©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Folding, Flavinylation, and Mitochondrial Import of 6-Hydroxy-

D

-nicotine Oxidase Fused to the Presequence of Rat Dimethylglycine Dehydrogenase (*)

(Received for publication, November 28, 1994; and in revised form, January 20, 1995)

Michaela Stoltz Petr Rysavy (1) Frantisek Kalousek (1) Roderich Brandsch (§)

From the Biochemisches Institut, Universität Freiburg, Freiburg, Federal Republic of Germany Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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)-8alpha-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 (Me(2)GlyDH), 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(2)-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 Me(2)GlyDH 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.


MATERIALS AND METHODS

Chemicals

FAD, FMN, riboflavin, glycerol 3-phosphate, trypsin, soybean trypsin inhibitor, and digitonin were purchased from Sigma (Deisenhofen, Federal Republic of Germany), rabbit reticulocyte lysate from Promega (Madison, WI) and [S]methionine was from Amersham (Braunschweig, FRG). All other chemicals were of highest purity available.

Plasmid Constructs

The 6-HDNO gene was cloned into pSPT20 BM (Boehringer Mannheim, Mannheim, FRG) as a NcoI-endonuclease restriction fragment isolated from pLM 1702(18) . A fusion between the presequence of Me(2)GlyDH and 6-HDNO was constructed by digestion of pSPT19-Me(2)GlyDH with NaeI/HindIII and replacement of the NaeI/HindIII Me(2)GlyDH fragment with the 6-HDNO gene. For convenience the chimeric construct was named pre-6-HDNO. Plasmids were multiplied in Escherichia coli strain JM109, and plasmid DNA was isolated from bacterial cells with the aid of a DNA isolation kit (Diagen, Hilden, FRG).

In Vitro Transcription and Translation

Transcription with SP6 polymerase from pSPT20-6-HDNO and pSPT19-pre-6-HDNO and translation of the transcripts in the presence of [S]methionine in rabbit reticulocyte lysate was done according to the suppliers instructions (Promega).

Processing of Pre-6-HDNO with Purified Mitochondrial Processing Peptidase (MPP) and Determination of the Processing Site

4 µl of [S]Met-labeled pre-6-HDNO translation product were incubated for 1 h at 27 °C with 0.3 µg of a rat liver MPP preparation (19) and analyzed on a 10% polyacrylamide gel by SDS-PAGE. The MPP processing site of the Me(2)GlyDH presequence fused to 6-HDNO was determined by amino-terminal radiosequencing of products from processing substrate synthesized in the presence of [^3H]leucine. Processing products were recovered and sequenced as described previously(20) .

6-HDNO-holoenzyme Formation and Test for Trypsin Resistance

Pre-6-HDNO was synthesized in rabbit RL supplemented with 10 µM FAD for 1 h at 30 °C as recommended by the supplier of the RL (Promega) followed by incubation with 10 mM glycerol-3-P for 30 min at 30 °C. 6-HDNO activity was measured as described previously(8) . Labeling of 6-HDNO and pre-6-HDNO holoenzymes with [^14C]FAD was performed by translating the proteins in the presence of 10 µM [^14C]FAD followed by incubation of the translation assays with 10 mM glycerol-3-P for 30 min at 30 °C. The translation products were analyzed on 10% polyacrylamide gels by SDS-PAGE. To test for the presence of holoenzyme, translation products were digested with 0.2 mg/ml trypsin for 15 min at 0 °C. Digestion was stopped by addition of 4 mg/ml soybean trypsin inhibitor.

