(Received for publication, November 28, 1994; and in revised form, January 20, 1995)
From the
We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsinresistant conformation characteristic of holo-6-HDNO.
Import of nuclear encoded precursor proteins of mitochondrially
located enzymes seems to depend on a loosely folded, import-competent
conformation of the polypeptide (for a review, see (1) ). This
conformation may be maintained by the interaction of the precursor
protein with cytosolic chaperones of the Hsp70 family and/or with
particular presequence binding protein factors(2, 3) .
However, some mitochondrial precursor enzymes synthesized in
reticulocyte lysate (RL) ()have been shown to fold into a
conformation that incorporates cofactor and exhibits enzyme activity (4, 5, 6) . Binding of the cofactor in
several enzymes takes place late in the folding pathway, when the
protein has reached a native-like conformation, and results in the
stabilization of the native structure of the holoenzyme (for a review
see(7) ). These findings raise the question whether precursor
proteins with bound cofactor are imported into mitochondria.
We are
investigating the biogenesis of enzymes with covalently attached FAD.
As a model protein we employed 6-hydroxy-D-nicotine oxidase
(6-HDNO), a bacterial enzyme with FAD attached to the polypeptide chain
by a histidyl(N3)-8-FAD linkage(8) . Folding of the
guanidinium hydrochloride unfolded protein in vitro requires
the activity of the chaperonins GroEL and GroES in the presence of
ATP(9) . The folding of the protein into its native
conformation is followed by the autoflavinylation of the enzyme and
attainment of enzyme activity(10) . Incorporation of FAD into
the apoprotein depends on a flavinylation competent conformational
state of the polypeptide which can be induced allosterically in
vitro by the presence of glycerol 3-phosphate
(glycerol-3-P)(10) .
In the eukaryotic cell there are
several enzymes known with covalently attached FAD(11) , all
located in subcellular organelles: monoamine oxidase on the outer
mitochondrial membrane, succinate dehydrogenase in the inner
mitochondrial membrane, dimethylglycine dehydrogenase
(MeGlyDH), and sarcosine dehydrogenase in the mitochondrial
matrix, L-gulono-
-lactone oxidase in the microsomal cell
fraction, the plant enzyme reticuline oxidoreductase (involved in the
synthesis of the alkaloid berberin) in a special vacuolar
compartment(12) . The list of enzymes exhibiting this covalent
modification is continuously growing. The mechanism and the
participation of organelle-specific factors in the flavinylation of
these enzymes has not yet been elucidated, and the effect of the
covalent modification on the mitochondrial import of precursor proteins
has not been established.
6-HDNO, L-gulono--lactone
oxidase(13) , reticuline oxidoreductase(12) , and a
recently characterized mytomycin C resistance enzyme from Streptomyces lavendulae(14) may form a subgroup of
covalently flavinylated enzymes. These enzymes lack the
NH
-terminal dinucleotide binding site characteristic of the
majority of flavoenzymes (15) and exhibit similarities in the
amino acid sequence surrounding the FAD-modified histidine (14, 16) and in secondary structure
predictions(17) .
In the work presented in this paper we
investigated several aspects of the biogenesis of covalently
flavinylated enzymes in the eukaryotic cell by fusing the mitochondrial
targeting sequence of rat MeGlyDH to 6-HDNO. We show that
1) the mitochondrial targeting sequence does not prevent folding,
flavinylation, or enzyme activity of the fusion protein synthesized in
the RL; 2) the fusion protein is imported into rat liver mitochondria
and correctly processed; 3) the compact, trypsin-resistant conformation
of the flavinylated precursor is unfolded upon interaction with the
mitochondrion; and 4) flavinylation of the precursor does not prevent
its import.
