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(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
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) ( 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 Covalent
cofactor attachment to mitochondrial and nonmitochondrial proteins can
be mediated by special enzymes. For example, heme is attached to
cytochromes c and c 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 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
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
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
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
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
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
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 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.
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4061-4067
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
)(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).-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. 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.-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).
Media, Strains, and Plasmids
Escherichia
coli and yeast strains and their growth media have been described
previously (Robinson et al., 1991). The plasmids, pSDH1 and
pS1H90S, encode the wild-type and a flavinylation-incompetent His-90
Ser Fp, respectively (Robinson and Lemire, 1992; Robinson et
al., 1994). The plasmid, pT7mFp, encodes an Fp subunit lacking its
presequence in the vector, pBluescript II KS- (Stratagene, La
Jolla, CA). By a polymerase chain reaction, a methionine codon was
introduced in-frame immediately preceding the Gln-29 codon; Gln-29
corresponds to the amino-terminal residue of the processed Fp precursor
(Bullis and Lemire, 1994). The plasmid pT7LSC encodes a chimeric
matrix-targeted precursor, LS, composed of a mitochondrial targeting
sequence fused to the large subunit of ribulose bisphosphate
carboxylase/oxygenase (Rospert et al., 1994). The SDH2 open reading frame was amplified by a polymerase chain reaction
and placed downstream of the T7 promoter to produce the plasmid
pSDHB39.
Preparation of Matrix Fractions
Matrix fractions
were prepared from mitoplasts (Jascur, 1991) suspended at 10 mg/ml
protein in Flavinylation Buffer (20 mM Hepes-KOH, pH 7.4, 5
mM ATP, 5 mM MgCl
, 50 µM MnCl, 50 µM ZnCl, 50
µM FAD, 10 mM succinate, 10 mM fumarate,
10 µg/ml oligomycin, and the protease inhibitors phosphoramidon
(1.25 µg/ml), N-p-tosyl-L-lysine
chloromethyl ketone (2.5 µg/ml), tosyl-L-phenylalanyl
chloromethyl ketone (5.0 µg/ml), 4-(2-aminoethyl)benzenesulfonyl
fluoride (2.5 µg/ml),
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (2.5
µg/ml), pepstatin A (6.5 µg/ml), aprotinin (52.5 µg/ml),
benzamidine (5 mM), leupeptin (51 µg/ml),
-macroglobulin (1 mg/ml), and phenylmethylsulfonyl
fluoride (1 mM)). Mitoplasts were disrupted by freezing in
liquid nitrogen and thawing while sonicating in a Branson 1200 water
bath (Glick et al., 1992b). This freeze-thaw cycle was
repeated 4 times, 5 mM 
-mercaptoethanol was added, and
the membranes were pelleted at 12,000 
g for 10 min and
washed once. Solubilized mitochondria or mitochondrial membranes were
prepared by resuspending in Flavinylation Buffer containing 1% Triton
X-100. 5 mM -mercaptoethanol was added after
solubilization or sonication.FAD Attachment Reactions and Assay
For
flavinylation reactions, 10 µl of rabbit reticulocyte lysate that
had been programmed with mRNA encoding the Fp precursor was added to 40
µl of matrix fraction or detergent-solubilized mitochondria. After
a 20-min incubation at 30 °C, the reaction was stopped by the
addition of 1 ml of 4% SDS, and the proteins were precipitated by the
addition of trichloroacetic acid to 11% final concentration. Samples
were incubated 10 min at room temperature and 10 min on ice and were
centrifuged for 5 min at 12,000 g. Samples were
reprecipitated with trichloroacetic acid and washed with acetone as
described in the accompanying article (Robinson and Lemire, 1996). The
final protein pellet was resuspended in 100 µl of 1% Triton X-100,
0.1 M Tris-HCl, pH 7.5. To determine the extent of proteolytic
processing, 4.5 µl of each sample was analyzed by SDS gel
electrophoresis and fluorography. Covalent FAD attachment was monitored
by immunoprecipitating modified Fp from 90 µl of each sample with
anti-FAD serum as described in the accompanying article (Robinson and
Lemire, 1996). All bands were quantified with a model BAS1000
Bio-Imaging analyzer (Fuji Photo Film Co., Ltd.).Native Immunoprecipitatons With Anti-hsp60
Serum
Flavinylation reactions incubated for 15 min at 30 °C
were further treated with 20 units/ml apyrase (Sigma, grade VIII) for
an additional 10 min to hydrolyze ATP and ADP to AMP. Native
immunoprecipitations with anti-hsp60 serum were performed as described
previously (Rospert et al., 1994) using 10 µl of serum for
12 µl of flavinylation reaction mix. Protein A-Sepharose bead-bound
proteins were removed by heating at 65 °C in 15 µl of IPLB (0.1 M Tris-HCl, 5% SDS, 5 mM EDTA, 0.005% bromphenol
blue, 25% glycerol, 5% -mercaptoethanol, pH 6.8) for 8 min. Beads
were removed by centrifugation and re-extracted with IPLB as above, and
the supernatants were combined.Immunodepletion of Matrix Fractions
60 µl of
matrix fraction at a protein concentration of 6 mg/ml was added to 120
µl of protein A-Sepharose beads that had been preincubated with 30
µl of either anti-native hsp60 serum or preimmune serum. The slurry
was incubated at 4 °C for 2 h with rocking, the beads were spun
down with a brief centrifugation, and the supernatant was collected.
