(Received for publication, September 12, 1995; and in revised form, December 6, 1995)
From the
Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial heterotetramer
containing a flavoprotein subunit with an
8-N(3)-histidyl-linked FAD cofactor. The covalent linkage
of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein
precursor in mitochondrial matrix fractions. Flavoprotein modification
does not depend on translocation across a membrane, but it does require
proteolytic processing by the mitochondrial processing peptidase prior
to flavin attachment. Since ATP depletion, N-ethylmaleimide,
or proteinase treatments of matrix fractions inhibit flavoprotein
modification, at least one additional matrix protein component appears
to be required. Having previously suggested that the flavoprotein
begins folding before FAD attachment occurs, we tested whether the
mitochondrial chaperonin, heat shock protein 60, might be necessary.
Co-immunoprecipitation of the flavoprotein and the chaperonin
demonstrate that the proteins do indeed interact. However,
immunodepletion of the chaperonin from matrix fractions does not
inhibit FAD attachment. Nonprotein components are also required for
flavoprotein modification. In addition to ATP, effector molecules such
as succinate, fumarate, or malate also stimulate modification.
Together, these results suggest that FAD addition is an early event in
succinate dehydrogenase assembly.
The assembly of many mitochondrial proteins can depend on the
activities of special proteins such as the mitochondrial processing
peptidase (Arretz et al., 1991; Glick et al., 1992a)
and the chaperonin, heat-shock protein 60 (hsp60) ()(Stuart et al., 1994). The processing peptidase removes cleavable
presequences, a step that for some proteins must occur before cofactor
insertion or assembly into multisubunit complexes can proceed
(Nicholson et al., 1989; Graham et al., 1993). hsp60
mediates the folding and assembly of some imported proteins in an
ATP-dependent reaction (Georgopoulos and Welch, 1993; Becker and Craig,
1994). Both mitochondrial processing peptidase and hsp60 are
indispensable for mitochondrial biogenesis, as they are both essential
for viability in Saccharomyces cerevisiae (Baker and Schatz,
1991).
We are using the S. cerevisiae succinate
dehydrogenase (SDH) or complex II as a model system for studying the
biogenesis of mitochondrial complexes. It catalyzes the oxidation of
succinate to fumarate and transfers the reducing equivalents directly
to the electron transport chain. One of the most notable features of
SDH and a closely related enzyme of the bacterial cytoplasmic membrane,
fumarate reductase (FRD), is the presence of a covalently attached FAD
cofactor. Both SDH and fumarate reductase are usually comprised of four
nonidentical subunits: a flavoprotein (Fp), an iron-sulfur protein
(Ip), and two hydrophobic membrane anchoring subunits (Cole et
al., 1985; Ackrell et al., 1992; Hederstedt and Ohnishi,
1992). In S. cerevisiae, the Fp, the Ip, and the membrane
subunits are encoded by the nuclear genes, SDH1, SDH2, SDH3, and SDH4, respectively (Lombardo et al., 1990; Chapman et al., 1992; Robinson and
Lemire, 1992; Bullis and Lemire, 1994; Daignan-Fornier et al.,
1994). The Fp contains the SDH active site and the FAD cofactor in an
8-N(3)-histidyl-FAD linkage. The small number of subunits
and the availability of the SDH genes for manipulation make the yeast
SDH an attractive and practicable system for examining cofactor
insertion and the assembly of mitochondrial complexes.
Covalent
cofactor attachment to mitochondrial and nonmitochondrial proteins can
be mediated by special enzymes. For example, heme is attached to
cytochromes c and c by heme lyases
(Nargang et al., 1988; Nicholson et al., 1989; Dumont et al., 1991), biotin is added to carboxylases by
holocarboxylase synthetase (Gross and Wood, 1984), and lipoic acid is
attached to
-keto-acid dehydrogenases by a lipoic acid activating
system (Schmidt et al., 1969). The addition of biotin and
lipoic acid also require ATP.
