Eur. J. Biochem. 97, 565-571 (1979)

Mitochondria1 ATPase Complex of Aspergillus nidulans and the Dicyclohexylcarbodiimide-Binding Protein Geoffrey TURNER, Carnal IMAM, and Hans KUNTZEL

Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen

(Received January 9, 1979)

The dicyclohexylcarbodiimide-binding protein of Aspergillus nidulans has been identified as the smallest subunit of the mitochondrial ATPase complex, and has a molecular weight of approximately 8000. It is extractable from whole mitochondria and from the purified enzyme in neutral chloroform/ methanol, contains 30% polar amino acids, and the N-terminal amino acid has been identified as tyrosine. Using a double-labelling technique in the absence and presence of cycloheximide, followed by immunoprecipitation of the enzyme complex with antiserum against Neurospora crassa F1 ATPase, it has been shown that this subunit is synthesized on cytoplasmic ribosomes.

Oligomycin-resistant mutants have been isolated in the filamentous Ascomycete Aspergillus nidulans [l ] and mutations in both nuclear and extranuclear ge- nomes have been shown to affect the oligomycin sensi- tivity of the mitochondria1 ATPase activity [2,3], suggesting the involvement of at least two subunits of the enzyme complex, one nuclearly coded and one extranuclearly coded, in the expression of oligomycin resistance. Furthermore, nuclear-extranuclear inter- actions have been observed in strains containing muta- tions in both genomes [3]. In contrast, only extra- nuclear oligomycin-resistant mutants in Saccharo- myces cerevisiae [4,5] and nuclear oligomycin-resistant mutants in Neurospora crassa [6] have been shown to alter the resistance of the ATPase activity of these organisms in vitro.

In order to identify the lesions caused by these mutants, and thus the gene-protein relationships, studies on the ATPase complex are essential. Oligo- mycin is known to inhibit oxidative phosphorylation by acting on the Fo or membrane components of the complex [7]. Dicyclohexylcarbodiimide acts at a nearby site in a similar manner, and shows irreversible binding to one of the FO components [8- 111. This hydrophobic polypeptide, referred to as the dicyclo- hexylcarbodiimide-binding protein, has now been ex- tracted from a number of sources, including the mito- chondria of beef heart [ll, 121, S. cerevisiae [13,14] and N . crassa 11.51, the chloroplasts of lettuce [16] and from membranes of Escherichia coli [17]. In all of these species the protein has a molecular weight of

Enzyme. ATP phosphohydrolase or ATPase (EC 3.6.1.3).

approximately 8000, and a low content of polar amino acids, usually between 20- 30 %, but as low as 16% in the case of E. coli [17]. Furthermore, it has been implicated in proton translocation across energy- transducing membranes [16].

The evolution of this protein and its gene is now of great interest, since it has recently been demon- strated that while in S. cerevisiae this protein is coded in mitochondrial DNA and synthesized on mitochon- drial ribosomes [14,18], the corresponding protein in N. crassa is nuclearly coded and cytoplasmically synthesized [6,19]. In addition, mutations in the gene for this protein are able to confer resistance to oligo- mycin [6,14].

Purification of the functional, oligomycin-sensi- tive ATPase of A . nidulans has already been reported, and the purified enzyme was characterized by sodium dodecylsulphate/urea/polyacrylamide gel electropho- resis [20]. To determine the site of synthesis of the dicyclohexylcarbodiimide-binding protein, antiserum against N. crassa F1 ATPase [19] has been used to precipitate the ATPase complex from small amounts of mitochondria which had been labelled in vivo in the absence and presence of cycloheximide [19,21].

MATERIALS AND METHODS

Growth of Mycelium and Isolation of Mitochondria

Crude mitochondria were prepared from strain pabaA2, biA1 as previously described [20]. Phenyl- methylsulphonyl fluoride (0.5 mM) was included in the isolation buffer, and after disruption of the hyphae with a grinding mill [22] the pH was immediately re-

566 Dicyclohexylcarbodiimide-Binding Protein of Aspergillus

adjusted to pH 7.5 with 2 M Tris base. For preparation of functional ATPase complex, mitochondria were further purified on sucrose gradients 1231.

