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(Received for publication, December 26, 1995; and in revised form, February 6, 1996) From the
Rat dimethylglycine dehydrogenase (Me
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) ( 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-
For expression of the Me
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-Me
Figure 1:
In vivo expression of Me
Human hepatoblastoma HepG2 cells, which do not
express Me
Figure 2:
In vitro folding and holoenzyme
formation of Me
The RL contains low levels of endogenous
FAD(24) , which may not be sufficient for Me The yield of
trypsin-resistant pMe 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
Me Me
Figure 3:
Import of the COOH-terminally deleted
pMe
Figure 4:
[
Cells were labeled with
[
Figure 5:
Mitochondrial import of
Me
Figure 6:
Processing of pMe
Figure 7:
Effect of riboflavin depletion of HepG2
cells on Me
The analysis of the biogenesis of the covalent modification
of Me In agreement with reports
on the folding of precursor molecules of mitochondrial (32, 33, 34, 35) and plastid
enzymes(36) , the presequence of Me 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 pMe 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 Me 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 Me An amino acid sequence comparison of Me It may be interesting to note that
the same type of covalent attachment of FAD to bacterial sarcosine
oxidase and mammalian Me
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9823-9829
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
GlyDH) 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 MeGlyDH
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
MeGlyDH from cell mitochondria, suggesting a decreased
efficiency of mitochondrial protein import.
)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) .
-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;
MeGlyDH), 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 MeGlyDH 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
MeGlyDH.
Chemicals
Cycloheximide, CCCP, digitonin, DMEM,
FAD, FMN, phenylmethysulfonyl fluoride, riboflavin, soybean trypsin
inhibitor, and trypsin were purchased from Sigma (Deisenhofen, FRG).
[
S]Methionine-[S]cysteine
mix was from Amersham Corp. (Braunschweig, FRG); dimethylglycine,
folate, geneticin G418, and protease inhibitors were from Boehringer
Mannheim (Mannheim, FRG); and RNase A was from Diagen (Hilden, FRG).
All other chemicals were of highest purity available.Plasmid Constructs
Plasmid pcD-MeGlyDH (11) was the starting DNA for subsequent clonings.
pSPT19-mMeGlyDH and pSPT19-pMeGlyDH were
constructed starting from pcD-MeGlyDH which contain the
MeGlyDH cDNA sequence in the vector pcD(12) . The
sequence 5`-GAATCCCACCATG-3` comprising an EcoRI restriction
site (GAATCC) and an eukaryotic ribosomal binding site (CCACC) (13) was introduced in front of the MeGlyDH start
codon (ATG) with the aid of a mutagenic oligonucleotide. This allowed
excission of the cDNA from pcD-pMeGlyDH by digestion with
the restriction enzymes EcoRI and PvuII and insertion
of the 2925-bp DNA fragment thus generated into pSPT19 (Boehringer
Mannheim) digested with the restriction enzymes EcoRI and SmaI. The ligated recombinant DNA was named
pSPT19-pMeGlyDH. pMeGlyDH transcripts could be
synthesized in vitro starting from the SP6 polymerase promoter
situated on the plasmid with the aid of SP6 RNA polymerase (Boehringer
Mannheim). Synthesis of transcripts was coupled with protein
translation of MeGlyDH in the rabbit RL system kit obtained
from Promega (Madison, WI). For the in vitro expression of the
mature form of MeGlyDH (mMeGlyDH) the sequence
5`-GAATCCACCATG-3` was introduced into the MeGlyDH cDNA
contained in pcD-pMeGlyDH in such a way that the ATG codon
replaced codon Ala-24(11) . Subcloning of the EcoRI/SmaI DNA fragment excised from
pcD-pMeGlyDH into pSPT19 gave rise to
pSPT19-mMeGlyDH. Transcription-translation of the plasmid
carrying the cDNA generated a protein corresponding to the mature form
of MeGlyDH shortened by two serine residues at the
amino-terminal end. The COOH-terminal deletions of the
pMeGlyDH designated 
SFU and XHO were achieved by
digesting plasmid pSPT19-pMeGlyDH with the restriction
enzymes SfuI/HindIII and XhoI/SalI,
respectively, and removing the excised MeGlyDH cDNA
fragment. The plasmid vector carrying the deleted cDNA was then blunt
ended with the Klenow fragment of DNA polymerase I and
religated(14) .GlyDH
proteins in eukaryotic cell lines, plasmids pSPT19-pMeGlyDH
and pSPT19-mMeGlyDH 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
MeGlyDH 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).In Vitro Transcription-Translation
Coupled
transcription-translation in the rabbit RL in the presence of
[S]Met was done according to the suppliers
instructions (Promega).Folding and Holoenzyme Formation of Me
[GlyDH
in the RLS]Met-labeled
MeGlyDH proteins were synthesized in the coupled
transcription-translation rabbit RL system for 30 min at 30 °C.