The mechanism of covalent FAD attachment for the Fp subunit of complex II has not been elucidated. Interestingly, 6-hydroxy-D-nicotine oxidase, a bacterial flavoprotein, autocatalytically attaches a covalent FAD (Brandsch and Bichler, 1991). The modification is independent of ATP and is stimulated by effector molecules such as glycerol 3-phosphate (Brandsch and Bichler, 1989; Brandsch and Bichler, 1991). Autocatalytic flavinylation has also been suggested for bovine monoamine oxidase and for Pseudomonas putida p-cresol hydroxylase (Weyler et al., 1990; Kim et al., 1994). Whether the formation of covalent protein-FAD bonds is autocatalytic for all flavoproteins remains to be documented.
Several observations indicate that proper
protein folding is a prerequisite for covalent FAD attachment. First,
unrelated enzymes with 8-N(3)-histidyl-FAD linkages, such
as 6-hydroxy-D-nicotine oxidase and the SDH Fp, show no
sequence identity (Lang et al., 1991), suggesting that protein
conformation may be the signal for FAD attachment. Second,
autocatalytic flavinylation of the 6-hydroxy-D-nicotine
oxidase is conformation-dependent (Brandsch and Bichler, 1992; Brandsch et al., 1993). Third, a number of observations suggest that
FAD attachment to the SDH Fp follows partial folding of the Fp
(Robinson and Lemire, 1996). These include increased FAD attachment to
the Fp upon expression of the Ip subunit in vivo and a
dependence of Fp modification on the addition of substrate or
substrate-like molecules. Both observations imply that folding is
important because interactions with the Ip or with substrate are
specific. Finally, carboxyl-terminal truncations of the Fp, which
should not affect flavinylation if the reaction proceeded with unfolded
Fp as substrate, completely block modification (Robinson and Lemire,
1996).
Fp modification can be monitored after import into isolated mitochondria (Robinson and Lemire, 1996). To further investigate the mechanism of Fp modification, we have developed a flavinylation system consisting of in vitro translated Fp precursor and a mitochondrial matrix fraction and assayed for flavin attachment by immunoprecipitation with an anti-FAD serum. Our results indicate that Fp translocation is not linked to FAD addition. Flavinylation is stimulated by the presence of Krebs cycle intermediates such as succinate and malate, is ATP-dependent, and N-ethylmaleimide- or proteinase-sensitive, suggesting that other proteins are involved. Interestingly, proteolytic processing of the presequence is mandatory for FAD attachment to the Fp. Finally, we show that the Fp interacts with hsp60, although the absence of hsp60 does not prevent its modification.
We first
tested whether mitochondrial integrity and import competence are
necessary for flavinylation of the Fp to occur. Isolated mitochondria
were solubilized with Triton X-100 in Flavinylation Buffer, which
contains ATP, MgCl, ZnCl
, MnCl
,
FAD, succinate, fumarate, and several protease inhibitors. We included
ATP and magnesium because FAD attachment might be an energy-dependent
reaction, succinate and fumarate because these molecules greatly
stimulate flavinylation of the Fp in mitoplasts (Robinson and Lemire,
1996), and the metals ions, Zn
and
Mn
, because they are necessary for mitochondrial
processing peptidase function. Fp precursor that had been translated in
rabbit reticulocyte lysate was incubated with the solubilized
mitochondria; precursor stability and processing were monitored by SDS
gel electrophoresis and fluorography, while cofactor attachment was
assayed by immunoprecipitation with the anti-FAD serum (Fig. 1).
Not only are detergent-solubilized mitochondria capable of
proteolytically processing the Fp precursor (lane 1) to its
mature size (lane 2), but they are also able to support
flavinylation of a significant fraction of that mature Fp (lane
5).
