(Received for publication, December 26, 1995; and in revised form, February 6, 1996)
From the
Rat dimethylglycine dehydrogenase (MeGlyDH) was used
as model protein to study the biogenesis of a covalently flavinylated
mitochondrial enzyme. Here we show that: 1) enzymatically active
holoenzyme correlated with trypsin resistance of the protein; 2)
folding of the reticulocyte lysate-translated protein into the
trypsin-resistant, holoenzyme form was a slow process that was
stimulated by the presence of the flavin cofactor and was more
efficient at 15 °C than at 30 °C; 3) the mitochondrial
presequence reduced the extent but did not prevent holoenzyme
formation; 4) covalent attachment of FAD to the Me
GlyDH
apoenzyme proceeded spontaneously and did not require a mitochondrial
protein factor; 5) in vitro only the precursor, but not the
mature form, of the protein was imported into isolated rat liver
mitochondria; in vivo, in stably transfected HepG2 cells, both
the precursor and the mature form were imported into the organelle; 6)
holoenzyme formation in the cytoplasm did not prevent the translocation
of the proteins into the mitochondria in vivo; and 7) lack of
vitamin B
in the tissue culture medium resulted in a
reduced recovery of the precursor and the mature form of
Me
GlyDH from cell mitochondria, suggesting a decreased
efficiency of mitochondrial protein import.
Since the first description of an enzyme with covalently
attached FAD (the flavoprotein subunit of succinate
dehydrogenase)(1) , an increasing number of flavinylated
enzymes have been discovered (for a review, see (2) ). Several
were added to this list recently: rat liver L-pipecolic acid
oxidase(3) , plant reticulin oxidoreductase(4) ,
streptomyces mitomycin resistance protein(5) , and penicillin
vanillyl-alcohol oxidase(6) . Using the bacterial
6-hydroxy-D-nicotine oxidase (EC 1.5.3.6; 6-HDNO) ()as a model enzyme, we showed that the covalent attachment
of FAD to His-71 of the polypeptide via an
FAD(8
)-(N
)histidyl linkage, the most common bond
encountered in this group of flavoenzymes, takes place
autocatalytically(7) .
In eukaryotic cells, enzymes bearing
this covalent modification are all compartimentalized: the fungal
enzyme vanillyl-alcohol oxidase in the glyoxisome, the plant enzyme
reticulin oxidoreductase in a special vacuolar compartment, in
mammalian cells L-gulono--lactone oxidase (involved in
vitamin C synthesis which is missing in humans) (8) in liver
microsomes, rat L-pipecolic acid oxidase in liver peroxisomes,
monoamine oxidase in the outer mitochondrial membrane, the flavoprotein
subunit of the succinate dehydrogenase complex in the inner
mitochondrial membrane, dimethylglycine dehydrogenase (EC 1.5.99.2;
Me
GlyDH), and sarcosine dehydrogenase in the mitochondrial
matrix. This particular cellular location raises questions regarding
the biogenesis of these enzymes. A holoenzyme synthetase within the
cell compartment could be required for the flavinylation of the
imported apoenzymes. Alternatively, attachment of FAD to the enzyme
could proceed spontaneously during or following folding of the
imported, mature form of the protein into its native conformation. It
is generally assumed that import of a precursor protein into
mitochondria requires its unfolding. Inside the organelle the precursor
presequence is then removed by a special peptidase and the protein
allowed to fold. One function of the presequence of mitochondrial
proteins seems to consist in its interaction with cellular chaperones
which keep the molecule in a loosly folded, import-competent
conformation (for a review, see (9) ). Given this scenario of
mitochondrial protein import, one may anticipate that spontaneous
attachment of FAD to the precursor will not take place since
autoflavinylation requires the folding of the protein into its native
conformation(7) . In addition, the covalently attached cofactor
may block the import of the protein into its place of
destination(9) . Previous work performed with the bacterial
enzyme 6-HDNO fused to the Me
GlyDH presequence showed that
import of the fusion protein into rat liver mitochondria in vitro was not inhibited by the bound FAD(10) . However, no
detailed data were available on the biogenesis of an authentic
mitochondrial flavinylated enzyme in eukaryotic cells. Here we present
results obtained in vitro in the rabbit reticulocyte lysate
(RL) and in vivo in stably transfected HepG2 cells on the
flavinylation, cofactor-dependent folding and mitochondrial import of
the mature and precursor form of rat liver mitochondrial
Me
GlyDH.