Translation was stopped by the addition of 80 µg/ml RNase A and 100
µg/ml cycloheximide. Folding of the translated proteins was then
monitored at 15 and 30 °C in the absence or presence of 10
µM FAD, 10 µM folate, and 10 µM dimethylglycine, added separately or in combination to the
incubation assays. Holoenzyme formation was tested by digestion of the
translation assay for 15 min at 0 °C with 0.2 mg/ml trypsin.
Digestion was stopped by the addition of 4 mg/ml soybean trypsin
inhibitor.In Vitro Mitochondrial Protein Import
Rat liver
mitochondria were prepared as described in Conboy et al.(15) . Mitochondrial import assays were performed by
incubating 4 µl of [S]Met-labeled RL
MeGlyDH translation product with freshly isolated rat liver
mitochondria at a final concentration of 8 mg/ml as reported in Stoltz et al.(10) . For flavin supplementation the isolated
mitochondria were incubated before performing the import assays with
either 12 µM FAD, 12 µM FMN, or 12 µM riboflavin at 27 °C for 5 min. Separation of mitochondria into
a soluble and a membrane fraction and densitometric quantification of
labeled protein bands on autoradiographs was performed as described in
Stoltz et al.(10) .Transfection of Cell Lines
Cells of the
hepatoblastoma cell line HepG2 were grown at 37 °C as monolayer
under 5% CO in DMEM supplemented with 10% fetal calf serum
(FCS). They were transfected with plasmid DNA
pRC-CMV-pMeGlyDH and pRC-CMV-mMeGlyDH by the
calcium phosphate procedure (16) . After 72 h, selection for
transfectants was carried out by replacing the medium with DMEM
containing 10% FCS and 500 µg/ml geneticin G418.
Geneticin-resistant clones were picked and expanded in the same medium.
After inital selection, the extent of translational expression of the
new transfected sequences within individual clones was determined by
Western blotting (see below). Transfectants with the highest level of
expression were used for subsequent studies.Pulse-Chase Experiments
Cells were grown to 90%
confluency in 10-cm plastic dishes (approximately 5 
10
to 10 10 cells per dish) and fed 12 h before
radiolabeling with fresh DMEM containing 10% FCS. Cell monolayers were
rinsed twice with phosphate-buffered saline; 4 ml of labeling medium
(DMEM with 10% dialyzed FCS without methionine and cysteine) were added
to each dish, and cells were incubated for 30 min at 37 °C. Fresh
labeling medium (4 ml) containing 200 µCi of a mixture of L-[S]methionine and L-[S]cysteine was added, and incubation
was continued at 37 °C. When CCCP was used, it was added to the
medium at a final concentration of 100 µM, 30 min before
the addition of the labeling medium and was included at the same
concentration during the labeling of the cells. Monolayers were labeled
for 1 h in the presence or absence of 100 µM CCCP, the
medium aspirated, the monolayers washed twice with phosphate-buffered
saline, and methionine-containing DMEM plus 10% FCS with or without
CCCP was added to the cells as indicated.Preparation of Cell Fractions
Release of the
cytosolic and mitochondrial fraction was performed essentially as
described in Janski and Cornell(17) . The cytosolic fraction
was held at 4 °C until immunoprecipitation or Western blotting was
performed. The pellet containing the mitochondrial fraction was washed
twice with 200 µl of fractionation medium (250 mM sucrose,
20 mM MOPS, 3 mM EDTA, pH 7.0) without digitonin and
resuspended in 100 µl of mitochondrial homogenization buffer (220
mM mannitol, 70 mM sucrose, 10 mM HEPES-KOH,
pH 7.6). The mitochondrial proteins were released by freezing the
pellet three or four times in liquid nitrogen. Lactate dehydrogenase
(EC 1.1.1.27) and citrate synthase (EC 4.1.3.7) activity was determined
in each cell fraction according to Vassault (18) and Shepherd