Figure 1: FAD attachment activity is located in the mitochondrial matrix. Fp precursor was translated in rabbit reticulocyte lysate (lane 1), and 10 µl of lysate was incubated for 20 min at 30 °C with either mitochondria (Mit; lanes 2 and 5), mitochondrial membranes (Mem; lanes 3 and 6), or a mitochondrial matrix fraction (Mat; lanes 4 and 7) prepared from an initial mitochondrial concentration of 10 mg/ml. Aliquots (4.5% of total) were analyzed by SDS gel electrophoresis and fluorography to quantify the Fp remaining and to determine the extent of proteolytic processing to the mature size (lanes 2-4). Immunoprecipitations with anti-FAD serum followed by SDS gel electrophoresis and fluorography were performed with 90% of the total sample to measure cofactor attachment to the Fp (lanes 5-7). Radioactivity in the Fp bands was measured with a model BAS1000 Bio-Imaging analyzer, and the ratios of flavinylated Fp to total Fp were calculated. The extent of flavinylation (% Holo-Fp) in the matrix fraction was normalized to 100%.
To determine whether the flavinylating activity is located in the mitochondrial matrix or whether it is associated with a membrane, we fractionated mitoplasts. Mitoplasts suspended in Flavinylation Buffer were disrupted by freeze-thawing and sonication, and the membrane fraction was pelleted and solubilized with Triton X-100 in Flavinylation Buffer. The supernatant fraction is referred to as the matrix fraction. Flavinylation reactions containing in vitro translated Fp precursor were incubated with either the membrane (Fig. 1, lanes 3 and 6) or the matrix (lanes 4 and 7) fractions, and the extent of FAD attachment was determined. Both the membrane and the matrix fractions supported processing of the Fp precursor to the mature size (lanes 3 and 4, respectively). However, modification of the Fp with FAD was significantly more efficient in the matrix fraction (lane 7) than in the membrane fraction (lane 6). We believe that the flavinylation activity associated with the membrane fraction may reflect contamination by residual matrix components. The small apparent increase in the extent of flavinylation in reactions with matrix fractions over those with solubilized mitochondria could stem from competition for binding and precipitation by the anti-FAD serum by preassembled Fp present in mitochondrial membranes or from the presence of detergent. Flavinylation of the SDH Fp therefore requires matrix components.
We further examined the dependence of the flavinylation reaction on matrix components by preparing matrix fractions with different protein concentrations (Fig. 2). Fp precursor (lane 1) was incubated in Flavinylation Buffer without matrix fraction (lanes 2 and 6) or with matrix fractions prepared from mitochondria that were at 5, 10, or 20 mg/ml (lanes 3-5 and 7-9, respectively). Proteolytic processing (lanes 2-5) and FAD attachment (lanes 6-9) occur only in the presence of matrix fraction, demonstrating that these activities are not attributable to the reticulocyte lysate or to the Flavinylation Buffer. Furthermore, the extent of modification is directly proportional to the concentration of the fraction used. In all further experiments, we used matrix fractions prepared from mitochondria at 10 mg/ml.
Figure 2: Flavinylation is proportional to the concentration of the matrix fraction. In vitro translated Fp precursor (lane 1) was incubated 20 min at 30 °C in Flavinylation Buffer without matrix fraction (lanes 2 and 6) or with matrix fractions at concentrations of 5 (lanes 3 and 7), 10 (lanes 4 and 8), or 20 mg/ml (lanes 5 and 9). After incubation, the samples were analyzed by SDS gel electrophoresis and fluorography to monitor Fp levels and processing (lanes 2-5) or by immunoprecipitation with the anti-FAD serum to assay for cofactor attachment (lanes 6-9). Flavinylation in the 10 mg/ml sample was normalized to 1, and other samples were compared with it.
