate (diamonds), grown on glucose (triangles), and Sdh1Ad1 carrying pCuSDH1 and pKCuSDH2 grown on glucose (squares).



In Vitro Flavinylation of the Yeast Fp

To test whether modification by FAD could occur in vitro and thus provide a more convenient and manipulatable system for further investigations, we examined Fp flavinylation during import into both isolated whole mitochondria and mitoplasts (mitochondria with their outer membranes disrupted). In vitro translated wild-type or His-90 Ser precursor proteins were incubated with isolated mitochondria or mitoplasts, the organelles were reisolated, and cofactor attachment was assayed by immunoprecipitation with the anti-FAD serum (Fig. 4). If import is blocked by the ionophore, valinomycin, modification of the surface-bound Fp is not detected (lanes 2 and 3, alpha-FAD row), indicating that FAD is not attached prior to import. We found a significant proportion of the wild-type Fp precursor could be imported into mitoplasts, processed (5% of imported Fp row), and modified by FAD (lane 5, alpha-FAD row), demonstrating that the FAD could be attached to the Fp in vitro. In these experiments, mitoplasts imported, and hence modified, larger quantities of the Fp than intact mitochondria (compare lanes 4 and 5; Hwang et al., 1989). However, the fraction of imported Fp that was flavinylated in mitochondria and mitoplasts is approximately the same. FAD attachment might be occurring either during or after import. To distinguish between these possibilities, we performed import reactions and examined the effect of proteinase treatment on flavinylation. Proteinase treatment removes proteins that have not been fully imported. In these experiments, the extent of FAD attachment is not affected by proteinase treatment (compare lanes 5 and 7), suggesting that flavinylation requires complete import of the Fp into the mitochondria or mitoplasts. The His-90 Ser Fp (lane 8), although imported to the same extent as the wild-type, was not immunoprecipitated by the anti-FAD serum.


Figure 4: In vitro import of the Fp and FAD attachment. Wild-type Fp (WT) and His-90 Ser (H90S) Fp precursors were produced by in vitro transcription and translation in rabbit reticulocyte lysate. The precursors were imported into mitochondria (M) or mitoplasts (MP), and the organelles were reisolated for analysis by SDS gel electrophoresis and fluorography (5% of imported Fp row) or by immunoprecipitation with anti-FAD serum (alpha-FAD row) as described under ``Materials and Methods.'' Valinomycin was used at a final concentration of 10 µM. The precursor and mature proteins have masses of 70 and 67 kDa, respectively.



To confirm that FAD attachment occurs after import, we determined whether carboxyl-terminal truncated Fp proteins could be modified. If modification of the Fp happens co-translocationally, then truncations of the Fp should be without effect on the extent of flavinylation. We performed import reactions with Fp molecules that had carboxyl-terminal truncations of 70 (Pvu) or 90 amino acids (Eco; see Fig. 5). Truncated precursors were efficiently synthesized (Fig. 6, lanes 2 and 3) and imported to a protease protected location (Fig. 6, lanes 5 and 6). However, cofactor attachment was undetectable with the truncated Fp molecules (lanes 8 and 9). Thus, Fp modification occurs post-translocationally and requires the carboxyl terminus of the protein.


Figure 6: Carboxyl-terminal truncates of the Fp are not flavinylated. Full-length Fp (Wt), or Fp with a carboxyl-terminal truncations at the PvuII site, removing 70 amino acids (Pvu), or at the EcoRI site, removing 90 amino acids (Eco), were translated in rabbit reticulocyte lysate (lanes 1-3), imported into mitoplasts (lanes 4-6), and analyzed for FAD attachment by immunoprecipitation with the anti-FAD serum (lanes 7-9). The slowest migrating species in each lane corresponds to the full-length translation products of the wild-type and truncated Fp proteins.



We next examined whether Fp modification was dependent on the addition of flavins. Since the conversion of FMN to FAD is performed by a cytosolic enzyme (Wu et al., 1995), mitochondria must be able to transport FAD. Thus, FAD may be lost from mitochondria during their isolation. Import reactions were performed in the absence of added flavin or in the presence of riboflavin, FMN, or FAD, and cofactor attachment to proteinase-protected Fp assayed by immunoprecipitation with the anti-FAD serum (Fig. 7). Import of the Fp into mitoplasts was not affected by the addition of any of the flavins (lanes 2-5). FAD attachment to the imported Fp in the absence of added flavin was minimal (lane 6) and the addition of either riboflavin or FMN did not increase protein modification (lanes 7 and 8, respectively). In contrast, cofactor attachment was substantially increased with the addition of FAD to the import mix (lane 9), suggesting that FAD is indeed transported across the mitochondrial inner membrane. Flavinylation was quantified and is expressed as the ratio of holo-Fp to total Fp present for each sample. For comparison between samples, the ratio determined for modification without added flavin was set at 1 (Fig. 7, Fold Increase row). These results suggest that FAD is transported into mitochondria and is the immediate substrate for covalent attachment and that riboflavin and FMN are incapable of supporting modification under these conditions.


