(Received for publication, April 15, 1994)
From the
We studied the role of FAD in the intramitochondrial folding and
assembly of medium-chain acyl-CoA dehydrogenase (MCAD), a
homotetrameric mitochondrial enzyme containing a molecule of
non-covalently bound FAD/monomer. In the MCAD molecule, FAD is buried
in a crevice containing the active center. We have previously shown
that upon import into mitochondria, newly processed MCAD is first
incorporated into a high molecular weight (hMr) complex and that the
hMr complex mainly consisted of MCAD-heat-shock protein 60 (hsp60)
complex (Saijo, T., Welch, W. J., and Tanaka, K(1994) J. Biol.
Chem. 269, 4401-4408). In the present study, we incubated in vitro synthesized precursor MCAD with mitochondria isolated
from normal and riboflavin-deficient rat liver for 10-60 min and
fractionated the solubilized mitochondria using gel filtration. The
amount of MCAD in the hMr complex was larger and that of tetramer was
smaller in riboflavin-deficient mitochondria than in control at any
time point. In addition, riboflavin-deficient mitochondria were
solubilized after 10-min import in a buffer containing ATP and were
chased in the presence of FAD, FMN, or NAD or without
any addition. The mitochondrial proteins were analyzed using gel
filtration or immunoprecipitated with anti-hsp60 antibody. After 60-min
chase in the presence of FAD, the majority of MCAD in the complex with
hsp60 was transferred to tetramer, whereas no such transfer occurred
after the chase in the absence of FAD. When chase was done in the
presence of FMN, a significant amount of MCAD was transferred from the
complex with hsp60 to tetramer, but the transfer was not as efficient
as in the presence of FAD. The chase in the presence of NAD
resulted in no transfer. These data suggest that isoalloxazine
ring of FAD plays a critical role, exerting nucleating effect, in the
hsp60-assisted folding of MCAD subunit into an assembly competent
conformation, probably assisting the formation of the core.
Medium chain acyl-CoA dehydrogenase (EC 1.3.99.3, MCAD) ()catalyzes the first reaction in the
-oxidation of
fatty acids with medium chain length(1, 2) . It
belongs to a gene family of five mitochondrial flavoproteins, the
acyl-CoA dehydrogenase family(3, 4) , sharing common
structural and functional features. These five enzymes, including MCAD,
catalyze the
,
-dehydrogenation of acyl-coenzyme A esters.
Human MCAD is encoded on chromosome 1 (5) and is synthesized in
the cytosol as a 47-kDa precursor protein containing a 25-amino-acid
leader peptide as an extension of the amino
terminus(6, 7, 8) . The precursor (p) MCAD is
imported into the mitochondrial matrix, and the leader peptide is
proteolytically cleaved producing the 44-kDa mature protein. The mature
monomers are then assembled into the native homotetrameric
form(6) . After import and leader peptide cleavage,
flavin-adenine dinucleotide (FAD) is incorporated into the apo-enzyme (9) by a mechanism that is still unknown at present. In the
completed native form, MCAD contains one molecule of non-covalently
bound FAD/monomer(2) .
It had previously been observed that riboflavin deficiency in vivo in rats resulted in a drastic decrease in the activities of various acyl-CoA dehydrogenases, including MCAD(10, 11) . FAD addition to the homogenate of riboflavin-deficient rat liver mitochondria restored only 10-25% of the lost activity, suggesting that the loss of activity was mainly due to the loss of proteins(10, 11, 12, 13) . Nagao and Tanaka (9) recently studied the effects of riboflavin deficiency on various stages of acyl-CoA dehydrogenase biogenesis. They confirmed using immunoblot analysis that acyl-CoA dehydrogenase proteins were indeed greatly reduced in riboflavin-deficient rat tissues. They further showed that in riboflavin-deficient rats, the rate of transcription of the acyl-CoA dehydrogenase genes and the amounts of acyl-CoA dehydrogenase mRNAs in tissues both increased 3-8.5-fold over controls. In the absence of FAD, translation of acyl-CoA dehydrogenase mRNAs was moderately inhibited, as was that of proteins which do not contain FAD. The newly synthesized acyl-CoA dehydrogenase precursors were imported into riboflavin-deficient mitochondria at normal rates. In the absence of FAD, however, the resulting mature acyl-CoA dehydrogenase proteins were markedly less stable than in the presence of FAD. They concluded that the instability of the acyl-CoA dehydrogenase apo-proteins in mitochondria was the major cause for the loss of acyl-CoA dehydrogenases in riboflavin deficiency(9) .
