(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 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.
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), ()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 8
-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 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
8
-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.
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.
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 (
-Fp row) or the anti-FAD serum (
-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).
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 (
-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.
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. ()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.
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 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 (Dumont et al., 1991; Nargang et al., 1988), for biotin
addition to carboxylases (Gross and Wood, 1984), and for lipoic acid
addition to
-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.