Figure 7: In vitro flavinylation requires added FAD. Wild-type Fp precursor, produced by in vitro transcription and translation, was imported into mitoplasts in the absence of additions(-), or in the presence of 50 µM riboflavin (Rb), FMN, or FAD. After import, the mitoplasts were re-isolated for analysis by SDS gel electrophoresis and fluorography (lanes 1-5) or by immunoprecipitation with anti-FAD serum (lanes 6-9). The ratios of holo-Fp to total Fp for each sample were calculated. For comparison, the ratio of holo-Fp to total Fp for the import sample without addition of any flavin was set at 1.



With the bacterial flavoprotein 6-hydroxy-D-nicotine oxidase, flavinylation can be influenced by small molecules that apparently act as allosteric effectors (Brandsch and Bichler, 1989). We investigated whether a similar phenomenon occurs with Fp flavinylation. In import reactions without the addition of any metabolites, FAD attachment is minimal, while the Krebs cycle intermediates, succinate, fumarate, and malate result in 4-6-fold stimulations (not shown, see Fig. 3of the accompanying paper (Robinson, and Lemire, 1996). Thus, FAD attachment to the Fp is also stimulated by some Krebs cycle intermediates.


DISCUSSION

Many of the details of protein import into mitochondria have been elucidated in recent years, including some aspects of the folding process that follows translocation. We have been utilizing the yeast SDH as a model system with which to address the problems of subunit assembly and cofactor insertion. One of the most distinguishing features of SDH enzymes is the presence of a covalently attached FAD coenzyme; it is with the addition of FAD that this work is concerned.

FAD attachment was monitored with the aid of an FAD-specific polyclonal antiserum that recognizes the holo-Fp subunit but not the apo-subunit (Robinson and Lemire, 1995). As a control, we have used a mutant Fp subunit that is capable of being transported into mitochondria, assembling into a membrane-bound enzyme, and binding FAD but only in a noncovalent manner.

FAD attachment occurs in mitochondria after the Fp is imported. Pulse-chase experiments (Fig. 1) demonstrated that FAD is attached in vivo after the presequence has been cleaved, a process that occurs in the mitochondrial matrix. Consistent with this view, we could not detect any flavinylated precursor protein when import in vivo is blocked by an uncoupler. (^2)Partly imported or surface-bound Fp, which can be distinguished from fully imported molecules by proteinase sensitivity, were not modified during in vitro import into isolated organelles (Fig. 4). Finally, if FAD attachment is a co-translocational process, we would expect the modification of carboxyl-terminal truncated Fp molecules, since these would appear identical to the full-length Fp until their import is almost complete. FAD addition is not detected in truncated Fps, suggesting that attachment occurs when the entire Fp is translocated and available for folding and assembly. The truncated Fps were also not modified when expressed in vivo.^2 Similarly, flavinylation studies on the 6-hydroxy-D-nicotine oxidase6-hydroxy-D-nicotine oxidase and on E. coli fumarate reductase A indicate that cofactor attachment requires the entire length of these proteins and therefore must occur post-translationally (Brandsch et al., 1993; Cecchini et al., 1985; Cole et al., 1985).

Proteolytic processing can be a mandatory step for cofactor attachment. For example, cytochrome c(1) is cleaved in two steps, first to an intermediate form and then to the mature size. Heme attachment precedes cleavage of the intermediate to the mature form of the protein (Nicholson et al., 1989). Our studies with the SDH Fp have shown that cofactor attachment occurs after presequence cleavage, although they do not address whether FAD addition could proceed if proteolytic processing were prevented.

The immediate substrate for flavinylation is likely FAD since only FAD and not the precursors, riboflavin or FMN, increase the amount of cofactor attached in vitro. Recent work has demonstrated that the yeast flavin synthase, which adenylates FMN to FAD, is located in the cytosol (Wu et al., 1995). Thus, if riboflavin or FMN were transported into mitochondria, they would not be converted to FAD. Neither riboflavin or FMN are apparently attached to the Fp as they do not increase the amount of Fp immunoprecipitated by the anti-FAD serum (Fig. 7), even though this serum recognizes these molecules (Robinson and Lemire, 1995). Studies with the 6-hydroxy-D-nicotine oxidase have led to a similar conclusion; the flavin moiety is attached to the protein as FAD rather than as riboflavin or FMN, which is subsequently converted to FAD (Brandsch and Bichler, 1991). Implicit in this model is the existence of a yeast mitochondrial FAD transporter (Wu et al., 1995).