In earlier
work, we noted that the flavinylation of imported Fp precursor could be
greatly enhanced by certain citric acid cycle intermediates such as
succinate, fumarate, or malate, but not by others such as citrate or
oxaloacetate or by glycerol-3-phosphate, a stimulator of
6-hydroxy-D-nicotine oxidase FAD attachment (Robinson and
Lemire, 1996). The inability of the latter three compounds to stimulate
FAD attachment in mitoplasts might be due to their slower uptake. We
reassessed the efficacies of these molecules in flavinylation reactions
performed with matrix fractions (Fig. 3). In the absence of
additives, very little holo-Fp could be immunoprecipitated with
anti-FAD serum (lane 2). Succinate, fumarate, and malate
greatly increased the amount of holo-Fp detected (lanes 3, 4, and 6, respectively). Flavin attachment was only
slightly or not at all stimulated by the competitive inhibitors,
malonate and oxaloacetate (lanes 5 and 7,
respectively), or with citrate and glycerol 3-phosphate (lanes 8 and 9, respectively). The His-90 Ser Fp, which
cannot be modified (Robinson et al., 1994), was also incubated
with matrix fraction in the presence of succinate (lane 10).
Only trace amounts of the His-90
Ser Fp are immunoprecipitated
with the anti-FAD serum, demonstrating the serum's specificity
for the cofactor. These results demonstrate that FAD attachment in
matrix fractions is also greatly stimulated by the same Krebs cycle
intermediates that stimulate in intact organelles.
Figure 3:
Cofactor
attachment is stimulated by effector molecules. In vitro translated Fp precursor (lane 1) was incubated with
matrix fractions without additions (lane 2) or with 10 mM succinate, fumarate, malonate, malate, oxaloacetate, citrate, or
glycerol 3-phosphate (lanes 3-9, respectively). His-90
Ser Fp was incubated in the presence of succinate (lane
10). To determine the amount of Fp remaining and the extent of
proteolytic processing, a portion of each sample was analyzed by SDS
gel electrophoresis and fluorography (Protein row).
Flavinylation was assayed by immunoprecipitation with the anti-FAD
serum (
-FAD row). The level of flavinylation in the
extract without additives (lane 2) was normalized to 1, and
other samples were compared with it. It is unclear why malate results
in an apparent increase in the Fp remaining after the incubation (lane 6).
The requirement for ATP in Fp flavinylation was examined (Fig. 4). We increased the amount of ATP present in the matrix fractions by either supplementing the flavinylation reaction with additional ATP after the reaction had proceeded for 10 min (lanes 3 and 7) or by including an ATP-regenerating system (lanes 4 and 8). Neither of these changes stimulated flavinylation over untreated matrix fraction (lanes 2 and 6). Another sample was pretreated with apyrase to hydrolyze both ATP and ADP to AMP (Glick, 1991). ATP depletion reduced processing of the precursor (lane 5) and cofactor attachment by about 6-fold (lane 9). In this experiment, Fp recovery was low, but this was not reproducible (lane 5); the percentage of Fp flavinylation was not affected by recovery. Addition of either NADH or the ionophore, valinomycin, did not affect the amount of Fp modified (data not shown). Although not necessarily directly involved in the reaction, ATP is required for in vitro FAD attachment.
Figure 4: Flavinylation requires ATP. Untreated matrix fractions (lanes 2 and 6), fractions to which had been added an additional aliquot of 5 mM ATP after the first 10 min of incubation (lanes 3 and 7), or an ATP-regenerating system consisting of pyruvate kinase and phosphoenolpyruvate (lanes 4 and 8), or fractions pretreated with apyrase (20 units/ml for 10 min at 30 °C; lanes 5 and 9), were incubated with in vitro translated Fp precursor (lane 1). A portion of each sample was analyzed by SDS gel electrophoresis and fluorography (lanes 2-5) or by immunoprecipitation with the anti-FAD serum (lanes 6-9), and the ratios of flavinylated to total Fp were calculated. The level of flavinylation in the untreated fraction was normalized to 100%.
The Ip subunit stimulates FAD attachment to the Fp subunit under respiratory conditions in vivo (Robinson and Lemire, 1996). To test whether the same is true in vitro, flavinylation reactions were supplemented with in vitro translated Ip. The extents of Fp processing or modification are not significantly changed by the presence of the Ip subunit (not shown).