and Garland (19) , respectively.Immunoprecipitation and SDS-PAGE
Cell monolayers
were washed with phosphate-buffered saline and then incubated 30 min at
4 °C with low salt lysis buffer (1% Triton X-100, 50 mM Tris, 10 mM EDTA, pH 8.0). The lysed cells were
centrifuged for 3 min at 12,000 g. Protein content was
determined by the method of Bradford(20) .GlyDH 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.Western Blotting
Total cell lysate and cytosolic
or mitochondrial fractions were resuspended in sample buffer, and
proteins were separated on 7.5% polyacrylamide gels by SDS-PAGE and
transferred electrophoretically onto a nitrocellulose membrane
(Optitran BA-S 85 Schleicher & Schuell, Dassel, Germany) using a
trans-blot semidry electrophoretic transfer cell (Bio-Rad,
München, Germany). The MeGlyDH protein
was visualized with the aid of specific antiserum and a
chemiluminescence Western blotting kit as described by the supplier
(Boehringer Mannheim).Nondenaturing Gel Electrophoresis
Nondenaturing
polyacrylamide gel electrophoresis was conducted according to Davis (22) using a 2.5% stacking gel, pH 6.7, and a 7% separating
gel, pH 8.9. Gels were stained for dehydrogenase activity by incubation
in a solution containing 0.2 M dimethylglycine, 0.1 M potassium phosphate, pH 7.2, 0.67% phenazine methosulfate, and 0.5
mM nitro blue tetrazolium at room temperature. Unreacted
tetrazolium dye was then removed by diffusion into water.
Activity Staining and Trypsin Resistance of
Me
When the cytosolic and the
mitochondrial fractions of rat liver were analyzed by Western blotting
for the presence of MeGlyDH Holoenzyme Expressed in Rat Liver Hepatocytes and
Stably Transfected HepG2 CellsGlyDH, a MeGlyDH
antibody-reactive band was detectable only in the mitochondrial
fraction but not in the cytosolic fraction (Fig. 1, Panel
A, lanes 1 and 2). Trypsin treatment revealed
that this band was protease resistant (Fig. 1, Panel A, lane 3). This mitochondrially located, trypsin-resistant,
mature form of MeGlyDH represented enzymatically active
holoenzyme as demonstrated by activity staining on nondenaturing
polyacrylamide gels (Fig. 1, Panel A, lanes 4 and 5). It exhibited a certain microheterogeneity
indicated by the presence of several enzymatically active bands. This
heterogeneity of MeGlyDH has been observed
before(23) .
GlyDH, 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 MeGlyDH. The mitochondrial fraction (lane
4) was separated after digestion with 0.2 mg/ml trypsin (lane
5) under nondenaturing conditions and activity stained for
MeGlyDH. Panels B and C, HepG2 cells
transfected with pRC-CMV-pMeGlyDH and
pRC-CMV-mMeGlyDH 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.
GlyDH (results not shown), were stably
transfected with pRC-CMV-pMeGlyDH and
pRC-CMV-mMeGlyDH, encoding the precursor and mature forms
of the protein, respectively. Analysis of the intracellular
distribution of the MeGlyDH 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
MeGlyDH protein isolated from rat liver mitochondria (Fig. 1, Panel B and C, lanes 4 and 5).Translation and Folding of Me
The stably transfected HepG2 cells showed
that trypsin resistance of MeGlyDH into
Holoenzyme in the RLGlyDH correlated with the
enzymatically active holoenzyme form. This observation allowed us to
test in the RL translation system the conditions required for folding
of the precursor and mature protein into the trypsin-resistant
conformation. The percentage of trypsin resistance was determined by
comparison of the intensity of the
[S]Met-labeled MeGlyDH band on the
autoradiogram of the nondigested sample with that of the
trypsin-digested sample. Fig. 2, Panel A, presents the
temperature dependence of MeGlyDH holoenzyme formation.