For expression of the MeGlyDH
proteins in eukaryotic cell lines, plasmids pSPT19-pMe
GlyDH
and pSPT19-mMe
GlyDH were linearized by digestion with EcoRI, the restriction site filled in with the Klenow fragment
of DNA polymerase I, followed by digestion with the restriction enzyme XbaI. The DNA fragment corresponding to the
Me
GlyDH cDNA was isolated and inserted by ligation into the
eukaryotic expression vector pRC-CMV (Promega) digested with HindIII, blunt ended with the Klenow fragment of DNA
polymerase I, followed by digestion with XbaI. Plasmids were
maintained in E. coli strain JM 109 and plasmid DNA was
isolated from bacterial cells by a DNA isolation kit (Diagen, Hilden,
FRG).
For
immunoprecipitation 20 µl of a suspension of 100 mg Protein
A-Sepharose/ml was incubated for 1 h at 4 °C with 10 µl
anti-MeGlyDH antiserum in 300 µl of Triton buffer (1%
Triton, 300 mM NaCl, 5 mM EDTA, 20 mM Tris,
pH 7.5). To the centrifuged and washed pellet 200 µg of protein of
either cell lysate or cytosolic or mitochondrial fraction and 300
µl of Triton buffer were added, and the mixture was incubated for 1
h at 4 °C. Following centrifugation and washing four times with
Triton buffer, the immunocomplexes were analyzed by SDS-PAGE according
to Laemmli(21) . The 7.5% gels were fixed, dried, and
autoradiographed.
Figure 1:
In vivo expression of MeGlyDH, activity stain and trypsin
resistance. Panel A, cytosolic (lane 1) and
mitochondrial fractions (lanes 2 and 3) of isolated
rat liver mitochondria were trypsin digested (lane 3),
analyzed on 7.5% SDS-PAGE, Western blotted, and decorated with
antibodies to Me
GlyDH. The mitochondrial fraction (lane
4) was separated after digestion with 0.2 mg/ml trypsin (lane
5) under nondenaturing conditions and activity stained for
Me
GlyDH. Panels B and C, HepG2 cells
transfected with pRC-CMV-pMe
GlyDH and
pRC-CMV-mMe
GlyDH were fractionated in cytosol (lane
1) and mitochondria (lanes 2 and 3) and analyzed
by activity staining (lanes 4 and 5) in the same way
as in Panel A.
Human hepatoblastoma HepG2 cells, which do not
express MeGlyDH (results not shown), were stably
transfected with pRC-CMV-pMe
GlyDH and
pRC-CMV-mMe
GlyDH, encoding the precursor and mature forms
of the protein, respectively. Analysis of the intracellular
distribution of the Me
GlyDH proteins on Western blots
showed that both the precursor as well as the mature form were imported
into mitochondria (Fig. 1, Panel B and C). The
imported protein showed trypsin resistance (Fig. 1, Panels B and C, lanes 3), and exhibited on nondenaturing
gels enzymatic activity and the same microheterogeneity as the
Me
GlyDH protein isolated from rat liver mitochondria (Fig. 1, Panel B and C, lanes 4 and 5).