Labelling of Mitochondria1 Proteins

For control label, t3H]leucine (50 Ci/mmol) was added to a 16-h culture (1 mCi/l) and growth con- tinued for a further 2 h before the mycelium was harvested and mitochondria prepared.

For specific labelling of mitochondrially-synthe- sized proteins, a double-labelling technique was used [19,21]. To a 16-h culture, [14C]leucine (300 Ci/mol) was added (0.125 mCi/l), and growth continued for a further 2 h, after which chloramphenicol was added (2 mg/ml) and growth continued for a further 30 min. The mycelium was then washed three times and resus- pended in fresh growth medium, and after 15-min recovery, cycloheximide was added (100 pg/ml). After 2 min, t3H]leucine was added (4 mCi/l), and 10 min later, a chase of unlabelled leucine (2 mM). After a further 60-min growth, the mycelium was harvested, and crude mitochondria prepared.

Purgication of the Functional A TPase Complex

ATPase was prepared from sub-mitochondrial particles by extraction with 0.3 "/, Triton X-100, fol- lowed by glycerol gradient centrifugation 1241 as described previously [20]. ATPase assays were carried out in 50 mM Tris-acetate, 5 mM MgS04, 3 mM ATP (pH 7.5) as described 1201.

Immunoprecipitation of ATPase Complex

Immunoprecipitation was carried out essentially as described 1191 using antiserum prepared against F, ATPase from N . crassa, which was obtained from Dr W. Sebald. Crude mitochondria were suspended in 10 mM Tris-acetate, pH 7.5, at a concentration of 2 mg/ml, and Triton X-1 00 was added to a final concen- tration of 1 %. After centrifugation (4 min, 10000 x g) , 0.5 ml clear supernatant was mixed with 0.5 ml of antiserum. These conditions were found to give maxi- mum precipitation. After 4 h at 0 "C, the precipitate was collected by centrifugation and washed twice by resuspension and recentrifugation in Tris-acetate buffer. The pellet was dissolved in electrophoresis sample buffer (25 pl).

Ex tract ion of the Dicyclohexylcarbodiimide-Binding Protein from Whole Mitochondria

Mitochondria labelled uniformly with [3H]leucine followed by [14C]dicyclohexylcarbodiimide labelling

as described were extracted with neutral chloroform/ methanol (2/1, v/v) as follows 1151. Mitochondria washed in 10 mM Tris-acetate (pH 7.5) were homo- genized in chloroform/methanol (211, v/v), 5 mg pro- tein/ml, and ether (5 vol.) was added. After 15 min at room temperature, the mixture was centrifuged (8000 x g, 10 min), and the pellet washed twice in the same manner. The pellet was then extracted five times with chloroform/methanol (2/1, v/v), 20 mg protein/ ml, for 10 min, centrifuged, and the combined extracts were precipitated with 4 vol. cold ether. The centri- fuged precipitate was dissolved in a minimal volume of chloroform/methanol (2/1, v/v), recentrifuged, and the supernatant re-precipitated with ether.

Gel Electrophoresis

Electrophoresis was carried out using a slab gel system [25] in 0.5% sodium dodecylsulphate, 0.1 M Tris-acetate, pH 8.0. Sample buffer contained 0.1 M Tris-acetate, 2 % sodium dodecylsulphate, 5 mer- captoethanol, pH 8.0. Gels were either stained with Coomassie blue or sliced for counting of radioactivity [19]. Mercaptoethanol was omitted when gels of immunoprecipitates were to be stained, in order to minimise dissociation of rabbit globulin.