Trypsin digestion of the precursor protein removed most of the
presequence of pMeGlyDH(10) , at a site closely
preceding the MPP processing site (compare lanes 1 and 2). Formation of the trypsin-resistant form of the protein was
more efficient at 15 °C than at 30 °C (compare lanes 2 and 4). Similar results were obtained with the mature
form of the protein (Fig. 2, Panel A,
mMeGlyDH). However, the yield of trypsin-resistant protein
after 5 h incubation at 15 °C was higher than in the case of the
precursor form (30% for mMeGlyDH and 12% for
pMeGlyDH on average from three independent folding
experiments).
GlyDH in the rabbit RL; dependence on
temperature, cofactor additions and mitochondrial matrix. Panel
A, coupled transcription-translation of MeGlyDH
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 mMeGlyDH 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 mMeGlyDH protein present after protease
treatment as compared to the amount of mMeGlyDH protein in
undigested control assays. Panel C, as in Panel B,
but performed with pMeGlyDH. Panel D, coupled
transcription-translation of pMeGlyDH and
mMeGlyDH 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
MeGlyDH proteins was expressed as percentage of
MeGlyDH 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.
GlyDH
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 MeGlyDH in the presence of externally added
FAD, folic acid and the MeGlyDH 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 MeGlyDH 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 mMeGlyDH was dependent
on the cofactor concentrations in the assays.GlyDH was lower when compared to that
obtained with mMeGlyDH. 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).In Vitro Translation, Mitochondrial Import, and Trypsin
Resistance of Wild Type and Carboxyl-terminally Deleted
Me
Incubation of the
[GlyDH ProteinsS]Met-labeled pMeGlyDH translation
product with isolated rat liver mitochondria resulted in the import of
the protein. The mature form of MeGlyDH, however, was not
imported (results not shown).GlyDH 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).GlyDH
contains a FAD and a HPteGlu 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
HPteGlu cofactor of MeGlyDH. In
addition, recent sequence comparison of bacterial sarcosine oxidase
with MeGlyDH 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
pMeGlyDH 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.
GlyDH/SFU and pMeGlyDH/XHO
proteins into rat liver mitochondria. Panel A,
[S]Met-labeled pMeGlyDH/SFU and
pMeGlyDH/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.
In Vivo Expression of pMe
HepG2
cells stably transfected with pRC-CMV-pMeGlyDH and
mMeGlyDH in Stably Transfected HepG2 CellsGlyDH and
pRC-CMV-mMeGlyDH were employed in pulse-chase assays to
analyze the distribution of the labeled proteins between cytoplasmic
and mitochondrial fractions (Fig. 4). After a 0-min chase the
MeGlyDH precursor as well as the mature form were recovered
in the cytoplasmic fraction (Fig. 4, Panels A and B, lanes 1 and 2). Surprisingly, after a
15-min chase both proteins were predominantly found in the
mitochondrial matrix (Fig. 4, Panels A and B, lanes 3 and 4), indicating that in vivo also
the mature MeGlyDH, lacking the mitochondrial presequence,
was imported into mitochondria.
S]-pulse-chase
labeling of HepG2 cells. HepG2 cells stably transfected with
pRC-CMV-pMeGlyDH (Panel A) or
pRC-CMV-mMeGlyDH (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 MeGlyDH
proteins were immunoprecipitated with specific antibody bound to
protein A-Sepharose and analyzed on 7.5% polyacrylamide gel by SDS-PAGE
and autoradiography.
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
pMeGlyDH as well as the mMeGlyDH 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
MeGlyDH, 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
MeGlyDH in vivo.
GlyDH in vivo. Panel A,
pRC-CMV-pMeGlyDH 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
pMeGlyDH. Panel B, same experiment as in Panel
A performed with HepG2 cells stably transfected with the mature
form of MeGlyDH.