Figure 1:
In vitro translation and holoenzyme
formation of 6-HDNO and pre-6-HDNO. Panel A, mRNAs transcribed
from of pSPT20-6-HDNO and pSPT19-pre-6-HDNO were translated in
the RL in the presence of [S]Met as described
under ``Materials and Methods.'' 5 µl of the translation
assays were analyzed on SDS-PAGE. Panel B, holoenzyme
formation of 6-HDNO and pre-6-HDNO in translation assays was determined
photometrically(8) . Lanes 1 and 4, 6-HDNO
and pre-6-HDNO translation assays without any additions; lanes 2 and 5, translation of 6-HDNO and pre-6-HDNO in the
presence of 10 µM FAD; lanes 3 and 6,
translation of 6-HDNO and pre-6-HDNO in the presence of 10 µM FAD followed by incubation with 10 mM glycerol-3-P (G3P) for 30 min at 30 °C. Panel C, covalent
incorporation of FAD into 6-HDNO (lane 1) and pre-6-HDNO (lane 2) translated in the RL in the presence of 10 µM [
C]FAD followed by incubation with 10
mM glycerol-3-P for 30 min at 30 °C. 25 µl of the
translation assays were analyzed on a 10% polyacrylamide gel by
SDS-PAGE and fluorography.
Figure 2:
Trypsin resistance of holoenzymes. 5
µl of [S]Metlabeled RL translation assays of
6-HDNO (lane 1) and pre-6-HDNO (lane 5) were digested
in lanes 2 and lane 6 with trypsin (0.2 mg/ml) at 0
°C for 15 min. Holoenzyme formation of both proteins was performed
as described under ``Materials and Methods.'' Lanes 3 and 7 represent 5 µl assays containing holoenzyme
previous to trypsin digestion; lanes 4 and 8, after digestion
with 0.2 mg/ml trypsin at 0 °C for 15 min. G3P,
glycerol-3-P.
Figure 3:
NH-terminal amino acid
sequence of the pre-6-HDNO fusion protein and processing by the MPP. Panel A, schematic representation of the Me
GlyDH
amino acid sequence fused to the 6-HDNO protein. MPP-cleavage site (arrow) is indicated as determined by radiosequencing (see
``Materials and Methods''). Positively charged amino acid
residues are indicated. Panel B, RL translation assays
containing [
S]Met-labeled 6-HDNO and pre-6-HDNO
were incubated for 20 min at 27 °C without (lanes 1 and 3) or with rat liver MPP (19) (lanes 2 and 4).
Figure 4:
Import of the pre-6-HDNO into rat liver
mitochondria. Panel A, [S]Met-labeled
translation products of 6-HDNO and pre-6-HDNO were incubated without (lanes 1 and 4) or with isolated rat liver
mitochondria (lanes 2 and 5) (see ``Materials
and Methods''), or digested with trypsin (lanes 3 and lane 6). The mitochondria were reisolated and the
[
S]Met-labeled 6-HDNO proteins analyzed by
SDS-PAGE and fluorography. Panel B, mitochondria were
preincubated without (lane 1 and 2) or with 12
µM FMN (lane 3 and 4), and import assays
were performed as described under ``Materials and Methods.''
Following import the isolated mitochondria were fractionated and the
matrix digested with 0.2 mg/ml trypsin at 0 °C for 15 min (lane
2 and 4). The products were analyzed by SDS-PAGE and
fluorography. Panel C, holoenzyme formation inside the
mitochondrial matrix following import of pre-6-HDNO was established by
densitometric quantification of the trypsin resistant fraction obtained
in the matrix of mitochondria not preincubated with FMN (lane
1) or incubated with FMN (lane 2) expressed as percentage
of imported protein. T.R., trypsin
resistance.
