Eur. J. Biochem. 40,201-206 (1973)

Cycloheximide-Resistant Synthesis of Mitochondrial-Membrane Components in Aspergillus nidulans

Geoffrey TURNER

Department of Bacteriology, University of Bristol

(Received July 10, 1973)

In the presence of cycloheximide, Aspergillus nidulam mycelium incorporated radioactive leucine preferentially into mitochondrial protein. Although cytoplasmic protein synthesis was not totally abolished in the presence of the drug, mitochondrially synthesized components could be identified by a double-labelling procedure. Labelled mitochondria were purified by centrifugation on sucrose gradients, and electrophoresis of mitochondrial membrane proteins on dodecyl- sulphate-polyacrylamide gels resulted in the identification of four mitochondrially synthesized components with molecular weights of 13000, 18000, 27000 and 40000.

It is well established that mitochondria possess their own protein synthesizing system, involving mitochondrial DNA, RNA and ribosomes [I -31. Although it is apparent that the synthetic capacity of mitochondria is very limited, it is not clear how many of the mitochondrial proteins are synthesized by the mitochondrial system, nor is i t certain what their exact function is. More recently, some evidence has accumulated to suggest that at least portions of the cytochrome oxidase [4,5], cytochrome b [6] and sub- units of mitochondrial ATP synthetase [7,8] may be synthesized by the mitochondrial protein-synthesiz- ing system. It has also been observed that there is some variation in the amount of mitochondrial DNA amongst eukaryotic microorganisms [l], though it has not yet been demonstrated that organisms containing more DNA are able to code for a greater number of mitochondrial components. It is therefore of interest to examine the protein-synthesizing capacity of a variety of eukaryotes over the evolu- tionary scale.

Recently, extrachromosomal mutants have been isolated in Aspergillus nidulans [9,10] and in order that their lesions might be more closely studied, this work was undertaken in an attempt to identify mitochondrially-synthesized proteins in this organism. Previous workers have undertaken this task with a variety of organisms, either in vitro [Ill, observing the proteins synthesized by isolated mitochondria, or in vivo [4,11,12], using an inhibitor such as cyclo-

Abbreviation. butyl-PBD, 2(4’-t-butylphenyl)-5(4”-bi-

Enzymes. Cytochrome oxidase or ferrocytochrome c: 0, phenylyl)-1,3,4-oxadiazole.

oxidoreductase (EC 1.9.3.1).

heximide to prevent synthesis of proteins on cyto- plasmic ribosomes. I n view of the uncertainties of labelling in vitro [13,14], and the ease with which isolated mitochondria may be damaged, the in vivo method was adopted for this work.

METHODS AND MATERIALS

Xtrain and Culture Media The strain of Aspergillus nidulans (Eidam) Winter

used in this work (R21 pabaAl, yA2, a p-amino- benzoic acid auxotroph) was kindly supplied by Dr C. F. Roberts of the University of Leicester. The organism was routinely maintained on solid complete medium [lo] and this medium was also used to pro- duce conidia for inoculation of liquid cultures. Mycelium used in all the experiments described was grown in liquid minimal medium [lo] with forced aeration (8 l/min). Minimal medium (16 1) was inoculated with conidia to a final concentration of IOe/ml, and the mycelium was harvested after 20 h growth by filtration through a single layer of fine muslin. The growth temperature was 37 “C in all cases.

Measurement of Protein Synthesis by Whole Mycelium The concentration of mycelium was adjusted to

approximately 10 mg dry weightlml of growth medium, and [14C]leucine was added to a concentration of 0.2 pCi/ml. At suitable time intervals, samples of the culture (5 ml) were added to an equal volume of trichloroacetic acid (120/,). The samples were collected on glass fibre filters (Whatman GF/C) by

Eur. J. Biochem. 40 (1973)

202 Mitochondrial-Membrane Components in Aspergillus

filtration, washed with 10 ml trichloroacetic acid (6O/,) followed by 20 ml acetic acid (lo/,). The filters were transferred to counting vials, and dried at 90 "C for 1 h prior to counting.

To assess the effect of cycloheximide on protein synthesis, mycelial suspension was pipetted into a number of vials (2 ml/vial) to which were added different concentrations of cycloheximide. After 30 min an equal volume of 12O/, trichloroacetic acid was added to the vials, and the samples were prepared for counting as described above. All incubations were a t 37 "C in a shaking water bath.

Preparation of Mitochondria1 Fractions Unless stated otherwise, all operations were

carried out a t 4 "C. After harvesting, the mycelium was washed with tap water and distilled water, then washed and resuspended in 5vol. isolation buffer (I0 mM Tris-HC1, 1 mM EDTA, 0.5 M sucrose, pH 7.0). The mycelium was then disrupted by hand- homogenization in a ground-glass homogenizer (40 ml, Fisons, England) and the resulting homogenate was filtered through 8 layers of muslin to remove un- broken mycelium and large pieces of hyphal wall. The filtrate was centrifuged a t 10000 x gav for 30 min, and the supernatant retained. The pellet was resuspended in I ml of isolation buffer by gentle pipetting. Linear sucrose gradients (20 ml) were formed from 0.5 M-2 M sucrose containing 10 mM Tris-HC1, 1 mM EDTA, pH 7.0, and after layering the crude mitochondrial fraction on to the gradient, i t was centrifugated at 70000 x gav for 1 h. Following centrifugation, the mitochondrial band was collected by means of a syringe (5 ml), resuspended in 40 ml isolation buffer, and centrifuged a t 35000 x g,, for 1 h to obtain a pellet.