N- Terminus Analysis

The protein was suspended in a known small volume of 0.2 M NaHC03, pH 9.5, and lyophilized. The dry material was resuspended in the same volume of 50 aqueous dimethylformamide containing 5 mg dansyl chloride/ml, incubated for 30 min at 37 "C, then lyophilized. The dansylated protein was washed twice with a large volume of toluene/ether ( l / l , v/v), then a large volume of acetone/formic acid (19/1, v/v) to remove any free dansylated amino acids, centrifuged and dried. Hydrolysis was carried out with 6 M HCl at 105°C for up to 20 h. The hydrolysate was dried, washed in toluene, dried, and extracted in chloroform/ methanol (119, v/v). The extract was centrifuged, the supernatant dried and redissolved in a small volume of acetone/formic acid (19/1, v/v). The liberated dansylated amino acids were analyzed by two-dimen- sional thin-layer chromatography [20], first dimension 3 "/, formic acid, second dimension benzene/acetic acid (9/2, v/v).

Amino Acid Analysis

This was carried out as previously described [20].

Chemicals

['4C]Leucine and [3H]leucine were obtained from the Radiochemical Centre, Amersham, England. ['4C]- Dicyclohexylcarbodiimide was a gift from Dr W. Sebald.

G. Turner, G. Imam, and H. Kiintzel 567

55000

36000

15000 -

12000 -

- 55000

33000

-22500 20000

-11000

-7000

Fig. 1 . Sodiun? dodecylsulphatrlacrylamid~ gel electrophoresis of.functional A TPase and immunoprecipitates. After electrophoresis, gels were stained with Coomassie blue (a) Fi ATPase of Neurosporu crassa; (b) immunoprecipitate of mitochondria dissolved in 1 :,<, Triton; (c) immunoprecipitate of clarified, 0.3 % Triton extract of sub-mitochondrial particles; (d, e) functional ATPase; (0 chloroform/rnethanol/ water extract of functional ATPase

RESULTS

Immunoprecipitation of the A TPase Complex

When antiserum against N. crassn Fl ATPase was incubated with Triton-X-100-solubilized mitochondria of A . nidulans, a white precipitate was formed, which under optimal conditions represented about 7 % of total mitochondrial protein. Control serum gave no observable precipitate under the conditions normally used with the test serum.

Since preliminary results suggested that the elec- trophoresis system previously used for analysis of ATPase from Aspergillus [20] resulted in incomplete dissociation or aggregation of the mitochondrial prod- ucts, and in order to facilitate comparison with the ATPase of N . crassa [19], the gel system of Sebald et al. [25] was used throughout this work.

Sodium dodecylsulphate electrophoresis of this precipitate, followed by staining with Coomassie blue, resulted in the gel shown in Fig. 1 b, which may be compared with a preparation of the functional enzyme (Fig. I d ) and F1 ATPase of N. crassa (Fig.1a). An immunoprecipitate of the centrifuged, 0.3 % Triton X-100 extract of sub-mitochondrial particles prior to glycerol gradient centrifugation [20] is also shown (Fig. 1 c). The band of M, 22500 and all those of lower molecular weight correspond in all preparations. How- ever, the immunoprecipitates include one or two extra bands of M , 23000-25000, and numerous small bands of hfr40000-50000, which may not be part of ATPase complex. A band of M, 23500 is present in all immunoprecipitates, and may be the L chain of immunoglobulin from the rabbit antibody. The c1 and

p bands of the F1 portion of the ATPase (M, 53000 and 58 000) are not resolved because of the overloading necessary to visualize the weaker bands. Bands of M , 12000 and 15000 in the immunoprecipitate corre- spond to 6 and E bands of N . CYUSSU F1, though no band corresponding to the y band of Neurospora is seen. Either it is lost in the immunoprecipitate, or the y band of Aspergillus is the one of M , 33000, being rather smaller than that of Neurospora (M, 36000). High- molecular-weight protein close to the top of the gel is the undissociated rabbit immunoglobulin.

