|
|
|
|||||||
|
|||||||||
(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), ( 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 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
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
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
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
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
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. ( Proteolytic processing
can be a mandatory step for cofactor attachment. For example,
cytochrome c 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
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4055-4060
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)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. 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). 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.
Strains, Media, and Plasmids
Yeast strains used
are described in Table 1. Yeast media, the Escherichia coli strains, UT580 and DH5, and the plasmids, pSDH1 and pS1H90S,
have been described previously (Robinson et al., 1991;
Robinson and Lemire, 1992; Robinson et al., 1994). The
plasmid, pSfRHAC, which encodes the SDH1 gene without any
5`-untranslated sequence, was created in two steps. pSDH1 was cut with SfaN1 and the ends were blunted with the Klenow fragment of
DNA polymerase and digested with EcoRI (see Fig. 5).
The SfaN1/EcoRI fragment encoding the Fp amino
terminus was cloned into EcoRV- and EcoRI-digested
pBluescript II SK- (Stratagene, La Jolla, CA), placing the
partial SDH1 coding sequence under the expression of the T7
promoter and creating pSfR1. To reconstruct the entire SDH1 gene, pSDH1 was digested with NdeI, the ends were
blunted, and it was cut with EcoRI, and the NdeI/EcoRI fragment encoding the Fp carboxyl terminus
was cloned into EcoRI- and SmaI-digested pSfR1. The SDH1 gene from pSfRHAC, was inserted into the multicopy
vector, YEplac195 (Gietz and Sugino, 1988) and placed under the control
of the copper metallothionein promoter (CUP1) obtained from
the vector, Yep96 (Ellison and Hochstrasser, 1991), to create the
plasmid, pCuSDH1. To place SDH2 under CUP1 control,
the SDH2 open reading frame was amplified by a polymerase
chain reaction and cloned into the vector, pTCu, which is YEplac112
(Gietz and Sugino, 1988) bearing the CUP1 promoter, to create
pCuB. The integrity of the SDH2 gene was established by
complementation of an SDH2 disruption mutant. To select for
the SDH2 gene in the yeast strain Sdh1Ad1, the LYS2 gene (Fleig et al., 1986) was inserted into pCuB, making
the plasmid pKCuSDH2.
In Vivo Labeling
Labeling of cellular proteins was
based on a published procedure (Brandt, 1991). Cells were grown
overnight on semisynthetic media containing 0.15% yeast extract as well
as the necessary auxotrophic markers and a carbon source of either 2%
glucose, galactose, or lactate. MH125 and related strains were
supplemented with 0.1% glucose when grown on galactose. Strains
carrying plasmids with the CUP1 promoter were labeled without
induction. When the A of the culture was between
0.8 and 1.0, it was quickly harvested and resuspended in prewarmed
labeling buffer containing auxotrophic markers and 2% carbon source.
Radiolabeled methionine was added (Tran
S-label, 81
µCi/ml; 1037 Ci/mmol; ICN Biomedicals, Mississauga, Ontario), and
the cells were labeled for 3 min at 30 °C with vigorous shaking.
Cycloheximide and cold methionine were added to 100 µg/ml and 2
mM, respectively, and the chase continued at 30 °C with
vigorous shaking. At the indicated times, 0.5-ml aliquots were taken,
lysed with NaOH and 2-mercaptoethanol, and precipitated with
trichloroacetic acid (Yaffe and Schatz, 1984). Lysates were pelleted
(14,000
g for 10 min), resuspended in 1 ml of 2% SDS
and 1% trichloroacetic acid. 0.1 ml of 55% trichloroacetic acid was
added to reprecipitate proteins, and the sample was incubated at room
temperature for 10 min and then on ice for an additional 10 min. We
found it necessary to wash the precipitates in SDS and to reprecipitate
with trichloroacetic acid for the anti-FAD serum to efficiently
immunoprecipitate holo-Fp. Lysates were pelleted, washed with 1 ml of
ice-cold acetone, resuspended in 115 µl of 2% SDS, 100 mM Tris-HCl, pH 7.5, and heated to between 65 and 90 °C for 10
min. Insoluble material was removed by centrifugation at 14,000 g for 10 min, and the supernatant was used for
immunoprecipitations. The efficiencies of the labeling and the chase
were monitored as described previously (Brandt, 1991).In Vitro Transcription, Translation, and
Import
Template mRNA was produced from the plasmids, pSDH1,
pS1H90S, or pSfRHAC using T7 RNA polymerase and translated in rabbit
reticulocyte lysate as described by the supplier (Promega Corp.).
Carboxyl-terminal truncated Fps were produced from the plasmid,
pSfRHAC, that had been digested with the restriction enzymes, EcoRI or PvuI1, before transcription and translation.