One of the most interesting
questions about the covalent FAD attachment to flavoproteins is whether
the reaction is catalyzed by an enzyme or whether an autocatalytic
mechanism is employed. To determine whether a matrix protein is
responsible for Fp modification activity, matrix fractions were treated
in several ways that either inactivate proteins or remove small
molecules (Fig. 5). Boiled matrix fractions are incompetent for
flavinylation. Treatment with N-ethylmaleimide, a
sulfhydryl-modifying reagent, or digestion with proteinase K also
inhibit FAD attachment, thus implying that a protein component is
involved in Fp flavinylation. Addition of ovalbumin, which might
nonspecifically stabilize the Fp in protease-treated matrix fractions,
does not restore FAD attachment activity, suggesting that protease
treatment removes at least one specific protein. Depletion of small
molecules from the matrix fraction with a Sephadex G-25 spin column
results in an almost complete loss of flavinylation and a reduction of
processing activity. Readdition of ATP, Mg,
succinate, fumarate, Zn
, Mn
, and
FAD can partially restore FAD attachment activity to the depleted
lysate. Collectively, these data strongly argue that the modifying
activity found in the matrix lysate is a protein component, although
small molecules such as divalent cations, ATP, and FAD are also needed.
Figure 5:
Flavinylation requires a matrix protein
component. Fp precursor was incubated with matrix fraction that had
been untreated, boiled for 10 min, incubated with 2.5 mMN-ethylmaleimide on ice for 30 min followed by the
addition of excess cysteine (NEM), treated with 2 µg/ml
proteinase K for 20 min at room temperature, followed by the addition
of protease inhibitor mixture with (Prot+Oval) or without (Protease) the subsequent addition of 400 µg of chicken
ovalbumin or applied to a Sephadex G-25 spin column with (G25+ATP) or without (G25) the
subsequent addition of 5 mM MgCl, 5 mM ATP, 50 µM FAD, 10 mM succinate, 10 mM fumarate, 50 µM ZnCl
, and 50 µM MnCl
to the void volume fractions. The level of
flavinylation in the untreated sample was normalized to
100%.
Figure 6:
Proteolytic processing is required for FAD
attachment. In vitro translated Fp precursor (lane 1)
was incubated with matrix fractions that had been prepared in ATP and
divalent cation-free Flavinylation Buffer supplemented with 50
µM MgCl and treated in the following ways:
untreated (lanes 2 and 6); incubated with 0.5 mM EDTA for 10 min in the presence of 5 mM ATP followed by
the addition of 0.1 mM MnCl
and 0.6 mM ZnCl
(lanes 3 and 7); incubated with
0.5 mM EDTA for 10 min in the absence of ATP followed by the
addition of 0.1 mM MnCl
and 0.6 mM ZnCl
(lanes 4 and 8); or incubated
with 0.5 mM EDTA for 10 min in the absence of ATP followed by
the addition of 0.6 mM MgCl
(lanes 5 and 9). Samples were analyzed for protein content and processing (lanes 2-5) and the amount of FAD attachment (lanes
6-9).
Since the presequence appears to inhibit cofactor attachment, we engineered an amino-terminally truncated Fp that does not contain a presequence, called pseudomature Fp (mFp). The wild-type and the pseudomature Fps were translated (Fig. 7, lanes 1 and 2, respectively), incubated with the matrix fractions (lanes 3 and 4) and subjected to immunoprecipitations with the anti-FAD serum (lanes 5 and 6). Although the pseudomature Fp is efficiently translated and is not degraded by the matrix fraction, cofactor attachment is undetectable. Therefore, FAD attachment requires the proteolytically processed Fp.