[ 4C ]Dicyclohexylcarhodiimide-labelled Immunoprecipitate

ATPase activity in isolated mitochondria was inhibited 80 % by 2 pg dicyclohexylcarbodiimide/mg protein. This concentration was used in radioactive binding experiments, since it i s known that excess concentration of the inhibitor can result in non- specific binding [ll].

Mitochondria labelled homogeneously with [3H]- leucine were suspended in 10 mM Tris-acetate (pH 7.5), and [14C]dicyclohexylcarbodiimide (10' counts min- ' pg-I) was added as a methanolic solution to a final concentration of 2 pg/mg protein (1 methanol). After 4-h incubation at 0 "C, a sample of the incubation mix- ture (1 mg protein) was dissolved in Triton X-100 and immunoprecipitated as described. The washed precip- itate was dissolved in sample buffer and applied to a gel for electrophoresis (Fig. 2). Dicyclohexylcarbo- diimide bound to the immunoprecipitate corresponded to 2.5 nmol/mg protein. It can be seen that the dicyclo-

568

1400

Dicyclohexylcarbodiimide-Binding Protein of AspergiNus

~

53000 I 1

/l i i i

200

0 I0 20 30 40 50 60 70 80 90 100 0

Gel slice

Fig. 2. Electrophoresis qS immunoprecipitate from mitochondria labelled homogeneously in vivo with [3H]leucine I.---.) and in vitro by incubation with [ ''C]dicyclohexylcarbodiirnide (o--o). The numbers in the figure are molecular weights

hexylcarbodiimide has bound specifically to a low- molecular-weight component ( M , 7000, estimated by co-electrophoresis with insulin) corresponding to the final, major 3H peak observed in the gel. As already observed in Neurospora, the peaks of I4C and 3H do not exactly coincide, and it has been suggested that the binding of dicyclohexylcarbodiimide to the protein may reduce its mobility [15]. There is evidence that in the ATPase complex the dicyclohexylcarbodiimide- binding protein exists as a hexamer and that modifica- tion of one of the six molecules by dicyclohexyl- carbodiimide is sufficient to inhibit enzyme activity maximally [12,18]. Dicyclohexylcarbodiimide bound to the final peak corresponds to about 18 nmol/mg protein (equivalent to 0.125 mol/mol protein).

Cycloheximide- Resistan t Labelling

In order to determine the site of synthesis of the dicyclohexylcarbodiimide- binding protein, ATPase complex was immunoprecipitated from mitochondria labelled in the absence and presence of cycloheximide, and analyzed on a sodium dodecylsulphate gel (Fig. 3). 14C label shows the position of all major bands, and 3H label (cycloheximide-resistant) should indicate mitochondrially-synthesized products. The clearest mitochondria1 product is that of M , 20000. Other minor peaks are apparent of M , 11000 and in the region M , 40000- 50000. However, it is apparent that the smallest component in the gel ( M , 7000) is not labelled in the presence of cycloheximide, even though control label indicates that incorporation into this band is high in the absence of the drug. Double labelling using phenylalanine instead of leucine gave a similar pattern.

Characterization of the Extracted Dicyclohexylcarbodiimide-Binding Protein

The protein extracted from whole mitochondria was analyzed by sodium dodecylsulphate gel (Fig. 4). The major peak at M , 7000 is associated with the 14C label, and this corresponds to 22 nmol dicyclohexyl- carbodiimide/mg protein, similar to the figure obtained for the 7000-M, peak of the immunoprecipitate. In addition, three minor bands are present which do not have any associated I4C label. Although the dicyclo- hexylcarbodiimide-binding protein is extracted from Aspergillus whole mitochondria in a rather specific manner by this method, the product is not quite as pure as that reported for Neurospora [15]. End group analysis of the isolated protein revealed tyrosine as the major N-terminal amino acid. Several weaker spots not corresponding to the positions of any amino acids were also present. No methionine was detectable before or after incubation of the protein at 37°C for 3 h with 0.1 M HC1 in methanol prior to dansylation.