The same import experiments were performed with pre-holo-6-HDNO (Fig. 5). Since the pre-6-HDNO holoenzyme form is protease-resistant (Fig. 5, panel A, lane 2), it was necessary to differentiate between a protein species that was only associated with mitochondria and a protein species located inside the mitochondria. Additionally, as the presequence of pre-holo-6-HDNO is removed by incubation with trypsin, it is not possible to distinguish between cleavage outside the mitochondria by trypsin or intramitochondrial processing by MPP. In order to make this distinction, we inhibited MPP by treatment with the chelators EDTA and o-phenanthroline. Following import the assay was treated with trypsin. Under these conditions the precursor protein is not cleaved (Fig. 5, panel A, lane 7) and therefore must be located intramitochondrially. Additional proof that the trypsin-resistant 6-HDNO form is intramitochondrially located is the finding that pre-holo-6-HDNO, upon contact with import incompetent mitochondria, is unfolded and becomes trypsin-sensitive (Fig. 5, panel A, lane 6). The matrix location of the processed pre-holo-6-HDNO was also supported by the fractionation of the mitochondria after import. The data showed that the processed 6-HDNO species was situated in the mitochondrial matrix (Fig. 5, panel B, lane 1). Trypsin digestion of the mitochondrial fractions demonstrated that the 6-HDNO protein recovered in the matrix was 80% protease-resistant and represented holoenzyme (Fig. 5, panel B, lane 2). Thus, pre-holo-6-HDNO with FAD covalently attached to the polypeptide was imported into mitochondria. Preincubation of mitochondria with FMN had no effect on import and recovery of the holoenzyme in the matrix fraction (Fig. 5, panel B, lane 4, and panel C).
Figure 5:
Import of pre-6-HDNO holoenzyme into rat
liver mitochondria and its location in the mitochondrial matrix. Panel A, holoenzyme pre-6-HDNO was prepared as described under
``Materials and Methods.'' Translation assays containing
[S]Met-labeled pre-6-HDNO holoenzyme (lane
1) were digested with trypsin (lane 2). Import assays
with 4 µl of translation product were incubated with intact rat
liver mitochondria (lanes 3 and 4) followed by
trypsin digestion (lane 4). Parallel import assays were
performed with
-depleted mitochondria (see ``Materials
and Methods'') (lanes 5 and 6) and treated with
trypsin (lane 6). Lane 7, import assay performed with
mitochondria preincubated with 5 mM EDTA and 1 mMo-phenanthroline followed by trypsin digestion. Panel
B, mitochondria were preincubated with 12 µM FMN (lanes 3 and 4) or not (lanes 1 and 2) and the import assays performed as described under
``Materials and Methods.'' Following import the isolated
mitochondria were fractionated and the matrix digested with 0.2 mg/ml
trypsin at 0 °C for 15 min (lanes 2 and 4). The
[
S]Met-labeled 6-HDNO proteins were analyzed by
SDS-PAGE and fluorography. Panel C, holoenzyme formation
inside the mitochondrial matrix following import of pre-6-HDNO was
established by densitometric quantification of the trypsin-resistant
fraction obtained in the matrix of mitochondria not incubated with FMN (lane 1) or incubated with FMN (lane 2) expressed as
percentage of imported protein. T.R., trypsin
resistance.
Figure 6:
6-HDNO holoenzyme formation in the
mitochondrial matrix. 4 µl of [S]Met-labeled
6-HDNO translation product were incubated with 100 µg of
mitochondrial matrix for 30 min at 30 °C. The assay was divided and
one part trypsin digested. The [
S]Met-labeled
6-HDNO contained in the two samples was separated by SDS-PAGE,
fluorographed, and the intensity of the 6-HDNO bands on the film was
quantified by densitometry. The amount of trypsin-resistant 6-HDNO is
represented as percentage of the amount of 6-HDNO in the sample not
treated with trypsin. Lane 1, control assay of 6-HDNO
translation product from the RL; lane 2, percentage of trypsin
resistant 6-HDNO in the matrix without any additions; lane 3,
percentage of trypsin resistant 6-HDNO in the matrix supplemented with
10 µM FAD; lane 4, percentage of trypsin
resistant 6-HDNO in the matrix supplemented with 10 mM glycerol-3-P (G3P); lane 5, percentage of
trypsin resistant 6-HDNO in the matrix supplemented with 10 µM FAD and 10 mM glycerol-3-P.