The supernatant from the 100OOxg spin was recentrifuged a t 35 000 x g,, for 1 h, and the super- natant retained as the "cell sap" or non-mitochondri- a1 proteins. Aliquots of mitochondrial and non- mitochondrial protein were added to trichloroacetic acid (6OJo final concentration) and the precipitates were prepared for radioactive counting as described for whole mycelium.

For electrophoresis, the mitochondrial pellet was resuspended in 0.01 M sodium phosphate buffer, pH 7.0 (approximately 0.1 mg proteinlml) and homo- genized by hand. The membrane fraction was col- lected by centrifugation a t 35000 x g,, for 1 h.

501, (v/v) 2-mercaptoethanol, loo/, (v/v) glycerol, O.O5O/, (v/v) bromophenol blue, 0.01 M sodium phosphate pH 7.0, by heating a t 90 "C for 5 min. Approximately 50-200 pg mitochondrial protein was applied to each gel, and electrophoresis was carried out a t 8 m a per gel for 5 h using a Shandon disc electrophoresis apparatus. The gels were either stained with Coomassie blue followed by electro- phoretic destaining [15] or were sliced prior to radio- active counting.

Standards of cytochrome c, chymotrypsin, lyso- zyme, myoglobin, y-globulin and pepsin were also subjected to electrophoresis in order to calibrate the gels for molecular weight.

Counting of Radioactivity Dried samples on glass fibre filters were placed in

counting vials, and 6 ml scintillation fluid (0.4O/, w/v 2(4'-t-butylphenylyl)-5(4"-biphenyl)- 1,3,4-0xadiazole in toluene) was added.

Gel slices were placed in counting vials, and 0.2 ml of hydrogen peroxide (100 vol.) was added. Following incubation a t 60 "C for 2 h in order to dissolve the fractions, 0.5 ml H,O was added, follow- ed by 6 ml scintillation fluid (0.4O/, w/v butyI-PBD in 2: 1, v/v, toluene/Triton X-100).

Each sample was counted for 10 min in a Packard Tri-Carb scintillation counter. When counting 3H and 14C in double-labelled samples, corrections were made for overlap.

Assays Cytochrome oxidase was assayed spectrophoto-

metrically by following the rate of reoxidation of reduced mammalian cytochrome c at 550 nm. The assay mixture contained 25pM cytochrome c in I0 mM phosphate buffer, pH 7.0.

Protein was assayed by the method of Lowry et al. [16] with bovine plasma albumin as standard.

Chemicals Most of the chemicals used were AnalaR or reagent

grade obtained from BDH Chemicals Ltd. The protein standards for electrophoresis and Coomassie brilliant blue were obtained from Sigma Chemical Co., cycloheximide (actidione) from Koch-Light Laboratories Ltd, [U-l4C]leucine (300 mCi/mmol) and [4,5-3H]leucine (I Ci/mmoI) from the Radio- chemical Centre (Amersham).

Polyacrylamide- Gel Electrophoresis Electrophoresis was performed according to the

method of Weber and Osborn [15] using 1O0/, acryl- amide gels (0.5 x 10 cm). Prior to electrophoresis, the mitochondrial membrane fraction was dissolved a t a concentration of approximately 4 mg/ml in sample buffer, i.e. 5 (w/v) sodium dodecylsulphate,

RESULTS Isolation Of the Mitochondriaz Fraction

Fig.1 shows the distribution of cytochrome oxi- dase activity and protein obtained following density gradient centrifugation of the crude mitochondrial

Purification

Eur. J. Biochem. 40 (1973)

G. Turner

0.3 1 300 1 '

203

- . m

E - -

u"

' 0 4 0 12 16 20 24

Fraction no. Fig. 1. Centrifugation of mitochondria on sucrose density gradient. Crude mitochondrial fraction (approximately 2 mg protein) was centrifuged for 1 h a t 70000 x gsv. Fractions (1 ml) were collected by piercing the centrifuge tube, and each fraction was assayed for cytochrome oxidase activity ( X-X) protein content (o+) and density (A----A)

I I I

10 100 1000 Cyclohexirnide (Kg /rnl)

Fig.2. Effect of cycloheximide on the incorporation of (14C]- lewine by whole mycelium. The mycelium was preincubated with cycloherimide for 5 min before addition of the tracer (0.2 pCi/ml)

fraction. It can be seen that most of the cytochrome oxidase activity banded a t a median density of 1.19 glml. The mitochondria appeared to be isopycnic after 1 h, since centrifugation for longer time periods failed to alter this pattern. A pellet, formed a t the bottom of the tube, contained little cytochrome oxi- dase activity, and appeared to consist of pieces of hyphae and hyphal walls.