Removal of the minor, higher-molecular-weight bands observed in gels could be achieved by column chromatography in 80% formic acid on P-30 Biogel, and an amino acid analysis of this preparation is given in Table 1. The molecular weight is estimated as 8500 and the polarity as 30% [26].

Extraction of the purified ATPase complex with aqueous chloroform/methanol yields a protein which is seen as a single, low-molecular-weight band on sodium dodecylsulphate gel electrophoresis, corre- sponding to the smallest subunit of the complex [20] (Fig. 1 d,e). End group analysis of this preparation before and after treatment with 0.1 M HCl in methanol revealed tyrosine as the only detectable N-terminal

569

I 400 E c ._

0 . - 5 300- 9 I

g 200- 73 c

,I 100-

0

G . Turner, G. Imam, and H. Kuntzel

40C

-

e. 8

30C

- (I .~ E . v) L

5 20c u I

0 * -

100

53r 20yoo

200

150

I c ._ E . “7

00 g s I

I

33

1

Fig. 3. Electrophoresis of’immunoprecipitate from double-labelled mitochondria. Mitochondria were labelled in vivo with [14C]leucine (o~-o) in the absence of cycloheximide followed by 10-min labelling with [3H]leucine (0----0) in the presence of cycloheximide and a chase with unlabelled leucine (see Methods). The numbers in the figure are molecular weights

amino acid, indicating a very specific extraction of the dicyclohexylcarbodiimide-binding protein.

DISCUSSION

A specific cross reaction between antiserum against Neurosporu FI ATPase and the ATPase complex of A . niduluns is apparent from both the gel pattern, which resembles that of the functional enzyme, and the observation that the antiserum precipitates the dicyclohexylcarbodiimide-binding protein. The dis- tribution of radioactivity in gels of both cycloheximide- resistant-labelled and [‘4C]dicyclohexylcarbodiimide- labelled immunoprecipitates closely resemble those observed for N. CYUSSU 115,191, suggesting close simi- larity in structure and synthesis of this complex in the two organisms. Although both are Ascomycetes, they

are in different sub-classes, and the mitochondria1 DNA of N . crussu ( M , 40 x lo6) [27-291 is almost twice the size of that of A . niduluns ( M , 21 x lo6)

Jack1 and Sebald [19] concluded that two of the subunits, M , 19000 and 11 000, were mitochondrially synthesized, and as in Aspergillus, the 19000-M, band was much more highly labelled than the 11000-M, band in the presence of cycloheximide. A number of minor, high-molecular-weight bands were also ob- served in N. crussu immunoprecipitates from mycelium labelled for 60 min in the presence of cycloheximide, though these were not observed after shorter labelling periods followed by a chase of unlabelled amino acid.

It is quite clear that the smallest-molecular-weight component of the Aspergillus ATPase complex, which binds dicyclohexylcarbodiimide, is of cytoplasmic

[30,31].

570 Dicyclohexylcarbodiin~ide-Binding Protein of Aspergillus

Table 1. Amino acid composition qf the dicyclohe.wylcarhodiimide- binding protein of A. nidulans Amino acid analysis was carried out as described [20]. The minimum number of amino acid residue was cakukdted with threonine set equal to 1.0. Averages of three independent determinations are given after hydrolysis for 20 h

Amino acid Amount Number of residues

mo1/100 mol

Aspartic acid 7.28 6 Threonine 1.20 1 Serine 8.64 7 Glutarnic acid 6.72 6 Proline 5.63 5 Glycine 14.38 12 Alanine 14.08 12 Valine 6.16 5 Methionine 4.74 4 Isoleucine 6.13 5 Leucine 9.74 8 Tyrosine 3.95 3 Phenylalanine 5.20 4 Lysine 2.13 2 I listidine 0.65 (1) 4i-pinine 3.14 3

Total 99.71 83 .~ . ~ - __ - Polarity [26] 30 2,

origin, since its synthesis is inhibited in the presence of cycloheximide, as is the synthesis of the a and p bands of FI ATPase. Aspergillus dicyclohexylcarbo- diimide-binding protein is similar in this respect to that of Neurospova, and contrasts with the homologous subunit of S. cerevisiae, whose synthesis is cyclo- heximide-resistant [14,18].