In our recent study (14) of the intramitochondrial biogenetic pathway of MCAD using in vitro synthesized pMCAD and isolated rat liver mitochondria, we have shown that after import into mitochondria and subsequent leader peptide cleavage, the monomeric mature MCAD subunit was folded into a proper conformation and assembled into the native homotetrameric form via processes mediated sequentially by heat-shock protein (hsp)70 and hsp60. Hsp70 and hsp60 belong to a group of proteins called molecular chaperons, which assists folding of proteins(15) . Hsp70 is considered to first bind to proteins emerging from the inner mitochondrial membrane and to maintain the proteins in a loosely folded state(16, 17, 18) . After being released from hsp70, proteins bind to hsp60, a toroidal ring heptamer of a 60-kDa subunit (19) . Upon consumption of ATP, the bound proteins are folded and released from hsp60(15, 20) . However, the role of hsp60 in oligomeric assembly is still unclear.
Recent x-ray
crystallographic studies by Kim et al.(21, 22) indicate that porcine MCAD tetramer is actually a dimer of
two dimers. The monomeric MCAD is folded into three major domains,
NH-terminal, middle, and COOH-terminal. Both
NH
-terminal and COOH-terminal domains mainly consist of
-helices, while the middle domain is composed of
-sheets. The
FAD molecule lays in an extended conformation. As previously predicted
by Ikeda et al.(23) from the enzymatic titration
study, the isoalloxazine ring is buried in the crevice that is formed
between the two
-helical domains (the NH
- and the
COOH-terminals) and the middle domain in a
monomer(21, 22) . The adenosine moiety makes contact
with the other subunit of the dimer and lies at the surface of the
tetrameric MCAD molecule(21, 22) .
In the x-ray crystallographic studies(21, 22) , several amino acids were shown to form hydrogen bonds with various parts of the FAD molecule. We reasoned that the interactive forces, created between FAD molecule and various amino acids in the MCAD protein, may play important roles in folding the loosely folded MCAD peptide chain into a conformation that is assembly competent and is capable of producing enzymatically active MCAD. We speculated that in riboflavin-deficient mitochondria, the lack of FAD may lead to failure of folding and to assembly incompetency of the newly processed subunit, resulting in the instability of MCAD. In this study, we studied the role of FAD on folding and assembly of the wild-type human MCAD in normal and riboflavin-deficient mitochondria using in vitro transcription-translation, isolated mitochondria, and fractionation of the imported MCAD on gel filtration. Such study of the MCAD biogenic pathway in the isolated rat liver mitochondria system would closely reflect the events in vivo.
In order to improve the
efficiency of transcription-translation, a new recombinant plasmid,
pGRPM2-WT, containing 55 bp of the 5`-flanking region of the Xenopus laevis -globin gene and the entire human pMCAD
cDNA encompassing 1 and 1270, was constructed by ligating the former to
the latter at a NcoI site that was artificially introduced in
the section surrounding the initiation codon of the latter. The
construction of this recombinant was achieved in several steps using
plasmid Delta-13Tb, containing the Xenopus
-globin gene
segment and cyclin B cDNA, as illustrated in Fig. 1. Delta-13Tb
had previously been constructed by M. Glotzer, University of California
at San Francisco, from cyclin-B sequence-containing Delta-13 (25) by ligating the 55 bp of the 5`-flanking region of the Xenopus
-globin gene to the NcoI site
artificially created at the initiation codon of sea urchin cyclin B
cDNA.
Figure 1:
Construction of pGRPM2-WT. The
construction of pGRPM2-WT (bottom), containing 55 bp of the
5`-flanking region of the X. laevis -globin gene
and the entire human pMCAD cDNA encompassing 1 and 1270, was achieved
using Delta 13Tb (top, right). In Delta-13Tb, a 55-bp
section of the 5`-flanking region of the Xenopus
-globin
gene is ligated to the NcoI site that is artificially created
at the initiation codon of sea urchin cyclin B cDNA in
Delta-13(28) . To create pGRPM2-WT, a 775-bp fragment of pMCAD,
encompassing nucleotides -13 and 762 of the pMCAD sequence, was
first amplified by PCR using as template pBPM1-WT (left, top), a pBluescript-based recombinant plasmid containing, at
downstream of the T7 promotor, a full-length human pMCAD cDNA
encompassing -13 and 1270(14, 24) . In the
upstream primer, A at position -2 was substituted with C, so that a NcoI site was introduced at the region
surrounding the initiation codon. The amplified fragment was digested
with BamHI/EcoRI, and the resulting 698-bp fragment
was ligated back into the pBPM1-WT at the BamHI/EcoRI
sites, from which the corresponding unaltered 698-bp fragment had been
excised, producing pBPM2-WT (left, middle).