Figure 7: Pseudomature Fp is not flavinylated. Wild-type (pFp and Wt) or pseudomature (mFp) Fp were translated in reticulocyte lysate (lanes 1 and 2, respectively), incubated with matrix fractions, and analyzed by SDS gel electrophoresis and fluorography (lanes 3 and 4), and by immunoprecipitation with the anti-FAD serum (lanes 5 and 6).
Figure 8: The Fp is co-immunoprecipitated with hsp60. The LS fusion protein and the Fp precursor were translated (lanes 1 and 2, respectively) and incubated in Flavinylation Buffer with (lanes 3-6, and 8-11) or without (lanes 7 and 12) matrix fraction. Each sample was incubated with 20 units/ml apyrase for 10 min at 30 °C. Where indicated, 50 µM FAD was added to the incubation mix. 20% of each sample was analyzed by SDS gel electrophoresis and fluorography (lanes 3-7). The remaining sample was analyzed by native immunoprecipitation with the anti-hsp60 serum (lanes 8-12). Note that after immunoprecipitation with the anti-hsp60 antibodies, LS migrates faster than usual on the gel because of co-migrating IgG heavy chains (Rospert et al., 1994).
Does the association of the Fp with hsp60 precede or follow covalent cofactor attachment? We first addressed this question by determining whether Fp found in association with hsp60 is already flavinylated. Co-immunoprecipitations of the Fp with the anti-hsp60 serum were used to separate Fp molecules into hsp60-bound and free fractions. In order to mimic the immunoprecipitation of the bound fraction with the anti-hsp60 serum, Fp in the free fraction was immunoprecipitated with anti-Fp serum. Both Fp fractions were removed from the protein A-Sepharose beads of the first immunoprecipitations and subjected to a second immunoprecipitation with the anti-FAD serum to determine the levels of FAD attachment in each fraction. The hsp60-bound and free fractions contain equal proportions of holo-Fp when compared with the amount of Fp recovered after the first immunoprecipitation (not shown). Thus, both the apo- and holo-Fp forms are found in association with hsp60.
To test whether Fp modification requires hsp60, FAD attachment reactions were performed with matrix fractions immunodepleted of hsp60. Western blot analysis with anti-hsp60 serum confirms that hsp60 has been quantitatively removed from the matrix fraction by the immunodepletion (Fig. 9, lane 2) but is still present in normal amounts in the sample immunodepleted with the preimmune serum (data not shown). Immunodepleted matrix fractions were tested in flavinylation reactions (Fig. 10, lanes 6 and 7). The extents of Fp modification in the two fractions were approximately the same, demonstrating that the removal of hsp60 does not inhibit FAD attachment. Thus, hsp60 is not required for Fp modification.
Figure 9: Immunodepletion of hsp60. Samples of mitochondria (lane 1), hsp60 immunodepleted matrix fraction (lane 2), and the immunoprecipitated hsp60 (lane 3), were separated by SDS gel electrophoresis and subjected to Western blot analysis using anti-hsp60 serum.
Figure 10: hsp60 is not essential for Fp modification. In vitro translated Fp precursor (lane 1) was incubated with matrix fractions that had either been incubated on ice for 2 h (lanes 2 and 5), immunodepleted with a preimmune serum (lanes 3 and 6), or immunodepleted with anti-native hsp60 serum (lanes 4 and 7). Samples were analyzed for protein content and processing (lanes 2-4) and the amount of FAD attachment (lanes 5-7).
During the biogenesis of SDH and fumarate reductase enzymes, a covalent FAD cofactor is added to the flavoprotein subunit. Aside from the site of covalent attachment, the Fp interacts with the FAD in at least two other sites, resulting in tight noncovalent binding (Blaut et al., 1989; Robinson et al., 1994). The covalent linkage of the cofactor is necessary to modify the redox midpoint potentials of the enzymes and to permit succinate oxidation (Blaut et al., 1989; Robinson et al., 1994). We have been studying the role of the covalent FAD and the mechanism by which this unusual cofactor is linked to the yeast SDH. We developed an assay for FAD addition that relies on an anti-FAD serum for immunoprecipitation of the modified protein following in vivo or in vitro import of the Fp precursor into mitochondria (Robinson and Lemire, 1996). In this work, we extended our assay system to include flavinylation reactions in mitochondrial matrix fractions. The absence of the mitochondrial membranes removes a permeability barrier between the external space to which Fp precursor is added and the matrix where FAD attachment occurs. Therefore, membrane-impermeable reagents can be tested and experiments can be performed under conditions that are incompatible with the translocation of proteins across the mitochondrial inner membrane.