Flavoenzymes may be classified into two groups according to the nature of the cofactor attachment to the protein. The majority exhibits the flavin moiety tightly but noncovalently bound to the polypeptide. The second group of over 20 enzymes contains the cofactor covalently bound to an amino acid side chain of the protein(11) . The covalently flavinylated enzymes in eukaryotic cells are compartmentalized. This observation raises the question as to when and how during the biogenesis of these enzymes the covalent modification takes place. For monoamine oxidase it has been shown that the apoenzyme becomes catalytically active, i.e. incorporates FAD, upon contact with the outer mitochondrial membrane, but prior to its ubiquitinylation and ATP-dependent insertion into this membrane (25) . From the expression of an enzymatically active bovine monoamine oxidase in yeast, it was inferred that covalent attachment of FAD to a cysteine residue of the enzyme proceeds autocatalytically(26) . Recently, the same conclusion was reached for the attachment of FAD to a tyrosine of p-cresol hydroxylase from Pseudomonas putida expressed in E. coli(27) . It was shown for succinate dehydrogenase that assembly of the enzyme complex did not require the flavinylation of the flavoprotein subunit(28, 29) . There are no more detailed data available on the mechanism of flavinylation of these enzymes and the possible importance of this covalent modification for their biogenesis.
Our results show that the 6-HDNO protein synthesized in the eukaryotic translation system was correctly folded and flavinylated. This fact demonstrates that in the heterologous RL system autoflavinylation of the enzyme proceeded unaltered. As in the case of the E. coli cell extract, supplementation of the RL with FAD only was not sufficient to transform all of the synthesized 6-HDNO into holoenzyme, but required the posttranslational addition of glycerol-3-P as well.
It is generally considered that mitochondrial
precursor proteins are maintained after completion of translation in a
loosely folded, import-competent state by the interaction with
molecular chaperones (1) . Evidence for the posttranslational
interaction of precursor proteins with Hsp70 in the RL is still
debated(5) . A 28-kDa mitochondrial targeting factor and a
50-kDa presequence binding factor have been identified in RL,
however(30, 31) . A loosely folded conformation might
be expected to prevent trypsin resistance and enzymatic activity of a
precursor protein. Although the apoenzyme forms of 6-HDNO and
pre-6-HDNO are trypsin-sensitive and thus differ in their conformation
from the holoenzyme, they are not recovered by immunoprecipitation from
RL as complexes with Hsp70. Our results also demonstrate that
attachment of the mitochondrial presequence of MeGlyDH does
not prevent flavinylation of the 6-HDNO moiety of the fusion protein
and formation of a tightly folded enzymatically active and
trypsin-resistant conformation. Folding of mitochondrial precursor
proteins translated in RL into enzymatically active, native-like
conformations has been documented before for other
enzymes(5, 6, 32) . The mitochondrial
presequence apparently does not prevent folding of the precursor
protein but makes its targeting to the mitochondrial import complex
more specific(33) .
The MeGlyDH presequence
conferred import competence to the fusion protein (pre-apo-6-HDNO), but
only a fraction of the imported and processed apo-6-HDNO acquired
trypsin resistance in the mitochondrial matrix. Addition of FAD and
glycerol-3-P to the mitochondrial matrix fraction was required to
transform apo-6-HDNO into the protease-resistant form. This finding
suggests that the intramitochondrial flavin concentration was
insufficient for complete 6-HDNO holoenzyme formation and that the
flavinylation reaction was not performed by a mitochondrial protein
factor, but required the conformational change induced by the
allosteric effector. It was recently shown that import of flavin
precursors into rat mitochondria takes place at the level of FMN and
that FMN is transformed intramitochondrially into FAD(34) .