Separately, the HindIII/PstI fragment of Delta-13Tb
was excised and ligated into pGEM-4Z vector (right, middle
steps). The recombinant plasmid was digested with NcoI/SalI to remove the cyclin B cDNA (right, second from the bottom), which was replaced
with NcoI/SalI fragment of pBPM2-WT (left, second from the bottom), creating pGRPM2-WT (bottom).
See text for more details. The abbreviations for the restriction
enzymes are: B, BamHI; E, EcoRI; H, HindIII; N, NcoI; P, PstI; S, SalI.
In the construction of pGRPM2-WT, a 775-bp fragment of pMCAD
encompassing nucleotides -13 and 762 of the pMCAD sequence was
first amplified using polymerase chain reaction (PCR) with pBPM1-WT (left, top) as a template. In the upstream primer, Aat position -2 was substituted with C, so that a NcoI site was introduced at the
region surrounding the initiation codon. The altered fragment, that was
amplified, was digested with BamHI/EcoRI, and the
resulting 698-bp fragment was ligated back into the pBPM1-WT at the BamHI/EcoRI sites, from which the corresponding
unaltered 698-bp fragment had been excised (left, second
step), producing pBPM2-WT (left, third step).
Separately, the HindIII/PstI fragment of Delta-13Tb
was excised (right, second step) and cloned into
pGEM-4Z vector (Promega) (right, third step). The
recombinant plasmid was digested with NcoI/SalI to
remove the cyclin B cDNA (right, fourth step), which
was replaced with the NcoI/SalI fragment of pBPM2-WT,
creating pGRPM2-WT (bottom). Thus, pGRPM2-WT contained the
5`-flanking region of the -globin gene and its powerful
ribosome-binding site downstream of the T7 promotor. The
-globin
gene sequence was immediately followed by the initiation codon of pMCAD
cDNA.
In vitro transcription of pGRPM2-WT was performed
using T7 RNA polymerase (Promega), and translation of the pGRPM2-WT
transcripts was performed using rabbit reticulocyte lysate (Promega)
and [S]methionine (Amersham Corp.) according to
the manufacturer's instructions. When analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) and autofluorography, the
translation product of pGRPM2-WT transcripts contained approximately 10
times as much pMCAD as that of pBPM1-WT transcripts did, while the
amounts of other MCAD-related fragment bands remained the same (data
not shown). Thus, when the transcript of pGRPM2-WT was used, 50 µl
of the translation product was sufficient for one gel filtration
analysis of MCAD imported into mitochondria. Previously, when the
transcript of pBPM1-WT was used as template, 500 µl of the
translation product was necessary for the same purpose.
The amount of residual mitochondrial FAD was quantitated by measuring the ability of the mitochondrial extract to convert the apo-enzyme of D-amino acid oxidase to its active holo-enzyme as follows: intact or solubilized mitochondria homogenate were boiled for 5 min in 10 mM potassium phosphate buffer, pH 8.5. Mitochondrial extract was then cooled rapidly and centrifuged. Twenty µl of the extract was added to 10 µg of apo-D-amino acid oxidase, dissolved in 980 µl of 65 mM potassium phosphate buffer, pH 8.5, containing 4 mMD-phenylglycine. The mixture was assayed for D-amino acid oxidase activity according to the method of Oka and McCormick(27) .
For gel filtration analysis, a
Sephacryl S-200 HR (Pharmacia) column (1 45 cm) was used after
equilibration with 10 mM potassium phosphate, 0.5 mM EDTA buffer, pH 8.0. The column was first calibrated with a
protein standard mixture (Bio-Rad), containing thyroglobulin (670 kDa),
-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa).
The void volume of the column was estimated according to the elution of
blue dextran. With collection of 0.3 ml in each tube under the
conditions used, tetramer, dimer, and monomer of MCAD, assuming they
are globular, are predicted to elute in areas centering at tubes
61-62, 65-66, and 69-70, respectively, according to
the calibration curve. The position of unfolded monomer is unknown.