Fp modification occurs once the mature Fp molecule has adopted some structure but before enzyme assembly is complete. Three lines of evidence support this hypothesis; first, we demonstrated a decrease in the extent of flavin attachment to the Fp in vivo in an SDH2 disruption mutant. The yeast SDH Fp and Ip subunits are postulated to form an assembly intermediate since in the absence of one, the other is proteolytically degraded (Lombardo et al., 1990; Robinson et al., 1991; Schmidt et al., 1992). Thus, the Ip may stabilize the folding or assembly of the Fp and assist in cofactor attachment. However, the Ip is not essential for the process, and thus, Fp modification may precede assembly with the other SDH subunits. Similar conclusions have been reached in prokaryotic systems; FAD is attached to the SDH or fumarate reductase Fps independent of the other subunits (Cole et al., 1985; Hederstedt, 1980; Hederstedt et al., 1982).

Second, FAD attachment in vitro was greatly stimulated by the addition of Krebs cycle intermediates that may promote a conformation conducive to the process (see Fig. 3of the accompanying article (Robinson and Lemire, 1996). Interestingly, the most efficient effectors, succinate, malate, fumarate, and to a lesser extent malonate, are all known to bind to the SDH active site located in the Fp subunit (Kotlyar and Vinogradov, 1984). The effector molecules may be stimulating flavinylation by binding to and stabilizing an appropriate conformation at the active site. Correspondingly, the 6-hydroxy-D-nicotine oxidase requires allosteric effectors, in this case, phosphorylated three carbon molecules, for its flavinylation while prokaryotic SDH and fumarate reductase Fps showed a dependence on Krebs cycle intermediates for FAD attachment (Brandsch and Bichler, 1989). The need for effectors may explain why the rate of FAD attachment is reduced in cells grown on glucose; a condition that might result in lower concentrations of these molecules. Accordingly, the limiting factor for FAD attachment to the 6-hydroxy-D-nicotine oxidase when expressed in E. coli, is proposed to be the concentration of the effector molecules (Brandsch and Bichler, 1992). This requirement for effectors may provide an additional level of control over the biogenesis of mitochondrial enzymes.

Third, if flavinylation were a reaction that required a fully unfolded protein substrate, then one would not expect carboxyl-terminal truncations of the Fp to have any effect since mitochondrial precursors are transported in an unfolded state (Deshaies et al., 1988; Eilers and Schatz, 1986; Schwarz and Neupert, 1994; Stuart et al., 1993) and since the FAD-modified histidine (His-90) is at the amino terminus of the precursor protein. The truncations we tested, removed 70 or 90 residues, all of which are well beyond portions of the protein postulated to be involved in contacting the AMP moiety of the FAD (Fig. 5).

Thus, the evidence suggests that flavinylation of the Fp requires some structure be adopted. This requirement is consistent with either an autocatalytic reaction mechanism in which the Fp catalyzes the formation of the protein-FAD linkage or a mechanism that involves an additional enzyme to catalyze the linkage. Highly purified apo-6-hydroxy-D-nicotine oxidase, which has the same protein-cofactor linkage, can modify itself, arguing in favor of an autocatalytic mechanism. One argument against autocatalysis is the existence of specific enzymes for heme addition to such proteins as cytochrome c, and cytochrome c(1) (Dumont et al., 1991; Nargang et al., 1988), for biotin addition to carboxylases (Gross and Wood, 1984), and for lipoic acid addition to alpha-ketoglutarate dehydrogenase (Schmidt et al., 1969). A second argument is that the reduced rate of FAD attachment observed when the cells are grown on glucose could reflect diminished levels of a flavinylating enzyme whose expression is repressed. Alternatively, metabolites such as effector molecules or even FAD itself may be limiting and limit autocatalysis. Clearly, further work using purified components is required to clarify the mechanism of flavinylation of the yeast Fp and its relationship to protein folding and subunit assembly.


FOOTNOTES

*
This research was supported by the Medical Research Council of Canada Grant PG-11440 (B. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by studentships from the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada. Present address: Dept. of Biology 0347, University of California, San Diego, Pacific Hall, Rm. 2216, 9500 Gilman Dr., La Jolla, CA 92093-0347.

To whom correspondence should be addressed. Tel.: 403-492-4853; Fax: 403-492-0886; :lem-1{at}bones.biochem.ualberta.ca.

(^1)
The abbreviations used are: SDH, succinate dehydrogenase; Fp, flavoprotein; Ip, iron-sulfur protein.

(^2)
K. M. Robinson, unpublished results.


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