When we tried to increase the intramitochondrial FAD concentration by
incubation of isolated rat liver mitochondria with FMN prior to
pre-apo-6-HDNO import, there was no increase in the recovery of
trypsin-resistant 6-HDNO. Apparently the intramitochondrial FAD
concentration was still insufficient for the conversion of pre-6-HDNO
into the holoenzyme. This observation is different for what was shown
for the import of precursor molecules of acyl-CoA dehydrogenases (35) and the flavoenzyme subunit of succinate dehydrogenase (29) which were converted intramitochondrially into holoenzyme.
The reduced level of intramitochondrial 6-HDNO holoenzyme formation may
be explained by the absence of the specific allosteric effector from
the mitochondrial matrix. It could also be explained by a low affinity
of the 6-HDNO apoenzyme for FAD which would render the
intramitochondrial FAD concentration insufficient for holoenzyme
formation. Enzymes with a typical dinucleotide binding pocket, like
acyl-CoA dehydrogenases and the flavoprotein subunit of succinate
dehydrogenase, may more avidly bind FAD than flavoenzymes lacking this
particular FAD-binding domain and thus form holoenzymes at a lower
cofactor concentration.
The finding that the covalently flavinylated holo-pre-6-HDNO was imported into rat liver mitochondria was unexpected. The effect of cofactor attachment on the mitochondrial import of precursor proteins varies. It was shown in several instances that the bound cofactor blocks import of the precursor protein. Thus import of the cytoplasmic dihydrofolate reductase fused to a mitochondrial presequence was blocked when the ligands NADP and methotrexate, an antagonist of tetrahydrofolate, were attached to the protein(36) . Studies on mitochondrial aspartate aminotransferase have shown that formation of the holoenzyme prevented its import(5) . Import of cytochrome c, which does not require a presequence, is blocked by the covalent attachment of heme(4) . In contrast to these reports, but similar to findings described in this paper, biotinylated precursors are imported into 3T3-L adipocyte mitochondria(37, 38) . Recently it was shown that the phosphopantotheine prosthetic group of the acyl carrier protein does not prevent import of the precursor protein into spinach chloroplasts(39) .
During import into mitochondria precursor
proteins are unfolded(40) . This process seems to be mediated
by their interaction with mitochondrial Hsp70(4) . Besides
mitochondrial Hsp70 translocation of precursor molecules requires an
across the inner mitochondrial membrane(1) . In the
case of deenergized mitochondria, the interaction of the precursor with
the import apparatus of the inner mitochondrial membrane should be
inhibited. Thus a contact between the 6-HDNO precursor and
mitochondrial Hsp70 should not take place. Therefore the observed
unfolding of holo-pre-6-HDNO when incubated with
-depleted
mitochondria cannot be attributed to the postulated unfoldase activity
of mitochondrial Hsp70(4) . It was recently shown that the
interaction of mitochondrial proteins with phospholipid components of
the mitochondrial membrane may lead to their unfolding (41) .
These findings may explain our observation.
We would like to hypothesize on a possible physiological consequence of the interaction between a cofactor and its precursor enzyme. Import into mitochondria in vivo is assumed to take place posttranslationally, at least to a considerable extent, although evidence for a cotranslational import has been provided(42) . Folding of proteins inside the cell apparently commences as they are translated, and completion of folding of the released protein takes place within seconds(43) , whereas the residing time of a precursor protein in the cytoplasm is considered to be a few minutes(1) . Thus it might be assumed that the folding of binding domains for small molecular ligands like cofactors are completed while the polypeptide is still in the cytoplasm and that cofactors may be bound to the protein. Unfolding of the precursor upon contact with the mitochondrion would release noncovalently bound cofactors from the protein. This could lead to an increase in the local concentration of the cofactor at the cellular organelle and an increased diffusion-driven or carrier-mediated import of the cofactor. In this way a functionally meaningful intracellular cotransport of the cofactor and the precursor enzyme to the place of their activity could be established.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X55995 [GenBank]and X05999 [GenBank](for dimethylglycine dehydrogenase and 6-hydroxy-D-nicotine oxidase, respectively).