Mitochondrial Import Assays

Rat liver mitochondria were prepared as described(21) . Mitochondrial import assays were performed by incubating 4 µl of radioactive RL translation product with freshly isolated rat liver mitochondria at a final concentration of 8 mg/ml. After incubation (60 min, 27 °C) the mitochondria were sedimented by centrifugation, washed with 200 µl of HMS buffer (2 mM HEPES, pH 7.4, 220 mM mannitol, 70 mM sucrose), and pelleted. Import assays were digested with 0.2 mg trypsin/ml at 0 °C for 15 min. The digestion was stopped by the addition of soybean trypsin inhibitor (4 mg/ml) and the mitochondria washed and pelleted. The mitochondrial pellets were analyzed on SDS-PAGE. When required MPP was inhibited by incubation of mitochondria for 3 min at 27 °C with 5 mM EDTA and 1 mMo-phenanthroline prior to import. The membrane potential Delta was dissipated by the addition of 20 µM oligomycin, 0.5 µM valinomycin, and 0.8 µM antimycin A to the isolated mitochondria. For FMN supplementation, the isolated mitochondria were incubated before performing the import assays with 12 µM FMN for 3 min at 27 °C. Fractionation of mitochondria into matrix and membranes was performed by freezing and thawing the washed mitochondria in liquid nitrogen four times followed by centrifugation at 100,000 times g at 2 °C for 30 min. The purity of the submitochondrial fractions was tested on Western blots with the aid of antibodies specific for a marker protein of the mitochondrial matrix. Densitometric quantification of the fluorographs was performed with an Image Master DTS from Pharmacia LKB (Freiburg, FRG).

Holoenzyme Formation in the Mitochondrial Matrix

Mitochondrial matrix was prepared from isolated rat liver mitochondria by sonication for 3 min at 0 °C and 100 watts. Mitochondrial membranes and matrix fraction were separated by centrifugation for 10 min at 14,000 times g and 4 °C. 4 µl of [S]Met-labeled pre-6-HDNO apoenzyme was incubated with 100 µg of mitochondrial matrix for 30 min at 30 °C. For investigation of holoenzyme formation the assays were divided and half subjected to trypsin digestion (0.2 mg/ml) for 15 min at 0 °C.


RESULTS

Folding, Holoenzyme Formation, and [^14C]FAD Incorporation into 6-HDNO and Pre-6-HDNO Translated in the in Vitro RL System

Since the autocatalytic incorporation of FAD into 6-HDNO depends on the folding of the polypeptide into a flavinylation-competent conformation(10) , we asked whether 6-HDNO translated in the RL folds correctly and exhibits cofactor attachment and enzyme activity. The [S]methionine-labeled RL translation products of 6-HDNO and pre-6-HDNO are presented in Fig. 1, panel A. They show the expected size difference in molecular weight between 6-HDNO and pre-6-HDNO. When 6-HDNO activity was measured in the translation assay, it was apparent that the endogenous content of FAD in the RL was insufficient to promote 6-HDNO holoenzyme formation and that supplementation of the RL with FAD was required for 6-HDNO activity (Fig. 1, panel B, lane 2). As shown previously(22) , holoenzyme formation of E. coli-expressed 6-HDNO required, besides FAD, supplementation with glycerol-3-P. The same observation was made with the RL translated 6-HDNO: addition of FAD and glycerol-3-P was necessary to convert the synthesized 6-HDNO into holoenzyme (Fig. 1, panel B, lane 3). RL translation assays performed with pre-6-HDNO gave identical enzyme activities to those performed with 6-HDNO (Fig. 1, panel B, pre-6-HDNO). Thus the presequence of Me(2)GlyDH did not prevent the folding of the 6-HDNO fusion protein into the flavinylation competent conformation, the incorporation of FAD into the polypeptide, or enzyme activity. We have shown previously that 6-HDNO activity requires covalently bound cofactor(10) . The covalent binding of FAD to the RL-translated 6-HDNO and pre-6-HDNO was directly demonstrated by supplementation of the RL with [^14C]FAD and fluorography of the [^14C]FAD-labeled 6-HDNO proteins (Fig. 1, panel C).


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 [^14C]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.



Trypsin Resistance of the RL-translated 6-HDNO Polypeptides

Flavinylation of the apoenzyme and attainment of the native conformation of the holoenzyme is accompanied in vivo in E. coli cells by the achievement of protease resistance of 6-HDNO(23) . This observation applies to the 6-HDNO and pre-6-HDNO RL translation products as well. Holoenzyme formation of both 6-HDNO and pre-6-HDNO resulted in a protein conformation highly resistant to trypsin (Fig. 2), proteinase K, and chymotrypsin (not shown). Trypsin digestion of pre-holo-6-HDNO resulted in a molecular form that migrated on SDS-PAGE identical to the holo-6-HDNO form (Fig. 2, lanes 4 and 8, respectively). Thus only the presequence remained accessible to proteolytic attack, and apparently the trypsin cleavage site must be close to the precursor processing site recognized by the MPP. The appearance of the trypsin-resistant form was taken as an indication for FAD incorporation and 6-HDNO holoenzyme formation.