Matrix proteins are necessary for the flavinylation of the Fp subunit to proceed. The extent of Fp modification is directly proportional to the protein concentration of the matrix fraction (Fig. 2). Furthermore, treatment of matrix fractions by boiling or with N-ethylmaleimide, or digestion with proteinase K all inactivate the flavinylation activity (Fig. 5).
One protein component not involved in the flavinylation reactions in vitro is the Ip subunit. This is in contrast to Fp flavinylation in vivo (Robinson and Lemire, 1996). The yeast SDH Fp and Ip subunits are believed to interact with each other to form an assembly intermediate (Lombardo et al., 1990; Robinson et al., 1991; Schmidt et al., 1992). Similarly, the E. coli fumarate reductase subunits form an active heterodimer in the absence of the membrane subunits (Lemire et al., 1982). The inability of the Ip to stimulate Fp flavinylation in matrix fractions may be because its iron-sulfur clusters are not assembled under the conditions used here, leaving the Ip in an inappropriate conformation. Alternatively, the Ip may need to interact with the membrane subunits, which are missing in these experiments, prior to its association with the Fp. Accordingly, when the membrane subunits are present, the E. coli fumarate reductase Ip assembles with them first and subsequently with the Fp (Latour and Weiner, 1988). Similarly, the Bacillus subtilis SDH Fp and Ip subunits will not form a heterodimer in the absence of the membrane subunit (Hederstedt and Rutberg, 1980; Hederstedt et al., 1982).
Mitochondrial processing peptidase, which
removes the amino-terminal presequence from the Fp precursor, is
required to allow cofactor addition to proceed. Similarly, cytochrome c must be proteolytically processed to its
intermediate form by the mitochondrial processing peptidase before the
heme cofactor can be attached (Nicholson et al., 1989). In our
experiments, no modified precursor Fp is detectable even when the
activity of the peptidase is inhibited, and a significant fraction of
the added Fp remains as precursor (Fig. 6). It is unlikely that
the lack of detection is because the anti-FAD serum does not recognize
the modified precursor, since it detects covalently attached FAD in
completely unrelated flavoproteins, even when the flavin is present in
different linkages (Robinson and Lemire, 1995). The simplest
explanation is that the presequence prevents flavinylation by
inhibiting Fp folding, which we suggest is essential for FAD attachment
(Robinson and Lemire, 1996). It may do this by interacting with
proteins such as the presequence binding factor (Murakami et
al., 1988, 1992; Murakami and Mori, 1990) or the mitochondrial
import stimulation factor (Hachiya et al., 1993) or by
interacting with the remainder of the Fp molecule. By preventing
folding, the presequence may delay in vivo flavinylation until
after Fp import into mitochondria. Mitochondrial processing peptidase
by removing the presequence may initiate folding and events leading to
flavinylation. Whether other protein components in addition to
mitochondrial processing peptidase are needed for flavinylation remains
to be determined.