Import reactions containing 400 µg of isolated mitochondria or
mitoplasts in a volume of 320 µl were performed essentially as
described using 10 µl of lysate/import reaction (Gasser et
al., 1982; Robinson et al., 1994). Import reactions were
supplemented with 20 mM succinate, 10 mM malate, and
50 µM FAD unless stated otherwise. Import was allowed to
proceed for 1 h at 30 °C. After proteinase K treatments (50 µg
for 15 min at 0 °C), re-isolated mitochondria or mitoplasts were
resuspended in 1 ml of 2% SDS, 0.1 M Tris-HCl, pH 7.5, and
prepared for immunoprecipitation as described above. 90% of the sample
was used for immunoprecipitation, and 4.5% was separated by denaturing
gel electrophoresis to determine protein import.Immunoprecipitations
Immunoprecipitations were
performed as described previously (Brandt, 1991) with the following
modifications. 45 µl of in vivo cell lysate was added to 1
ml of BTNTE (2 mg/ml bovine serum albumin, 50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, and
0.02% NaN
) containing 5 µl of preimmune serum and 1
mM phenylmethylsulfonyl fluoride. Samples were incubated 1 h
at room temperature with rotation and centrifuged at 14,000 g for 10 min. 8 µl of protein A-Sepharose beads (Sigma;
binding capacity of 20 mg human IgG/ml) were added to the supernatants
incubated as above. The beads were removed by centrifugation, and 10
µl of either anti-FAD serum or anti-Fp serum (Robinson et
al., 1991) were added. The anti-FAD serum immunoprecipitates only
the modified or holo-Fp; the anti-Fp serum immunoprecipitates both the
modified and the apo-Fp. Samples were incubated overnight with rocking
at 4 °C. Aggregated protein was removed by centrifugation at 14,000
g for 10 min, and 16 µl of protein A-Sepharose
beads were incubated with the supernatants for 1 h at room temperature.
The beads were pelleted with a brief centrifugation, the supernatant
was removed, and the beads were washed twice with NaTNTE (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% Triton X-100, 5 mM EDTA), twice with TNTE (50 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1% Triton X-100, 5 mM EDTA), and finally
twice with NTE (150 mM NaCl, 10 mM Tris-HCl, pH 7.5,
5 mM EDTA). The beads were resuspended in 20 µl of IPLB
(0.1 M Tris-HCl, pH 6.8, 5% SDS, 5 mM EDTA, 0.005%
bromphenol blue, 25% glycerol, 5% 2-mercaptoethanol), heated for 10 min
at 65-90 °C, and spun briefly, and the supernatant was
resolved by SDS-polyacrylamide gel electrophoresis. The gels were
treated for fluorography, exposed to x-ray film, and, when desired, the
bands were quantified with a model BAS1000 phosphoimager (Fuji Photo
Film Co., Ltd.). To determine the extent of Fp modification, the amount
of Fp immunoprecipitated by the anti-FAD serum was divided by the
amount of Fp immunoprecipitated by the anti-Fp serum. For
immunoprecipitations from in vitro translation experiments, 90
µl of lysate was added to 1 ml of BTNTE containing 1 mM phenylmethylsulfonyl fluoride and 10 µl of protein A-Sepharose
beads. Samples were treated as above except that 20 µl of anti-FAD
serum and 30 µl of protein A-Sepharose beads were used.
In Vivo Flavinylation of the Yeast Fp
We have
previously shown that our anti-FAD serum only recognizes flavinylated
protein and that this recognition can be competed for with free FAD,
FMN, or riboflavin (Robinson and Lemire, 1995). The anti-Fp serum
immunoprecipitates both apo- and holo-Fp forms of the protein.
Therefore, the amount of FAD-modified Fp can be expressed as the amount
of Fp immunoprecipitated by the anti-FAD serum divided by the amount of
Fp immunoprecipitated by the anti-Fp serum. Quantitation is performed
with a phosphoimager. The in vivo rate of FAD attachment to
the Fp was examined with a pulse-chase experiment in the wild-type
strain, D273-10B (Table 1). Aliquots taken during the chase
period were analyzed by immunoprecipitation with the anti-FAD and the
anti-Fp sera. Only the 67-kDa mature Fp is detected in this experiment
since all of the Fp molecules rapidly reach the mitochondrial matrix
where they are proteolytically processed (Brandt, 1991). The amount of
labeled Fp is constant throughout the chase period, indicating that the
chase has been effective and that the mature Fp is stable over this
time period (Fig. 1, -Fp row). In contrast, the
amount of flavinylated Fp immunoprecipitated with the anti-FAD serum is
initially undetectable and increases rapidly with time (Fig. 1, -FAD row), demonstrating that the Fp is modified
post-translationally after its presequence has been removed. Although
it did not exceed 15%, flavinylation occurred with a half-time of about
5 min. The low efficiency is in part due to incomplete
immunoprecipitation by the anti-FAD serum; larger volumes of serum do
not significantly increase the amount of Fp precipitated. However,
reprecipitation of the supernatant of a first round of
immunoprecipitation indicates significant quantities of FAD-modified Fp
remain (KMR, unpublished observations).