To analyze labeled MCAD in the mitochondria, 200 µl of the
supernatant of the solubilized mitochondrial preparation were applied
to the column, and 0.3-ml fractions were collected. Proteins from every
other fraction were precipitated overnight at -20 °C after
the addition of 0.5 ml of acetone. Protein precipitates were collected
by centrifugation at 10,000 g for 30 min at 4 °C
and redissolved in 50 µl of SDS-PAGE sample buffer (0.6 M Tris pH 8.85, 2% SDS, 5%
-mercaptoethanol, 10% glycerol, and
bromphenol blue). A 25-µl aliquot was analyzed on SDS-PAGE. Labeled
MCAD band was visualized by fluorography with Autofluor (National
Diagnostics). The intensity of MCAD band was determined
densitometrically using a BioImage system (Millipore).
In the present study, we first determined the amount of total mitochondrial protein and intramitochondrial FAD content in normal and riboflavin-deficient rat in livers. Rats were sacrificed at the end of 6 weeks on riboflavin-replaced (control) or riboflavin-deficient diet, and mitochondria were isolated from the liver. The amount of total mitochondrial protein was 2.9 ± 0.4 (n = 4) and 2.7 ± 0.5 (n = 5) mg/g of wet tissue in control and riboflavin-deficient rats, respectively. Thus, in spite of some morphological changes, the amount of total mitochondrial protein in riboflavin-deficient rat liver was essentially not different from control.
The FAD concentrations in control and riboflavin-deficient rat liver mitochondria were 4.8 ± 0.2 (n = 4) and 1.3 ± 0.1 n = 5) µg/mg mitochondrial protein, respectively.
The patterns of MCAD elution from normal liver mitochondria are shown in Fig. 2A. After 10-min import, MCAD in control mitochondria eluted from the column in two major forms, high molecular mass (hMr) complex with a size ranging 500-600 kDa (fractions 53-57) and tetramer (fractions 61-67), in similar quantities. In the previous experiments, we have shown that the main component of the hMr complex was the complex of MCAD with hsp60, a heat-shock protein that is required for folding of proteins(14) . After import for 30 min, the amount of the tetramer moderately increased, while that of the hMr complex decreased in a similar degree. At 60 min, the amount of the tetramer greatly increased, while that of the hMr complex further decreased. The amount of decrease of MCAD in the hMr complex was approximately equal to that of increase of tetramer MCAD. These results on various molecular forms of MCAD in normal liver mitochondria were essentially identical to those found in the previous study using pBPM1-WT as template(24) .
Figure 2:
Gel filtration analysis of newly imported
and processed human MCAD in normal and riboflavin-deficient
mitochondria. In vitro translated wild-type pMCAD was
incubated with either normal or riboflavin-deficient rat liver
mitochondria at 30 °C for 10, 30, or 60 min. After incubation,
mitochondria were treated with trypsin and then with trypsin inhibitor
and were solubilized. The solubilized mitochondria were fractionated on
a Sephacryl S-200 HR column (1 45 cm) with a flow rate of 4.1
ml/min. After the void volume of the column was discarded, 0.3-ml
fractions were collected in microcentrifuge tubes. Proteins in every
other fraction were precipitated with acetone and were analyzed with
SDS-PAGE and fluorography. Intensity of the MCAD band was measured by
scanning densitometry. In order to determine the total amount of
imported MCAD, one-twentieth of the sample before gel filtration was
also analyzed on the same gel. The amount of MCAD in each fraction is
presented in percentage of the total amount of MCAD imported. A, normal mitochondria. B, riboflavin-deficient
mitochondria. Symbols are:
, 10-min import;
, 30-min import;
, 60-min import.
The results from gel-filtration analysis of riboflavin-deficient mitochondria were in a sharp contrast to those observed with normal mitochondria, with markedly different patterns of MCAD elution (Fig. 2B). After 10-min import into riboflavin-deficient mitochondria, MCAD eluted predominantly as the hMr complex. A small unresolved peak encompassing tubes 62-67 was observed. Judging from the skewed peak shape, the amount of monomer appeared to be greater than that of tetramer. At 30 min, the amount of MCAD eluted in tubes 61-67 increased, suggesting the appearance of tetramer, although it is still not well resolved from the monomer peak. The amount of tetramer was much smaller than that in control. After 60 min of import, the amount of tetramer in riboflavin-deficient mitochondria slightly increased, but it was still markedly smaller than that formed in the control mitochondria in the same period. The appearance of a small amount of tetramer is probably due to the residual FAD and is consistent with the previous observation that riboflavin-deficient rat liver mitochondria, produced under the same conditions, contained MCAD activity that was 21.4% of control(9) . The amount of hMr complex in riboflavin-deficient mitochondria was considerably larger than in control at 10 min and only slightly decreased thereafter. At 60 min, the amount of hMr complex in the deficient mitochondria was at least three times as large as in normal mitochondria.
Figure 3:
Gel filtration analysis of newly imported
and processed human ornithine transcarbamylase in normal and
riboflavin-deficient mitochondria. In vitro translated
wild-type precursor ornithine transcarbamylase was incubated with
either normal or riboflavin-deficient rat liver mitochondria at 30
°C for 10 or 60 min. After incubation, mitochondria were treated
with trypsin and then with trypsin inhibitor and were solubilized.
Solubilized mitochondria were fractionated on a Sephacryl S-200 HR
column (1 45 cm). The steps for gel filtration and detection of
ornithine transcarbamylase were identical with those in the legend to Fig. 2. A, normal mitochondria. B,
riboflavin-deficient mitochondria. Symbols are:
, 10-min import;
, 60-min import.
Figure 4:
Effects of FAD, FMN, and NAD on the
transfer of MCAD from the hMr complex to tetramer. In vitro translated wild-type pMCAD was incubated with riboflavin-deficient
rat liver mitochondria at 30 °C for 10 min. After incubation,
mitochondria were treated with trypsin and then with trypsin inhibitor
and were solubilized in a buffer containing 2.5 mM ATP without
any additional nucleotide (A), or with the addition of 0.1
mM FAD (B), FMN (C), or NAD (D) and
chased for 15 or 60 min. Subsequent steps for gel filtration and
detection of MCAD were the same as those in the Fig. 2legend.
Elution pattern of MCAD after 10-min import is shown by dotted
lines. Symbols are: , 15-min chase;
, 60-min
chase.
In the solubilized mitochondria chased for 15 min without either form of flavin nucleotides, newly imported MCAD eluted mostly as hMr complex (Fig. 4A). There was no distinct tetramer peak at tubes 61-62. After tube 62, there was a broad peak encompassing tubes 65 and 73, suggesting the possibility that in addition to a small amount of monomer, an equally small amount of dimer may have been present. However, it is possible that this broad peak was due to a mixture of monomers with different physical configurations. At 60-min chase, a small peak of tetramer became detectable, while the peak extending to tubes 69-73 greatly decreased, indicating that the monomer peak almost disappeared. However, there was no significant decrease in the amount of MCAD in the hMr complex fraction.
In the mitochondrial lysate which was chased for 15 min after the addition of 0.1 mM FAD, a large amount of tetramer was detected, while the amount of the hMr complex was noticeably smaller than that found after chase without FAD addition (Fig. 4B). At 60-min chase after the addition of FAD, the amount of tetramer further increased, while that of MCAD in the hMr complex decreased in an amount comparable to that of increase of tetramer. This result confirms that FAD was necessary for folding the newly imported MCAD subunit into an assembly competent conformation. It should be noted that in the absence of FAD or FMN, ATP alone was incapable of promoting transfer of MCAD from the hMr complex to tetramer, although ATP shares the adenosine and diphosphate moieties with FAD.
In the mitochondrial lysate chased for 15 min in the presence of 0.1 mM FMN, a considerable amount of tetramer was detected, although the amount was not as large as that formed in the mitochondrial lysate chased in the presence of FAD (Fig. 4C). The amount of hMr complex also decreased compared to that immediately after 10-min pulse. After 60-min chase in the presence of FMN, the amount of tetramer further increased, while that of MCAD in the hMr complex decreased. However, the changes in the amount of tetramer and that of MCAD in the hMr complex between 15 and 60 min were both small.
In order to examine whether FMN exerted the assembly enhancing effects directly or indirectly after it was converted to FAD during the chase period, we determined the concentration of FAD in the FMN-substituted mitochondrial lysate before and after the chase. Since FAD synthetase is known to be present in the cytosol(30) , the latter possibility was unlikely. Nonetheless, we found that the FAD concentration in the mitochondrial lysate before and after 15-min chase was 1.44 and 1.53 µg/mg of mitochondrial protein, respectively, in rat A. In rat B, the FAD concentration in the mitochondrial lysate before and after 60-min chase was 1.34 and 1.33 µg/mg of mitochondrial protein, respectively. The difference in the amount of FAD before and after 15 min chase in rat A was small and in all likelihood, it was insignificant. In order to confirm that the folding enhancing effects were exerted directly by FMN but not by the small nominally increased amount of FAD (0.09 µg/mg of mitochondrial protein = 0.9 µM in the chase buffer) after chase, we chased the mitochondrial lysate with the addition of 0.9 µM FAD. We found that essentially no increase of tetramer was observed (data not shown). Hence, these results indicate that FMN per se is capable of supporting intramitochondrial folding and assembly of MCAD into the tetrameric form, although its enhancing effect is not as potent as that of FAD.
In order to
further characterize the structural requirements for enhancing
folding-assembly of MCAD, we tested the effects of
NAD, performing the same chase experiment using
mitochondria from another riboflavin-deficient rat (rat C) as shown in Fig. 4D. NAD
shares with FAD the
adenosine diphosphate moiety, differing only in the chromophore. The
FAD concentration in the mitochondria from rat C was 1.42 µg/mg of
mitochondrial protein and was comparable to those in rats A and B. In
the mitochondria chased for 15 min in the presence of 0.1 mM NAD
, newly imported MCAD eluted mostly as hMr
complex. No distinct tetramer peak was detectable. After 60-min chase
in the presence of NAD
, the elution pattern of MCAD
remained similar to that after 15-min chase without any recognizable
increase of tetramer or significant decrease in the amount of MCAD in
the hMr complex fraction.
Figure 5:
Immunochemical determination of the
effects of FAD, FMN, and NAD on the release of MCAD from the complex
with hsp60. In vitro translated wild-type pMCAD was incubated
with riboflavin-deficient rat liver mitochondria at 30 °C for 10
min. After incubation, mitochondria were treated with trypsin and then
with trypsin inhibitor and were solubilized in a buffer containing 2.5
mM ATP. The sample was then divided into nine equal aliquots.
The sample in one tube was immediately subjected to immunoprecipitation
with anti-hsp60 antibody (10` import). The remaining eight
tubes were divided into four groups of two, and to each of three
groups, FAD, FMN, or NAD was added in the final concentration of 0.1
mM. No nucleotide was added to the fourth group. One tube from
each group was chased for 15 min (15` chase) and the other for
60 min (60` chase). After the chase, samples were
immunoprecipitated with anti-hsp60 antibody. Radiolabeled MCAD, that
was coimmunoprecipitated with hsp60, was determined using SDS-PAGE and
fluorography. Upper panel, relevant portion
of the autofluorogram. Lower panel, quantitative determination
of MCAD which coimmunoprecipitated with hsp60. Symbols are: ,
chase without any additional nucleotide;
, chase with FAD;
, chase with FMN;
, chase with
NAD.
The results from the present study provide important clues for the role of FAD on chaperonin-assisted folding and assembly of MCAD protein that takes place in mitochondria. We have shown that in riboflavin-deficient mitochondria, the amount of MCAD tetramer formed was markedly smaller than that formed in control, and that unlike in normal mitochondria, the majority of MCAD in riboflavin-deficient mitochondria was still remaining in the hMr complex. In contrast, folding and assembly in riboflavin-deficient mitochondria of ornithine transcarbamylase, a non-FAD-containing enzyme, was as efficient as in control, indicating that the impaired formation of MCAD tetramer in riboflavin-deficient mitochondria was specifically caused by the lack of FAD. When in vitro translated radiolabeled pMCAD molecules were imported into riboflavin-deficient mitochondria and the resulting mature MCAD peptides were chased in the presence of 0.1 mM FAD after solubilization of the mitochondria, more than 70% of the labeled MCAD molecules were released from the complex with hsp60 and were assembled into the native tetrameric form within 60 min, whereas in the same sample chased without the addition of FAD, MCAD almost entirely remained complexed with hsp60 after the same length of chase. These results indicated that FAD was required for the folding of MCAD peptide into an assembly competent conformation, while MCAD peptide is still complexed with hsp60. The use in these experiments of whole mitochondria for the import and processing of in vitro translated radiolabeled pMCAD, followed by gel-filtration analysis of the mitochondrial proteins, was crucial for providing the clues for the role of a ligand in chaperonin-assisted folding and assembly of a protein.
In the past, the effects of ligand on folding and assembly
of a number of proteins have been studied. The results are variable
from protein to protein. Most of the proteins previously studied were
those which are capable of self-folding-assembly in vitro without involvement of molecular chaperons, and the methods of
analysis used were various types of spectrometric analysis, except in
some which also utilized gel-filtration analysis. Thus, the previous
studies provided mainly information on the conformational changes
during spontaneous refolding of the protein, which was unfolded using a
high concentration of denaturant, and that on the stage at which the
ligand was attached in the refolding pathway. Studied this way, the
addition of ligand in the refolding mixture was found to have had no
effects on spontaneous refolding and assembly of many proteins. The
enzymes and ligands (shown in parentheses) in this category include
liver alcohol dehydrogenase (NAD)(31) ,
bacterial barnase (3`-GMP)(32) , Escherichia coli tryptophan synthase (pyridoxal phosphate) (33) , and pig
heart and rat and chicken liver mitochondria aspartate aminotransferase
(pyridoxal phosphate)(34, 35) . For some proteins
including E. coli aspartate aminotransferase(36) , Azotobacter lipoamide dehydrogenase (FAD) (37) and
yeast UDP-galactose-4-epimerase (NAD
)(38) ,
refolding did not require cofactor, but the binding of the cofactor was
the prerequisite for dimerization, suggesting that the binding of
cofactor caused some conformational change in the monomer, making it
assembly competent. In contrast, both Deal (39) and Jaenicke et al.(40) provided evidence that the rate of
reconstitution of glyceraldehyde-3-phosphate dehydrogenase was markedly
enhanced by NAD
and suggested that NAD
enhances the rate of reconstitution of this enzyme via nucleating
effect. In a study using gel-filtration analysis of culture media of
the wild-type and a Mo cofactor-deficient mutant of Rhodobacter
sphaeroides, Masui et al.(41) obtained results
suggesting that folding of dimethyl sulfoxide reductase was retarded in
the absence of Mo cofactor, an essential prosthetic group. Prokaryotic
expression studies also produced results suggesting the role of ligand
in the biogenesis of proteins. When Pseudomonas putida urocanase, a NAD
-containing dimeric protein, was
expressed in P. putida in niacin-depleted medium, unstable
apo-protein was obtained in a small amount, but no activation occurred
after the addition of NAD
(42) , suggesting
that its biogenesis cannot be completed in the absence of the cofactor.
They considered that normally the ligand was trapped by the nascent
protein and served as a scaffold for folding the protein in the
bacteria, but no direct evidence supporting this hypothesis was
presented. Recently, Brandsch et al.(43) performed
prokaryotic expression and in vitro refolding-assembly of Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase, a
monomeric enzyme containing covalently bound FAD. They found that
during 6 h of expression, the addition of FAD or ATP had little effects
on the ratio of apo- and holo-enzymes (normally, 40 and 60%,
respectively). They further demonstrated that in the presence of groESL
and ATP, the refolding of guanidinium hydrochloride-denatured
holo-6-hydroxy-D-nicotine oxidase proceeded rapidly during the
first minute of the reaction, followed by a slower progress, but such
refolding did not take place when any groES, groEL, and ATP was absent.
An allosteric effector, glycerol-3-phosphate further enhanced the
folding. In the refolding of denatured
apo-6-hydroxy-D-nicotine oxidase, holo-enzyme activity became
detectable taking a similar kinetics pattern, only when the incubation
was done in the presence of groESL, ATP, and FAD. They concluded simply
that FAD was covalently incorporated into the polypeptide in the rapid
phase of groESL-assisted refolding. The role of FAD on groESL-assisted
refolding was not considered or specifically studied, however. They
subsequently studied the effects of serine substitution of 4 different
cysteine residues in the 6-hydroxy-D-nicotine oxidase peptide
using prokaryotic expression(44) . There were no correlations
between the amount of the high molecular weight complex formed and the
degree of reduced activity-FAD incorporation. They concluded that for
altering the interaction with groEL, structural changes in the
amino-terminal part were more important than those in the
carboxyl-terminal part. Thus, the role of FAD in the
chaperonins-assisted biogenesis of 6-hydroxy-D-nicotine
oxidase remained to be elucidated.
With regard to the mechanism for
the retention of MCAD in the hsp60 complex in riboflavin-deficient
mitochondria observed in the present study, it should be recalled that
we and others recently showed that some mutated proteins tend to remain
bound to chaperonin for a longer period of time than their normal
counterparts, presumably because the variants are difficult to, or
cannot, be correctly folded. These aberrant proteins include K304E
variant of MCAD, the product of a highly prevalent mutation, A to G transition(14) , some of the Cys-to-Ser mutants of
6-hydroxy-D-nicotine oxidase (44) , and HeLa cell
proteins cultured in a media containing a proline analog, L-azetidine 2-carboxylic acid(45) . With the knowledge
of these previous findings, a prolonged association of the wild-type
MCAD and hsp60 in the absence of FAD suggested that correct folding of
MCAD peptide cannot be accomplished in the absence of FAD, and that the
interaction of FAD with the MCAD peptide during its hsp60-assisted
folding process is critical for the correct folding of the protein.
Alternatively, it may be argued that a MCAD subunit can be correctly
folded without FAD, but interaction between the FAD molecule and
another subunit is required for the tetrameric assembly. The failure of
tetramer assembly may lead to the accumulation of the MCAD
hsp60
complex. The latter possibility is unlikely, however, because the
impaired assembly of folded monomer should lead to the accumulation of
monomer rather than the accumulation of MCAD
hsp60 complex.
In
spite of the critical role FAD plays in the folding and assembly of
MCAD in mitochondria, it is important to note that once the peptide is
correctly folded and the tetrameric form is achieved, FAD is not
necessary for the maintenance of the essential tertiary and quaternary
structure of MCAD. The FAD-free apo-enzyme of rat and porcine MCAD can
be readily prepared by use of dye-ligand chromatography or by a mild
chemical treatment(2, 46) . The molecular mass of the
apo-MCAD was 180 kDa, indicating a tetrameric structure. ()The apo-MCAD can be almost instantaneously converted back
to fully active holo-enzyme upon addition of
FAD(2, 43) .
In the x-ray crystallographic study of
porcine MCAD(21, 22) , it has been shown that the FAD
molecule, through its various parts, interacts with a number of amino
acid residues in the MCAD peptide chain. The isoalloxazine ring of the
FAD is buried in the crevice that is formed between the COOH-terminal
domain and the middle -sheet domain, with its pyrimidine portion
surrounded by the residues from the loops between
-strands.
Evidence for some hydrogen bonds between the flavin ring and amino acid
residues were observed. These include bonds formed between: N-3 of the
flavin ring and the carbonyl oxygen of Tyr
; N-1 and O-2
of the flavin ring and the hydroxyl of Thr
; and N-5 and
the carbonyl oxygen of Trp
and the amide nitrogen of
Thr
. The adenosine moiety is located at the surface of
the tetrameric molecule and makes contact with the other subunit of the
dimer.
In order to understand which moiety(ies) of the FAD molecule
exerts crucial interaction with the MCAD peptide, we also tested the
effects of two other cofactors, FMN and NAD, which
share riboflavin phosphate and adenosine diphosphate moieties,
respectively, with FAD. It should also be noted that all chase
experiments were done in a buffer containing 2.5 mM ATP, a
nucleotide sharing adenosine diphosphate moiety with FAD. ATP is
required as an energy source for folding of protein within, and its
release from, the complex with hsp60. In the presence of FMN, MCAD was
efficiently folded and released from the complex with hsp60 and was
assembled into tetramer, although the efficiency was not as high as
with FAD. This was rather surprising in view of the fact that FMN
addition was incapable of restoring any activity of
apo-MCAD(2) . In the presence of NAD
or in the
presence of ATP alone, no MCAD was released from the hsp60 complex nor
was tetramer formed. These results indicate that for hsp60-assisted
folding of the MCAD peptide, its interaction with isoalloxazine ring
moiety is critical.
In the structure of native MCAD, the acyl moiety
of the coenzyme A ester substrate is also deeply buried inside the
crevice at the re-side of the flavin ring. In the pathway of
,
-dehydrogenation of acyl-CoA, proR-
-hydrogen of
acyl-CoA is abstracted as a proton by the
-carboxyl of
Glu
(47) , while proR-
-hydrogen is abstracted
by N-5 of isoalloxazine ring as a hydride ion(48) . The flavin
ring also interacts, presumably at its si-face(22) ,
with the obligatory electron acceptor, electron transfer flavoprotein.
Thus, the crevice represents the core in the structure and function of
MCAD. The evidence presented in the present report indicates that
isoalloxazine ring plays a critical role in the formation of the core
of MCAD in its hsp60-assisted folding pathway providing nucleating
effect. It should be reasonable to assume that the hydrogen bonds
formed between the flavin ring and the amino acids in the crevice would
serve as a scaffold in the folding MCAD monomer. In contrast, the role
of the adenosine diphosphate moiety of FAD in the MCAD biogenesis
appears to be secondary. Once the tetramer is completed, however, the
adenosine diphosphate moiety may be important for anchoring that end of
the FAD molecule on the protein, contributing to the activity producing
binding, as observed in the reconstitution study of holo-electron
transfer flavoprotein from its apo-form(49) .