That presequence cleavage is related to flavinylation is also demonstrated by the lack of modification of pseudomature Fp, which only differs from mature Fp by the addition of an amino-terminal methionine residue (Fig. 7). We propose that the pseudomature Fp is not modified because without a presequence to impede folding, it misfolds in the reticulocyte lysate and adopts a conformation incompatible with FAD attachment. A flavinylation-incompetent state has been observed with the 6-hydroxy-D-nicotine oxidase (Brandsch and Bichler, 1992; Brandsch et al., 1993). Curiously, the amino acid sequence predicts that the yeast SDH Fp is cleaved in two steps: first by the mitochondrial processing peptidase to an intermediate species, and then the remaining octapeptide is removed by the mitochondrial intermediate protease producing the mature sized Fp. Correspondingly, a yeast mitochondrial intermediate protease mutant has no SDH activity, suggesting that this protease is required to process at least one of the SDH subunits (Isaya et al., 1994). Perhaps flavinylation is restricted to the intermediate sized Fp and not to the mature or pseudomature Fps. However, we have never detected an intermediate Fp species and have not examined its role in modification.
Nonprotein components are also required for Fp flavinylation by matrix fractions. ATP may be required for cofactor activation as for biotin or lipoic acid (Schmidt et al., 1969; Gross and Wood, 1984), but this is unlikely because 6-hydroxy-D-nicotine oxidase flavinylation is independent of ATP (Brandsch and Bichler, 1991). Alternatively, the ATP may be required for Fp release from either cytosolic (Pfanner et al., 1990) or mitochondrial heat shock proteins (Cheng et al., 1989; Ostermann et al., 1989) that are necessary for protein folding. Certain Krebs cycle intermediates, especially succinate, fumarate, and malate, which are substrates for SDH, stimulate FAD attachment. Strangely, both oxaloacetate and malonate, which bind tightly to the enzyme active site located in the Fp, do not enhance FAD attachment. Perhaps the nascent active site has a different geometry from that of the fully assembled enzyme and does not recognize these molecules. Alternatively, FAD attachment may require a molecule that can also be oxidized or reduced (Decker, 1993). Clearly, the interconversion of Krebs cycle intermediates in the matrix fractions is not efficient enough to erase the different levels of stimulation we observed with individual intermediates. In contrast to our results with the yeast Fp, citrate and succinate were the most efficient stimulators of E. coli SDH Fp modification (Brandsch and Bichler, 1989). A better understanding the mechanism of effector molecule stimulation may require a more purified system.
Our results suggest that FAD attachment is one of the earliest steps in the SDH Fp assembly pathway. After presequence cleavage, the Fp begins folding with the aid of substrate-like molecules and possibly ATP-dependent chaperones such as mitochondrial heat shock protein 70. FAD itself may act as a nucleation site for Fp folding as it does for the medium chain acyl-CoA dehydrogenase, a noncovalent flavoprotein (Saijo and Tanaka, 1995). Since hsp60 is not required for Fp modification, it most likely interacts with the Fp following FAD attachment. Subsequent to these experiments, we determined that the rabbit reticulocyte lysate added to the matrix fractions contains sufficient FAD to support Fp modification (data not shown). Hence, our experiments do not address whether the Fp must associate with FAD noncovalently before it can be bound by hsp60 or if the chaperonin still recognizes the apo-Fp in the absence of FAD. Notably, the medium chain acyl-CoA dehydrogenase binds to hsp60 only when its flavin cofactor is present (Saijo and Tanaka, 1995). Since almost half of the SDH Fp present is bound by hsp60 (Fig. 8), this interaction is probably real and may reflect a role for hsp60 in assembling the Fp into the SDH holo-complex.
In summary, we have developed an in vitro flavinylation assay using mitochondrial matrix fractions. Translocation across a membrane is not a prerequisite for FAD attachment to the Fp. At least one matrix protein appears to participate in Fp flavinylation, the mitochondrial processing peptidase, which removes the presequence from the precursor. Curiously, although folding seems to be crucial for Fp modification, hsp60 is not required for cofactor attachment. The participation of mitochondrial processing peptidase in cofactor addition does not eliminate the possibility that bond formation is an autocatalytic process; rather, it may, as we believe, signify the need for an appropriate conformation before modification can proceed. Further insights into the mechanism of FAD addition may await the development of an assay with purified components.