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.''
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.
), 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.
In Vitro Flavinylation of the Yeast Fp
To test
whether modification by FAD could occur in vitro and thus
provide a more convenient and manipulatable system for further
investigations, we examined Fp flavinylation during import into both
isolated whole mitochondria and mitoplasts (mitochondria with their
outer membranes disrupted). In vitro translated wild-type or
His-90 Ser precursor proteins were incubated with isolated
mitochondria or mitoplasts, the organelles were reisolated, and
cofactor attachment was assayed by immunoprecipitation with the
anti-FAD serum (Fig. 4). If import is blocked by the ionophore,
valinomycin, modification of the surface-bound Fp is not detected (lanes 2 and 3,
-FAD row), indicating
that FAD is not attached prior to import. We found a significant
proportion of the wild-type Fp precursor could be imported into
mitoplasts, processed (5% of imported Fp row), and modified by
FAD (lane 5, -FAD row), demonstrating that the
FAD could be attached to the Fp in vitro. In these
experiments, mitoplasts imported, and hence modified, larger quantities
of the Fp than intact mitochondria (compare lanes 4 and 5; Hwang et al., 1989). However, the fraction of
imported Fp that was flavinylated in mitochondria and mitoplasts is
approximately the same. FAD attachment might be occurring either during
or after import. To distinguish between these possibilities, we
performed import reactions and examined the effect of proteinase
treatment on flavinylation. Proteinase treatment removes proteins that
have not been fully imported. In these experiments, the extent of FAD
attachment is not affected by proteinase treatment (compare lanes 5 and 7), suggesting that flavinylation requires complete
import of the Fp into the mitochondria or mitoplasts. The His-90
Ser Fp (lane 8), although imported to the same extent as the
wild-type, was not immunoprecipitated by the anti-FAD serum.
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.
)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). 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. (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.
))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
ABSTRACT
This article has been cited by other articles:
|
|
 |
|
  M. Marelli, J. J. Smith, S. Jung, E. Yi, A. I. Nesvizhskii, R. H. Christmas, R. A. Saleem, Y. Y. C. Tam, A. Fagarasanu, D. R. Goodlett, et al. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane J. Cell Biol., December 20, 2004; 167(6): 1099 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bafunno, T. A. Giancaspero, C. Brizio, D. Bufano, S. Passarella, E. Boles, and M. Barile Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria: INVOLVEMENT OF THE Flx1p CARRIER IN FAD EXPORT J. Biol. Chem., January 2, 2004; 279(1): 95 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Vergani, M. Barile, C. Angelini, A. B. Burlina, L. Nijtmans, M. P. Freda, C. Brizio, E. Zerbetto, and F. Dabbeni-Sala Riboflavin therapy: Biochemical heterogeneity in two adult lipid storage myopathies Brain, December 1, 1999; 122(12): 2401 - 2411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Oyedotun and B. D. Lemire The Saccharomyces cerevisiae Succinate-ubiquinone Oxidoreductase. IDENTIFICATION OF SDH3P AMINO ACID RESIDUES INVOLVED IN UBIQUINONE BINDING J. Biol. Chem., August 20, 1999; 274(34): 23956 - 23962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Miller and D. E. Edmondson Influence of Flavin Analogue Structure on the Catalytic Activities and Flavinylation Reactions of Recombinant Human Liver Monoamine Oxidases A and B J. Biol. Chem., August 13, 1999; 274(33): 23515 - 23525. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Przybyla-Zawislak, D. M. Gadde, K. Ducharme, and M. T. McCammon Genetic and Biochemical Interactions Involving Tricarboxylic Acid Cycle (TCA) Function Using a Collection of Mutants Defective in All TCA Cycle Genes Genetics, May 1, 1999; 152(1): 153 - 166. [Abstract] [Full Text]
|
|
|
|
|
|
B. P. Zhou, B. Wu, S.-W. Kwan, and C. W. Abell Characterization of a Highly Conserved FAD-binding Site in Human Monoamine Oxidase B J. Biol. Chem., June 12, 1998; 273(24): 14862 - 14868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Oyedotun and B. D. Lemire The Carboxyl Terminus of the Saccharomyces cerevisiae Succinate Dehydrogenase Membrane Subunit, SDH4p, Is Necessary for Ubiquinone Reduction and Enzyme Stability J. Biol. Chem., December 12, 1997; 272(50): 31382 - 31388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Robinson and B. D. Lemire A Requirement for Matrix Processing Peptidase but Not for Mitochondrial Chaperonin in the Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein J. Biol. Chem., February 23, 1996; 271(8): 4061 - 4067. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |