©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isoalloxazine Ring of FAD Is Required for the Formation of the Core in the Hsp60-assisted Folding of Medium Chain Acyl-CoA Dehydrogenase Subunit into the Assembly Competent Conformation in Mitochondria (*)

(Received for publication, April 15, 1994)

Takahiko Saijo Kay Tanaka (§)

From the Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Medium chain acyl-CoA dehydrogenase (EC 1.3.99.3, MCAD) (^1)catalyzes the first reaction in the beta-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 alpha,beta-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(2)-terminal, middle, and COOH-terminal. Both NH(2)-terminal and COOH-terminal domains mainly consist of alpha-helices, while the middle domain is composed of beta-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 alpha-helical domains (the NH(2)- 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.


MATERIALS AND METHODS

Construction of pGRPM2-WT for Efficient Transcription-Translation of the Wild-type pMCAD in Vitro

Previously, transcription-translation of pMCAD was carried out using a recombinant plasmid, pBPM1-WT, as template. This pBluescript-based plasmid contained, downstream of the T7 promotor, a full-length human pMCAD cDNA encompassing -13 and 1270. This region includes 13 bp in the 5`-non-coding region, the entire coding region, and 4 bp in the 3`-non-coding region. Such experiments yielded 46.6-kDa pMCAD and several other truncated MCAD fragments, which were produced via translation that was initiated from various internal methionine codons (14, 24) . The amount of pMCAD band was only approximately one-tenth of the total MCAD-related proteins synthesized. Therefore, the transcription-translation experiments using pBPM1-WT as template were inefficient.

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 beta-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 beta-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 beta-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 beta-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 beta-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 beta-globin gene and its powerful ribosome-binding site downstream of the T7 promotor. The beta-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.

Preparation of Riboflavin-deficient Rat and Isolation of Mitochondria

Weanling male Wistar rats (Charles River), weighing 50-60 g, were randomly divided into two groups. One group was fed a riboflavin-deficient diet (ICN Biomedicals) ad libitum for 6 weeks (riboflavin-deficient group). The other group received the same diet, to which 22 mg of riboflavin was added per kg, and served as control. At the end of 6 weeks rats were sacrificed, and mitochondria were isolated from livers using the method of Loewenstein et al.(26) .

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) .

In Vitro Translation of pMCAD Using FAD-depleted Rabbit Reticulocyte Lysate

In order to study the effects of FAD on folding/assembly of in vitro synthesized MCAD, it was necessary to remove a small amount of free FAD that was present in rabbit reticulocyte lysate. For this purpose, we used apo-enzyme of glucose oxidase that was prepared as described previously(9) . When apo-glucose oxidase was added to rabbit reticulocyte lysate, free FAD becomes depleted as glucose oxidase was reconstituted from apo-form to the holo-form, incorporating the existing free FAD. For depleting free FAD, 35 µl of rabbit reticulocyte lysate (Promega) were incubated with 5 µl of 80 µM apo-glucose oxidase at 30 °C for 30 min. This preparation was immediately used for the translation reaction in a total volume of 50 µl as described above.

Gel Filtration Analysis of Newly Imported and Processed Human MCAD in Normal and Riboflavin-deficient Mitochondria

The isolated normal or riboflavin-deficient rat liver mitochondria were suspended in a final concentration of 20 mg protein/ml in HMS buffer (2 mM HEPES, pH 7.4, 220 mM mannitol, and 70 mM sucrose). Import of the in vitro translated pMCAD into the mitochondria was performed in duplicates in order to obtain sufficient amount of products. Each of the duplicate tubes contained 25 µl of in vitro translation product, 100 µl of rat liver mitochondria, 2.5 mM MgATP, 3% bovine serum albumin, 80 mM KCl, and 1 mg/ml NADH in a total volume of 250 µl. After an incubation at 30 °C for a desired length of time, 4 µl of 4 mg/ml trypsin were added to each tube and incubated for 10 min at 4 °C to digest pMCAD which remained outside of mitochondria. Ten µl of 10 mg/ml trypsin inhibitor were then added, and the mitochondria were isolated by centrifugation and washed twice with HMS buffer. The mitochondria in the duplicates were then combined. In some experiments, the combined mitochondrial pellet was instantly frozen in an alcohol/dry ice bath and was stored at -70 °C for 1-2 days until use. The combined mitochondrial pellet was solubilized in 220 µl of solubilization buffer (10 mM potassium phosphate, pH 8.0, 0.5 mM EDTA, 1% Triton X-100, and 0.4 mg/ml trypsin inhibitor) and centrifuged at 10,000 times g for 20 min.

For gel filtration analysis, a Sephacryl S-200 HR (Pharmacia) column (1 times 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 times 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% beta-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).

Study of the Effects of FAD, FMN, and NAD on the Transfer of MCAD from the hMr Complex to Tetramer

After 10-min import of pMCAD, riboflavin-deficient mitochondria were treated with trypsin and were isolated as described above. The combined mitochondrial pellet was solubilized in 220 µl of chase buffer (50 mM potassium phosphate, pH 8.0, 0.5 mM EDTA, 1% Triton X-100, 0.4 mg/ml trypsin inhibitor, and 2.5 mM MgATP) containing FAD, FMN, or NAD (0.1 mM each), or no nucleotides. Solubilized mitochondria were incubated at 30 °C for 15 or 60 min and centrifuged. 200 µl of the supernatant were applied to the gel filtration column and every other fractions were analyzed for labeled MCAD using SDS-PAGE and fluorography.

Immunochemical Determination of the Effects of FAD, FMN, and NAD on the Release of MCAD from the Complex with Hsp60

pMCAD import into riboflavin-deficient rat liver mitochondria was performed in total volume of 250 µl as described above. After the import at 30 °C for 10 min, the mixture was treated with trypsin followed by the addition of trypsin inhibitor. Isolated mitochondria were solubilized in 200 µl of the chase buffer and were centrifuged at 10,000 times g for 20 min. The supernatant was divided into nine 20-µl aliquots. The sample in one tube was immediately subjected to immunoprecipitaton with anti-hsp60 antibody as described below to measure the amount of MCAD complexed with hsp60. The remaining eight tubes were divided into four groups of two, and to each group 180 µl/tube of the chase buffer containing one of FAD, FMN, or NAD, or no nucleotides were added. The final concentration of each nucleotide was 0.1 mM. One tube in each group was chased at 30 °C for 15 min, and the other for 60 min. After the chase, the volume was adjusted to 0.5 ml with TETN buffer (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 250 mM NaCl, and 0.1% Triton X-100), and 10 µl of anti-hsp60 antiserum (28) were added. The samples were allowed to react at 4 °C for 30 min by rotating end-over-end. 10 µl of 10% Staph A cells (Life Technologies, Inc.) were then added. After further 15-min incubation at 4 °C, Staph A cells were sedimented by centrifugation and washed twice with TETN buffer, followed by two washes with 10 mM Tris-HCl, pH 7.5, and 5 mM EDTA. Staph A-immunoglobulin-antigen complexes were dissociated by boiling for 3 min in 30 µl of the sample buffer for SDS-PAGE, and proteins in the supernatant were analyzed by SDS-PAGE and autofluorography. The amount of labeled MCAD that was co-immunoprecipitated with hsp60 was determined densitometrically.


RESULTS

The Amount of Total Mitochondrial Protein and FAD Content in Control and Riboflavin-deficient Rat Liver Mitochondria

With regard to the general morphological features and oxidative status, Hoppel and associates (10, 29) previously reported that by day 53 on riboflavin-deficient diet, hepatic mitochondria exhibited some morphological changes such as central array of parallel cristae and cup-like shape, but maintained normal respiratory control and ADP/O ratios, suggesting that riboflavin-deficient mitochondria were capable of generating ATP normally. Hence, it is reasonable to assume that riboflavin-deficient mitochondria are capable, in general, of carrying out hsp60-assisted protein folding that requires ATP.

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.

Molecular Forms of Newly Imported MCAD in Control and Riboflavin-deficient Mitochondria

In order to study the molecular forms of newly imported MCAD in normal and riboflavin-deficient mitochondria, human pMCAD was synthesized via in vitro transcription/translation and was incubated with either control or riboflavin-deficient rat liver mitochondria for varying periods of time. After incubation, mitochondria were solubilized with Triton X-100. The supernatant of the solubilized mitochondria was analyzed on gel filtration to determine the molecular size of the newly imported MCAD in the manner similar to the previous study for folding and assembly of the wild-type and lysine 304 to glutamate (K304E) variant MCAD in normal rat mitochondria(24) . In the present experiment, however, in vitro transcription-translation was carried out using a new recombinant plasmid, pGRPM2-WT, as a template. As discussed Under ``Materials and Methods,'' transcription-translation using this new recombinant produced pMCAD 10 times as efficiently as with pBPM1-WT that was used in the previous experiment(14, 24) .

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 times 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: bullet, 10-min import; circle, 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.

Assembly of Human Ornithine Transcarbamylase in Riboflavin-deficient Mitochondria

In order to test whether or not the low availability of FAD was directly responsible for the impaired assembly of MCAD, a FAD-containing enzyme, in riboflavin-deficient mitochondria, we carried out similar experiments on ornithine transcarbamylase, a non-FAD-containing, trimeric mitochondrial enzyme, that is involved in the urea cycle. The amount of ornithine transcarbamylase in the hMr complex and that of trimer found in riboflavin-deficient mitochondria (Fig. 3B) were both comparable to those detected in control mitochondria (Fig. 3A) at any time point, indicating that riboflavin-deficient mitochondria were capable of folding and assembling a non-FAD-containing protein. Thus, these data support the contention that the impaired assembly of MCAD in riboflavin-deficient mitochondria was caused by unavailability of FAD.


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 times 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: bullet, 10-min import; , 60-min import.



Effects of FAD, FMN, and NAD on the Transfer of MCAD from the hMr Complex to Tetramer

We then studied the effects of FAD restitution and FMN substitution on the folding-assembly of MCAD in riboflavin-deficient mitochondria (Fig. 4, A-C). The transcription-translation mixture containing radiolabeled pMCAD was incubated with riboflavin-deficient rat liver mitochondria for 10 min at 30 °C (``pulsed''). After solubilization and addition of 0.1 mM FAD or FMN, the mitochondria were further incubated at 30 °C for 15 or 60 min (``chased'') in a buffer containing 2.5 mM ATP. We have previously shown that ATP was essential for the folding of MCAD (14) as in that of other proteins. It has been considered that ATP hydrolysis is a prerequisite as an energy source for the correct folding of proteins(20) . The 15- and 60-min chase experiments were performed using mitochondria preparations from two different riboflavin-deficient rats (A and B, respectively). The initial FAD concentrations in the two deficient mitochondria preparations were comparable (1.44 and 1.34 µg/mg mitochondrial protein, respectively).


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: bullet, 15-min chase; box, 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.

Immunochemical Determination of the Effects of FAD, FMN, and NAD on the Release of MCAD from the Complex with Hsp60

We then directly determined the effects of the three nucleotides, FAD, FMN, and NAD, on the release of MCAD from the complex with hsp60 by quantitatively measuring the amount of MCAD that remained complexed with hsp60 after chase. In this experiment, riboflavin-deficient mitochondria from rat C were used. After 10-min import of MCAD, deficient mitochondria were solubilized and were chased in ATP-containing buffer for 15 or 60 min in the presence of FAD, FMN, or NAD, or without any cofactor. Hsp60 protein in each sample before and after the chase was immunoprecipitated using specific antibody against hsp60, and the amount of labeled MCAD that was co-precipitated with hsp60 was determined (Fig. 5). In the samples chased with ATP alone or those chased in the presence of ATP and NAD, MCAD almost entirely remained as the complex with hsp60 at the end of 60-min chase (96 and 92%, respectively, of the amount at 10-min import). In the sample chased in the presence of FAD and ATP for 60 min, in contrast, only 25% of MCAD remained complexed with hsp60, while in the sample chased in the presence FMN and ATP, 58% of MCAD remained complexed with hsp60, indicating the effectiveness of FAD and FMN in promoting the hsp60-assisted MCAD-folding process and bringing it to completion.


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: bullet, chase without any additional nucleotide; circle, chase with FAD; , chase with FMN; , chase with NAD.




DISCUSSION

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 MCADbullethsp60 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 MCADbullethsp60 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. (^2)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 beta-sheet domain, with its pyrimidine portion surrounded by the residues from the loops between beta-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 alpha,beta-dehydrogenation of acyl-CoA, proR-alpha-hydrogen of acyl-CoA is abstracted as a proton by the -carboxyl of Glu(47) , while proR-beta-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) .


FOOTNOTES

*
This work was supported by a National Institutes of Health Grant DK38154. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprints request should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., P. O. Box 3333, New Haven, CT 06510. Tel.: 203-785-2659; Fax: 203-785-3363.

(^1)
The abbreviations used are as follows: MCAD, medium chain acyl-CoA dehydrogenase; hMr, high molecular weight; hsp, heat-shock protein; p, precursor; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s).

(^2)
K. Ikeda, Y. Ikeda, and K. Tanaka, unpublished observations.


ACKNOWLEDGEMENTS

Drs. William J. Welch and M. Glotzer, University of California, San Francisco, CA, kindly provided anti-hsp60 antibody and Delta-13Tb, respectively, for this study. Ornithine transcarbamylase cDNA was a gift from Dr. Wayne Fenton of this department. We thank Drs. Elly M. Tanaka and David N. Drechsel, University of California, San Francisco, CA, for their advice for the use of Delta-13Tb, and Julie Kim of this laboratory for reading the manuscript.


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