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.



Assignment of the MPP Cleavage Site of the Me(2)GlyDH Presequence in the Pre-6-HDNO Protein

In order to establish the MPP cleavage recognition site of the Me(2)GlyDH signal sequence fused to 6-HDNO, we performed NH(2)-terminal amino acid sequencing on the in vitro [^3H]L-leucine-labeled translation product, processed with purified MPP. From the amino acid sequence deduced from the Me(2)GlyDH-cDNA, the MPP processing site was predicted between Pro and Ser, which would leave Arg in position -2 with respect to the cleavage site (Fig. 3, panel A, arrow). This site would agree with the Arg rule established for the mitochondrial MPP recognition site(24) . It also predicted that the first leucine residue, which is also the first residue of the 6-HDNO fusion partner, would appear in the fourth cycle of the amino acid sequencing reaction. This was indeed the case (not shown). Fig. 3, panel B, shows the MPP digestion product of the RL translated pre-6-HDNO.


Figure 3: NH(2)-terminal amino acid sequence of the pre-6-HDNO fusion protein and processing by the MPP. Panel A, schematic representation of the Me(2)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).



Import of Pre-6-HDNO into Rat Liver Mitochondria

Next we analyzed whether the Me(2)GlyDH presequence confers import competence to the fused 6-HDNO partner. [S]Methionine-labeled, RL-translated pre-apo-6-HDNO and apo-6-HDNO were incubated with isolated rat liver mitochondria and tested for intramitochondrial location of the 6-HDNO proteins by trypsin digestion (Fig. 4, panel A). Apo-6-HDNO incubated with mitochondria remained, as expected, protease-sensitive (Fig. 4, panel A, lane 3). Pre-apo-6-HDNO, however, upon incubation with mitochondria, was processed to a lower molecular weight, trypsin-resistant, and, therefore, intramitochondrially located form (Fig. 4, panel A, lane 6). From this experiment we conclude that the fusion protein between the Me(2)GlyDH presequence and 6-HDNO was imported into rat liver mitochondria and correctly processed by the MPP. Only a fraction of the imported pre-apo-6-HDNO (approximately 20%) was transformed inside the mitochondria into the trypsin-resistant form characteristic for the holoenzyme, however (Fig. 4, panel B, lane 2). Does the intramitochondrial transformation of the imported apo-6-HDNO into holoenzyme require the simultaneous import of the flavin cofactor? To answer this question we performed import assays with rat liver mitochondria preincubated with FMN. The amount of trypsin resistant 6-HDNO formed in the mitochondrial matrix after import of pre-apo-6-HDNO did not increase after preincubation of mitochondria with FMN (Fig. 4, panel B, lane 4), nor when mitochondria were preincubated with FAD or riboflavin (not shown).


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 Delta-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.



6-HDNO Holoenzyme Formation in the Matrix Fraction of Rat Liver Mitochondria

Incubation of apo-6-HDNO with mitochondrial matrix resulted in only partial formation of holo-6-HDNO as indicated by trypsin digestion of the protein (Fig. 6, lane 2). Addition of FAD only to the matrix increased the holoenzyme level (Fig. 6, lane 3); however, addition of both FAD and glycerol-3-P was required to induce a further increase in trypsin resistance of the 6-HDNO protein (Fig. 6, lane 5). Apparently the intramitochondrial FAD and effector concentration was insufficient to support flavinylation of the imported precursor.


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.




DISCUSSION

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 Me(2)GlyDH 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 Me(2)GlyDH 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 Delta 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 Delta-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.


FOOTNOTES

*
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (to R. B.) and National Institutes of Health Grant DK-09527 (to F. K.). 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.

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).

§
To whom correspondence should be addressed: Biochemisches Institut, Hermann-Herder-Str. 7, 79104 Freiburg, Germany. Tel.: 49-761-203-5231; Fax: 49-761-203-5253.

(^1)
The abbreviations used are: RL, reticulocyte lysate; glycerol-3-P, glycerol 3-phosphate; 6-HDNO, 6-hydroxy-D-nicotine oxidase (EC 1.5.3.6); MPP, mitochondrial processing peptidase; Me(2)GlyDH, dimethylglycine dehydrogenase (EC 1.5.99.2); PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. W. Voos for fruitful discussions and A. Hebrok for critical support during the experiments.


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