The protein A-Sepharose beads were rinsed with 10 µl of
Flavinylation Buffer, which was added to the first supernatant
fraction. The immunodepleted matrix fractions (10 µl) were removed
and analyzed with anti-hsp60 serum by Western blotting. An additional
aliquot of ATP to 5 mM was added to the remaining
immunodepleted matrix fraction. Rabbit reticulocyte lysate (10 µl)
that had been incubated with mRNA encoding the Fp precursor was added
immediately, and the FAD attachment assays were performed as described
above.Miscellaneous
In vitro translated Fp
precursor was prepared in rabbit reticulocyte lysate as described by
the supplier (Promega Corp.) in the presence of radiolabeled methionine
(TranS-label; ICN Biomedicals). The anti-FAD and anti-Fp
antisera have been described previously (Robinson et al.,
1991; Robinson and Lemire, 1995). All protease inhibitors and other
reagents are from Sigma.
Flavinylation Requires Mitochondrial Matrix
Fractions
FAD attachment to the Fp occurs during in vitro import into isolated mitochondria or mitoplasts (Robinson and
Lemire, 1996). Investigation of the flavinylation mechanism with import
experiments is limited to conditions that are compatible with protein
translocation. Furthermore, the mitochondrial inner membrane is a
barrier that prevents full access to the matrix, the site of FAD
attachment (Robinson and Lemire, 1996). To determine whether import and
Fp modification are coupled and to remove the experimental limitations
of maintaining import competent organelles, we developed an in
vitro flavinylation system using matrix fractions., 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).
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.
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).
,
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.
, 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%.
Proteolytic Processing Is Required for FAD
Attachment
Cofactor attachment to the mature but not the
precursor form of the Fp is detected both in vivo and in
vitro (Robinson and Lemire, 1996), although the fraction of Fp
present in the precursor form is usually quite small unless import or
processing are inhibited. Hence, mitochondrial processing peptidase, a
mitochondrial matrix metalloprotease, is a candidate for the matrix
protein component required for Fp modification (Arretz et al.,
1991). We treated matrix fractions with EDTA in the presence or absence
of ATP to inactivate the mitochondrial processing peptidase. We
reasoned that the mitochondrial processing peptidase might not be
inactivated in the presence of ATP since hsp60 might stabilize it. The
peptidase's solubility and function are intimately connected to
the hsp60 function (Glick et al., 1992b; Hallberg et
al., 1993). Matrix fractions were prepared in Flavinylation Buffer
without ATP and divalent cations, treated with EDTA for 10 min to
chelate endogenous Zn, Mn
, and
Mg
ions, and then resupplied with one or more of the
cations. As a control, Fp precursor (Fig. 6, lane 1) was
incubated with a matrix fraction that had been prepared in the normal
way (lane 2). This resulted in substantial processing to the
mature size (lane 2) and flavinylation of the mature Fp (lane 6). Fractions treated with EDTA in the presence of added
ATP showed similar levels of Fp processing (lane 3) but
reduced levels of flavinylation (lane 7). Fractions treated in
the absence of ATP and resupplied with all three divalent cations were
able to process (lane 4) and inefficiently flavinylate the Fp (lane 8). Only in fractions treated in the absence of ATP and
resupplied with Mg
was a significant increase in the
levels of unprocessed Fp seen (lane 5). When the flavinylation
products of this reaction were analyzed, only the mature-sized Fp was
immunoprecipitated with the anti-FAD serum (lane 9). Even
though substantial Fp precursor remains at the end of the flavinylation
reaction (lane 5), it has not been flavinylated (lane
9), suggesting that the precursor is incompetent for FAD
attachment.
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).
hsp60 Binds Fp
The requirement for proteolytic
processing of the Fp prior to flavinylation could explain the need for
a matrix protein component, but it does not account for an ATP
requirement. We have suggested that FAD addition is to a folded Fp
molecule (Robinson and Lemire, 1996). For this reason, we investigated
whether the mitochondrial chaperonin, hsp60, which assists in the
ATP-dependent folding of proteins, has a role in Fp flavinylation
(Becker and Craig, 1994). We first needed to determine whether the Fp
interacts with hsp60. Co-immunoprecipitation with specific anti-hsp60
antibodies is a reliable method of monitoring association with hsp60
(Rospert et al., 1994). The fusion protein, LS, which contains
a matrix targeting signal fused to the large subunit of Chlamydomonas ribulose bisphosphate carboxylase/oxygenase, was
used as a control for hsp60 binding (Rospert et al., 1994). In vitro translated LS or Fp precursor proteins were incubated
in matrix fractions for 15 min before the addition of apyrase to
hydrolyze ATP and inhibit the release of bound proteins from hsp60.
Samples were divided, and the proteins were analyzed by denaturing gel
electrophoresis and fluorography (Fig. 8, lanes
3-7) or by immunoprecipitations with anti-native hsp60 serum
to determine hsp60 association (lanes 8-12). As a control to
show that the anti-hsp60 serum does not recognize the Fp, a sample
consisting of Fp in Flavinylation Buffer without matrix fraction was
also subjected to the immunoprecipitation protocol (lane 12).
Both the LS and Fp precursors were efficiently translated (lanes 1 and 2, respectively), proteolytically processed to mature
forms (lanes 3 and 5, respectively), and
co-immunoprecipitated with hsp60 (lanes 8 and 10,
respectively). In the absence of matrix fraction, the Fp was neither
proteolytically processed (lane 7) nor immunoprecipitated with
the anti-native hsp60 serum (lane 12). The fractions of LS and
Fp bound to hsp60 under these conditions are 15 and 43%, respectively.
The addition of FAD to the matrix fractions did not affect Fp
processing (lane 6), Fp association with hsp60 (lane
11), or results with the LS precursor (lanes 4 and 9). These results demonstrate that the Fp interacts with
mitochondrial hsp60.
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.
)
We thank Sabine Rospert and Jeff Schatz for kindly
providing the native anti-hsp60 serum and a plasmid encoding the LS
precursor. We also thank Richard Hallberg for anti-hsp60 serum.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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