Further confirmation of the site of synthesis of this protein is given by the fact that the N terminus is not formylmethionine, as is the case for the dicyclohexyl- carbodiimide-binding component of yeast [14]. Re- cently, it has been reported that the dicyclohexyl- carbodiimide-binding protein of rat liver mitochondria is a mitochondria1 product, though the protein was not clearly resolved on gels, and end group determi- nation was not carried out as a check for purity [32]. It is possible that a low-molecular-weight mitochon- drial translation product(s), extractable from a tri- chloroacetic acid precipitate of whole mitochondria by chloroform/methanol and similar to that of A . nidu- lam 1201 has not been separated from the dicyclo- hexylcarbodiimide-binding protein. The N-terminal amino acid of beef heart dicyclohexylcarbodiimide- binding protein is aspartate [12]. Although this sug- gests a cytoplasmic origin for the protein, further labelling studies are required to firmly establish its synthetic origin in higher organisms.

As observed in N . crassa [15] and S. cerevisiae [14], the dicyclohexylcarbodiimide-binding protein of A. Midulans may be extracted from whole mitochondria

with some degree of specificity by dry, neutral chloro- form/methanol, and has a low content of polar amino acids. Aqueous chloroform/methanol has been used to extract this protein from whole mitochondria of beef heart [11,12] and E. coli [17], though a number of major components are simultaneously extracted, and further extensive purification was necessary. Aqueous chloroform/methanol extracts a low-molec- ular-weight mitochondrial translation product from A. nidulans [20] in addition to the dicyclohexylcarbo- diimide-binding protein, though the function of this component is unknown, since it is not extracted from the purified oligomycin-sensitive ATPase by the same solvent.

Sequencing studies have shown a considerable degree of homology between the dicyclohexylcarbo- diimide-binding proteins of N . crassa and S. cerevisiae, where approximately 50%) of the positions are occu- pied by identical amino acids, and a lesser degree of homology between these two organisms and E. coli (25% and 2073, suggesting a rather slow evolution rate for this protein [33]. In addition, the amino acid sequence of the dicyclohexylcarbodiimide-binding pro- tein is altered in nuclearly inherited, oligomycin- resistant mutants of N . crassa [6]. A number of nuclear, oligomycin-resistant mutants of Aspergillus with in- creased resistance at the level of the mitochondrial ATPase have previously been described, and all nuclear mutations so far tested map very close to- gether on chromosome VII [1,3]. It seems likely in view of the many similarities so far observed between the ATPase complexes of these two organisms that the mutations may also be located in the nuclear gene which codes for the dicyclohexylcarbodiimide pro- tein, but sequencing of the Aspergillus protein of wild type and mutants will be necessary to confirm this hypothesis.

This work was supported by the Science Research Council, U K., and by a European Molecular Biology Organisation Long- Term Fellowship (G.T.). The authors are grateful to Dr Sebald for gifts of antiserum and ['4C]dicyclohexylcarbodiimide, and for useful discussion.

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G. Turner, Department of Bacteriology, University of Bristol Medical School, University Walk, Bristol, Great Britain, BS8 ITD

G . Imam, Faculty of Science, Mansura University, Mansura, Egypt

H. Kuntzel, Abteilung Chemie, Max-Planck-Institut fur Experimentelk Medizin, Hermdnn-Rein-StraBe 3, D-3400 Gottingen, Federal Republic of Germany