Processing of pMe
When pMeGlyDH in the Cytoplasm of
HepG2 CellsGlyDH translated in the RL was
incubated with the cytoplasmic fraction of HepG2 cells, the precursor
protein was processed to a smaller molecular weight species. Initially
we assumed that contamination of the cytosol with MPP originating from
mitochondria broken during the isolation procedure was responsible for
the observed effect. Closer inspection by SDS-PAGE of the molecular
weight of the protein form processed by the cytosolic fraction of HepG2
cells and of the molecular weight of the mature protein generated by
the MPP revealed that the processed form exhibited a molecular weight
that was intermediate between the precursor form and the mature, MPP
processed form of the enzyme (Fig. 6, Panel A).
Analysis of the specificity of the HepG2 cytoplasmic protease activity
by protease inhibitors revealed an inhibition spectrum characteristic
for thiol proteases (Fig. 6, Panel B). Since this
molecular weight MeGlyDH species did not accumulate in the
cytoplasm of labeled HepG2 cells, we conclude that it was imported into
mitochondria. We assume that the proteolytic processing removed part of
the mitochondrial presequence.
GlyDH in the
cytosolic fraction of HepG2 cells. Panel A, in vitro synthesized pMeGlyDH (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
MeGlyDH is shown in lane 3. Panel B, in vitro synthesized pMeGlyDH 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 pMeGlyDH (lane
1) and mMeGlyDH (lane
2).
Effect of Riboflavin Depletion on Me
HepG2 cells stably transfected with
pRC-CMV-pMeGlyDH
Biogenesis in HepG2 CellsGlyDH and pRC-CMV-mMeGlyDH were
grown in riboflavin-free cell culture medium supplemented with dialyzed
FCS. Riboflavin deficiency became evident after 5 days by a decrease in
cell growth. Fig. 7shows that when riboflavin-deficient cells
were pulse labeled with [S]Met on the 5th day,
the uptake of the labeled pMeGlyDH into the mitochondria
was decreased as compared to that from mitochondria of nondeprived
cells (Fig. 7, Panel A, compare lanes 2 and 4). When the same experiment was performed with HepG2 cells
stably transfected with pRC-CMV-mMeGlyDH, cells kept under
riboflavin deficiency accumulated [S]Met-labeled
mMeGlyDH in the cytosol (Fig. 7, Panel B,
compare lanes 1 and 3). Nevertheless, a certain level
of mitochondrial import of both the precursor and the mature form could
be observed (Fig. 7, Panel A and B, lanes
2 and 4). This observation may be explained by a less
efficient mitochondrial MeGlyDH import in
riboflavin-deficient cells.
GlyDH biogenesis. Panel A, cells stably
transfected with pRC-CMV-pMeGlyDH 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
mMeGlyDH.
GlyDH 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 MeGlyDH 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 MeGlyDH
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
MeGlyDH(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 MeGlyDH, 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) .GlyDH 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 pMeGlyDH into the protease-resistant conformation was
increased in the presence of ATP may be explained by this finding. GlyDH and
mMeGlyDH 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 pMeGlyDH
since no change in molecular weight of the mMeGlyDH
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 mMeGlyDH 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
MeGlyDH species behaves in all respects relevant to the
aspects investigated in this work as the authentic mMeGlyDH
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 mMeGlyDH 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 MeGlyDH presequence and the
bacterial enzyme 6-HDNO(10) . Import of the mature form of
mitochondrial enzymes has been shown
previously(34, 38, 39, 40) . GlyDH.
This may indicate a suboptimal FAD supply for flavinylation reactions
in the matrix.GlyDH 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. GlyDH with
bacterial and mammalian enzymes of C1-metabolism revealed similarities
among these enzymes in both the FAD binding as well as the
HPteGlu
binding domain(26) .
MeGlyDH 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 MeGlyDH represent independent
folding units. However, the results obtained with MeGlyDH
deletion proteins suggest that the two domains do not fold
independently from one another.GlyDH 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 MeGlyDH, 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.Acknowledgment
We thank Castor Menendez for fruitful
discussions and critically reading the manuscript.
)GlyDH, dimethylglycine dehydrogenase; MOPS,
3-N-morpholinepropane sulfonic acid; MPP, mitochondrial
processing peptidase; RL, reticulocyte lysate; PAGE, polyacrylamide gel
electrophoresis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