Figure 2:
In vitro folding and holoenzyme
formation of MeGlyDH in the rabbit RL; dependence on
temperature, cofactor additions and mitochondrial matrix. Panel
A, coupled transcription-translation of Me
GlyDH
proteins was carried out in rabbit RL as described under
``Materials and Methods.'' Translation was stopped and
incubation continued at either 15 or 30 °C (lanes 1 and 3, respectively). After 5 h, half of each assay was subjected
to trypsin digestion (lanes 2 and 4). The labeled
proteins were separated by SDS-PAGE, and the dried polyacrylamide gels
were autoradiographed. Panel B, coupled
transcription-translation of mMe
GlyDH was carried out at
30 °C in the absence of cofactors (
), in the presence of 10
µM FAD (
), in the presence of 10 µM FAD
and 10 µM folate (
), or in the presence of 10
µM FAD, 10 µM folate, and 10 µM dimethylglycine (
). After 30 min cycloheximide and RNase A
were added, and incubation was continued at 15 °C. At the indicated
time points equal aliquots were taken and submitted to trypsin
digestion and analyzed by SDS-PAGE. Shown is the percentage of
trypsin-resistant mMe
GlyDH protein present after protease
treatment as compared to the amount of mMe
GlyDH protein in
undigested control assays. Panel C, as in Panel B,
but performed with pMe
GlyDH. Panel D, coupled
transcription-translation of pMe
GlyDH and
mMe
GlyDH was stopped after 30 min, 2 µl of the assays
were trypsin-digested; incubation of the remaining assays was continued
at 15 °C in the presence or absence of 25 µg of mitochondrial
matrix extract. Aliquots were taken, trypsin-digested, and analyzed as
in Panel B. The amount of trypsin-resistant
Me
GlyDH proteins was expressed as percentage of
Me
GlyDH present in parallel assays not treated with
protease, at 0 h of incubation (black bars) without or with
mitochondrial matrix or after 5 h of incubation (white bars)
without or with mitochondrial matrix.
The RL contains low levels of endogenous
FAD(24) , which may not be sufficient for MeGlyDH
holoenzyme formation. The second cofactor of the enzyme,
H
PteGlu
is unstable; but the protein also binds
folic acid. We analyzed the folding kinetic of the RL translated mature
and precursor Me
GlyDH in the presence of externally added
FAD, folic acid and the Me
GlyDH substrate, dimethylglycine (Fig. 2, Panels B and C). After 20 h the
highest yield of the native, trypsin-resistant holoenzyme form was
obtained when both cofactors were present, the stimulating effect of
the cofactors being strongest during the initial phase of folding (2.5
h). After 20 h, assays without external additions reached the same
level of trypsin-resistant Me
GlyDH as that obtained in the
presence of FAD only (Fig. 2, Panel B,
and
). The presence of the substrate dimethylglycine did not change
the kinetics of folding (Fig. 2, Panel B,
).
Thus, the efficiency of folding of mMe
GlyDH was dependent
on the cofactor concentrations in the assays.
The yield of
trypsin-resistant pMeGlyDH was lower when compared to that
obtained with mMe
GlyDH. Within the first 2.5 h folding was
more efficient in the presence of cofactors and substrate (Fig. 2, Panel C,
and
) than without
additions (Fig. 2, Panel C,
). The final yield of
trypsin-resistant protein varied between 20 and 40%. Apparently the
presequence hampered the folding of the protein into the
trypsin-resistant conformation. Binding of RL chaperons to the
presequence could be responsible for the slowed and less efficient
folding of the precursor. We analyzed the folding of the two proteins
in the presence of added ATP without noting any effect on the
efficiency of folding of the mature protein (not shown). However,
folding of the precursor protein into the trypsin-resistant form was
stimulated in the presence of ATP (33% trypsin resistance after 5 h as
compared to 20% in the absence of ATP).
The experiments presented thus far did not exclude the possibility that a holoenzyme synthetase present in the mitochondrial matrix may stimulate folding of the mature protein into its trypsin resistant conformation and thus FAD attachment. Addition of various concentrations of mitochondrial protein extract (10, 25, and 50 µg per assay) to the folding reactions had, however, no effect (Fig. 2, Panel D, shows results at 25 µg of mitochondrial protein).
Incubation of mitochondria with
flavins prior to import increased the yield of trypsin-resistant
MeGlyDH holoenzyme in the matrix, the highest increase
being observed with FAD (70% trypsin resistance with as compared to 45%
without preincubation of mitochondria with FAD).
MeGlyDH
contains a FAD and a H
PteGlu
binding domain.
This conclusion is inferred from the primary sequence of the protein,
which shows a typically dinucleotide binding motif
(Gly
XGlyXXGly
) situated at
the NH
-terminal part of the protein (11) and a
positively charged sequence rich in Lys residues
(Lys
-XX-Lys-XXXXXX-Lys-XXX-Lys-XX-Lys-XLys-XX-Lys-ArgArg
)
at the COOH-terminal part of the protein. Based on the comparison with
the folate polyglutamate cofactor binding site of other proteins (25) this sequence may represent the binding site of the
H
PteGlu
cofactor of Me
GlyDH. In
addition, recent sequence comparison of bacterial sarcosine oxidase
with Me
GlyDH revealed a significant amino acid sequence
similarity among the enzymes in the FAD-binding domain(26) .
These considerations prompted us to examine whether the
NH
-terminal FAD-domain of the protein folds by itself, in
an FAD-dependent manner, into a trypsin-resistant conformation. Two
COOH-terminal deletions were studied, the first reduces the 95-kDa
pMe
GlyDH to a 68-kDa protein (
SFU) and the second to a
52-kDa protein (
XHO). When these deleted proteins were translated
in the RL, they were in a trypsin-sensitive form. They were imported
into rat liver mitochondria (Fig. 3, Panel A), but
remained trypsin-sensitive inside the mitochondrial matrix (Fig. 3, Panel B, lanes 1 and 2).
Preincubation of mitochondria with various flavins previous to import,
did not change the trypsin sensitivity of the imported deletion
proteins (Fig. 3, Panel B, lanes 3-8).
These results suggest that the flavin domain does not fold
independently into a trypsin-resistant conformation, but that folding
and therefore FAD attachment requires the entire polypeptide chain.
Figure 3:
Import of the COOH-terminally deleted
pMeGlyDH/
SFU and pMe
GlyDH/
XHO
proteins into rat liver mitochondria. Panel A,
[
S]Met-labeled pMe
GlyDH/
SFU and
pMe
GlyDH/
XHO proteins were synthesized in the rabbit
RL and translation assays were analyzed by SDS-PAGE without (lane
1) or following trypsin digestion (lane 2). Aliquots of
the translation assays were incubated with isolated rat liver
mitochondria (lane 3) and import estimated by trypsin
digestion (lane 4). Panel B, mitochondria were
preincubated prior to import without flavins (lanes 1 and 2), with 12 µM FAD (lanes 3 and 4), with 12 µM FMN (lanes 5 and 6) or with 12 µM riboflavin (lanes 7 and 8). Following import the mitochondria were reisolated and
fractionated, and the soluble fraction were either not treated (lanes 1, 3, 5, 7) or treated with
trypsin (lanes 2, 4, 6, 8). The
proteins were analyzed by SDS-PAGE and
autoradiography.
Figure 4:
[S]-pulse-chase
labeling of HepG2 cells. HepG2 cells stably transfected with
pRC-CMV-pMe
GlyDH (Panel A) or
pRC-CMV-mMe
GlyDH (Panel B) were pulse labeled for
60 min with [
S]Met. Fractionation into cytosol (lanes 1 and 3) and mitochondria (lanes 2 and 4) was performed either immediately or following a
15-min chase with cold methionine. The labeled Me
GlyDH
proteins were immunoprecipitated with specific antibody bound to
protein A-Sepharose and analyzed on 7.5% polyacrylamide gel by SDS-PAGE
and autoradiography.
Cells were labeled with
[S]Met, and mitochondrial import was blocked by
the respiratory chain inhibitor CCCP for 60 min in order to allow the
formation of the holoenzyme in the cytoplasm. Following a chase of 15
min with cold methionine in the absence of the inhibitor, both the
pMe
GlyDH as well as the mMe
GlyDH were
translocated into the organelle and recovered from the mitochondrial
matrix fraction in the protease-resistant form (Fig. 5, Panels A and B, compare lanes 2, 3,
and 4, 5). When the chase was performed in the
presence of CCCP, mitochondrial import was inhibited and the proteins
were recovered mainly from the cytoplasmic fraction (Fig. 5, Panels A and B, compare lanes 6, 7 and 8, 9). The precursor and mature form of
Me
GlyDH, which accumulated in the presence of CCCP in the
cytoplasm, folded during the 60 min of CCCP treatment into the
trypsin-resistant conformation (Fig. 5, Panels A and B, lane 7). These results indicated that formation of
holoenzyme did not prevent the mitochondrial import of
Me
GlyDH in vivo.
Figure 5:
Mitochondrial import of
MeGlyDH in vivo. Panel A,
pRC-CMV-pMe
GlyDH transfected HepG2 cells were labeled with
[
S]Met in the presence of CCCP for 60 min and
then chased for 15 min in the absence (lanes 2, 3, 4, and 5) or in the presence of the inhibitor (lanes 6, 7, 8, and 9). Cytosol (lanes 2, 3, 6, and 7) and
mitochondria (lanes 4, 5, 8, and 9)
were isolated, trypsin-digested, immunoprecipitated, separated on 7.5%
polyacrylamide gel by SDS-PAGE, and autoradiographed. Lane 1 shows the in vitro translation product of
pMe
GlyDH. Panel B, same experiment as in Panel
A performed with HepG2 cells stably transfected with the mature
form of Me
GlyDH.
Figure 6:
Processing of pMeGlyDH in the
cytosolic fraction of HepG2 cells. Panel A, in vitro synthesized pMe
GlyDH (lane 1) was incubated
either with cytosol of HepG2 cells (lane 2), cytosol of HepG2
cells and MPP (lane 4), or cytosol of hepatocytes (lane
5). As a reference the migration behavior of the mature
Me
GlyDH is shown in lane 3. Panel B, in vitro synthesized pMe
GlyDH was incubated with cytosol
prepared from HepG2 cells for 1 h at 27 °C (lanes 3 and 11) and in the presence of the following additions: 5 mM 5,5`-dithiobis-(2-nitrobenzoic acid) (lane 4), 1 mM aprotinin (lane 5), 1 mM pepstatin (lane
6), 1 mM antipain (lane 7), 1 mM leupeptin (lane 8), 1 mM phenylmethylsulfonyl
fluoride (lane 9), and 1 mM protease-inhibitor mix (lane 10). Lanes 1 and 2 show as references
the in vitro synthesized pMe
GlyDH (lane
1) and mMe
GlyDH (lane
2).
Figure 7:
Effect of riboflavin depletion of HepG2
cells on MeGlyDH biogenesis. Panel A, cells stably
transfected with pRC-CMV-pMe
GlyDH were grown for 5 days in
DMEM without riboflavin and with 10% dialyzed FCS (lanes 3 and 4). They were labeled for 60 min with
[
S]Met, fractionated into cytosol (lanes 1 and 3) and mitochondria (lanes 2 and 4), separated on SDS-PAGE, and autoradiographed. Lanes 1 and 2 present as controls transfected HepG2 cells grown
in standard DMEM medium. Panel B illustrates the same
experiment with HepG2 cells expressing the
mMe
GlyDH.
The analysis of the biogenesis of the covalent modification
of MeGlyDH demonstrated that, as shown for many proteins (27) , the holoenzyme conformation was stabilized by the
incorporation of the cofactor and was trypsin-resistant. Significantly,
folding of the RL-translated Me
GlyDH into the
trypsin-resistant conformation proceeded more efficiently at 15 °C
than at 30 °C and was independent of mitochondrial protein. The
experimental data support the conclusion that Me
GlyDH
holoenzyme formation in the RL in vitro takes place
spontaneously in line with an autoflavinylation process demonstrated
first for the bacterial enzyme 6-hydroxy-D-nicotine oxidase
carrying the same histidyl(N
)-(8
)FAD linkage as
Me
GlyDH(7) . There exists now strong support for
the notion that covalent FAD attachment progresses autocatalytically
and depends on a conformation of the protein favorable for the
interaction of the isoalloxazine ring with the reactive group of the
enzyme. Besides for 6-HDNO and Me
GlyDH, this was shown to
be the case with mammalian monoamine oxidase which contains the
cofactor attached by a cysteinyl(S)-(8
)FAD
linkage(28, 29) , bacterial trimethylamine
dehydrogenase characterized by a cysteinyl(S)-(6)FMN bond(30) ,
and bacterial p-cresol-methylhydroxylase exhibiting a
tyrosyl(O)-(8
)FAD bond(31) .
In agreement with reports
on the folding of precursor molecules of mitochondrial (32, 33, 34, 35) and plastid
enzymes(36) , the presequence of MeGlyDH slowed
down the folding of the protein into a conformation similar to that of
the mature form. The decreased folding efficiency of precursor proteins
has been attributed to the interaction of chaperones present in the
rabbit RL with the presequence. A specific presequence-binding protein
from rabbit RL that interacts in an ATP-dependent manner with the
presequence of mitochondrial precursor proteins keeping them in a
``loosly'' folded conformation has been
described(37) . The observation that the efficiency of folding
of pMe
GlyDH into the protease-resistant conformation was
increased in the presence of ATP may be explained by this finding.
It is interesting to note that the results obtained in
vivo, in stably transfected HepG2 cells, differed in several
respects from those observed in the in vitro rabbit RL system.
In the HepG2 cell cytosol the pMeGlyDH and
mMe
GlyDH proteins synthesized during the
[
S]Met pulse rapidly became trypsin-resistant,
indicating that folding in vivo needed less time than in the
rabbit RL. Also noteworthy is the observation that HepG2 cells contain
a cytosolic protease that seems to remove part of the
NH
-terminal amino acid sequence of pMe
GlyDH
since no change in molecular weight of the mMe
GlyDH
expressed in HepG2 cells was observed. Although we have found no
indication of this protease in hepatocytes, fibroblasts or Cos 7 cells
and it may therefore be a particular feature of this hepatoblastoma
cell line, import of mitochondrial precursor proteins in HepG2 cells
seemed not to be impaired. Factors present in the HepG2 cytosol but
absent from the RL may mediate this import. This possibility is
documented in the case of mMe
GlyDH which is imported into
mitochondria in vivo but not in vitro. The mature
form of the protein expressed in HepG2 cells lacks two serine residues
at the NH
terminus. Deletion of these two amino acids did
not affect enzyme activity, the FAD binding domain, or trypsin
resistance of the protein. We therefore conclude that this
Me
GlyDH species behaves in all respects relevant to the
aspects investigated in this work as the authentic mMe
GlyDH
generated by the MPP. It is not yet clear what sequences within the
mature protein may be responsible for the observed in vivo import. Translocation of the mMe
GlyDH accumulated in
the cytoplasm of HepG2 cells following CCCP treatment apparently took
place with the covalently bound flavin cofactor since the protein was
in its protease-resistant form characteristic for the holoenzyme. This
conclusion is in agreement with results obtained with the fusion
protein consisting of the Me
GlyDH presequence and the
bacterial enzyme 6-HDNO(10) . Import of the mature form of
mitochondrial enzymes has been shown
previously(34, 38, 39, 40) .
Mitochondria seem to be able to take up riboflavin and/or FMN and to
synthesize FAD(41) . Indeed, preincubation of mitochondria with
flavins increased the amount of trypsin-resistant MeGlyDH.
This may indicate a suboptimal FAD supply for flavinylation reactions
in the matrix.
Mitochondria isolated from rats kept on a
riboflavin-deficient diet showed an increased protease sensitivity of
acyl-CoA dehydrogenases and electron transfer flavoprotein as compared
to mitochondria isolated from rats fed a diet with sufficient
riboflavin(24) . The MeGlyDH proteins expressed
during pulse-chase experiments in HepG2 cells grown in
riboflavin-deficient medium did not exhibit a significant increase in
protease sensitivity, but a decreased mitochondrial uptake. Since
riboflavin deficiency may have multiple effects in the cell, it is
difficult to make a specific correlation between riboflavin supply and
import of flavoenzymes, but such a relationship cannot be excluded.
An amino acid sequence comparison of MeGlyDH with
bacterial and mammalian enzymes of C1-metabolism revealed similarities
among these enzymes in both the FAD binding as well as the
H
PteGlu
binding domain(26) .
Me
GlyDH as well as the related sarcosine dehydrogenase of
rat liver mitochondria (42) may have been generated by the
fusion of two primordial genes(26) . This hypothesis could
imply that the two domains of Me
GlyDH represent independent
folding units. However, the results obtained with Me
GlyDH
deletion proteins suggest that the two domains do not fold
independently from one another.
It may be interesting to note that
the same type of covalent attachment of FAD to bacterial sarcosine
oxidase and mammalian MeGlyDH and sarcosine dehydrogenase
was conserved during evolution. The same observation applies to the
flavoprotein subunit of succinate dehydrogenase(43) . The
biochemical reason behind the conservation of this particular
protein-cofactor interaction has not yet found an
explanation(44) . In mammals Me
GlyDH, sarcosine
dehydrogenase, and succinate dehydrogenase are mitochondrially located.
According to the theory of bacterial origin of mitochondria (see (9) for a discussion) one may speculate that these enzymes and
their particular type of cofactor linkage were introduced into the
ancestor of the eukaryotic cell by the bacterial endosymbiont.