Effect of Cycloheximide on Protein Synthesis in Whole Mycelium

Fig. 2 shows the effect of increasing concentrations of cycloheximide on the uptake of [14C]leucine by whole mycelium. Although more than 90 inhibition occurred in the presence of 10 pg/ml cycloheximide, concentrations of up to 1 mg/ml did not achieve

600 -

- c ._ E . c -400-

8 r 3 - > > - ._ ._ 2 200 - 0 -0 ._

B

I

20 40 60 0

0 Time (min)

Fig. 3. Uptake of /14C]leucine into non-mitochondria1 proteins in the presence of 100 ,ug/ml cycloheximide. Mycelium (8 g wet weight) was resuspended in 80 ml growth medium, and after preincubation with 100 pg/ml cycloheximide for 5 min, 0.2 pCi/ml [14C]leucine was added to the culture. At suitable time intervals, 10-ml samples were withdrawn, and non-mito- chondrial fraction was prepared. Aliquots of protein (1.4mg) from each sample were counted

complete inhibition of uptake, and in all subsequent experiments, a concentration of 100 pg/ml was used. Previous workers [17,18] have demonstrated that at least part of the cycloheximide-insensitive synthesis reflects mitochondrial protein synthesis. However, it is also known that cycloheximide does not always entirely block cytoplasmic protein synthesis [19]. To test this possibility, incorporation of [14C]leucine into "cell sap" proteins was measured in the presence of cycloheximide (Fig. 3). Prolonged preincubation with the drug did not abolish this uptake. It is clear that the drug does not entirely prevent incorporation of amino acids into cytoplasmic proteins, and a double-labelling technique wag therefore adopted in order to ascertain whether mitochondrial proteins were preferentially labelled in the presence of cyclo- heximide.

Em. J. Biochem. 40 (1973)

204 Mitochondrial-Membrane Components in Aspergillus

Table 1. Uouble labelling of mitochondrial and non-mito- chondrial proteins in the absence and presence of cycloheximide Whole mycelium was incubated for 30 min in the presence of [*H]leucine (1 pCi/ml) then washed and resuspended in medium containing cycloheximide (100 pg/ml). After 5-min preincubation the mycelium was incubated for a further 60 rnin with [14C]leucine (0.2 pCi/ml). Mitochondrial and cell sap fractions were isolated, and samples equivalent to 1.4 mg protein were counted for radioactivity

Table 2. Distribution of label between soluble and insoluble mitochondrial proteins following labelling of whole mycelium in the presence of cycloheximide. Whole mycelium was incubated for 60 min in the presence of cycloheximide and [14C]leucine (0.2 pCi/ml) following 5-min preincubation in the presence of the drug. Mitochondria were isolated, and fractionated into water-soluble and water- insoluble portions, which were then assayed for protein and radioactivitv

Fraction 3H 14c l4CI3H

counts/min

Mitochondria 553 1293 2.34 Cell sap 1311 474 0.36

Emetine, a drug which has been used instead of cycloheximide in mammalian systems [ll, 191, was found to be ineffective in Aspergillus nidulans.

Effect of Cycloheximide on Incorporation in vivo of [3H]Leucine and [14C]Leucine into Mitochondrial and Non-Mitochondria1 Proteins

Whole mycelium (5 g wet weight, equivalent to 0.5g dry weight, in 50 ml growth medium) was incubated a t 37 "C in the presence of 1 pCi/ml [3H]leucine for 30 min, and washed with growth medium to remove excess tracer. Following resuspen- sion of the mycelium in 50 ml fresh medium, cyclo- heximide was added to a concentration of 100 pg/ml, and the mycelium was incubated for 5 rnin prior to addition of [14C]leucine (0.2 pCi/ml). The culture was incubated for a further 60 min and the mycelium was washed with growth medium. Mitochondrial and cell sap protein fractions were prepared as described previously, and aliquots of protein were counted for sH and I4C. The results are shown in Table 1. Although labelling of nun-mitochondria1 proteins continued in the presence of cycloheximide, i t can be seen that preferential labelling of mitochondrial protein oc- curred.

Distribution of Label between Water-Soluble and Water- Insoluble Proteins of the Mitochondrial Fraction

Previous studies have suggested that water- soluble mitochondrial proteins are not significantly labelled as a result of mitochondrial protein synthesis [11,20]. The purpose of this work was to examine the membrane proteins of the mitochondria and, prior to electrophoresis, the mitochondrial fraction was homogenized in phosphate buffer, followed by re- centrifugation, to remove soluble proteins. Although it is likely that such a procedure would not remove all the soluble protein following bursting of the mito- chondria, techniques such as sonication and freezing and thawing of mitochondrial preparations were avoided in order to obtain consistent patterns on

Specific protein radioactivity activity

x min-1 mg

x mg protein-'

Fraction Total Total

counts/min counts

Insoluble 3.0 1790 597 Soluble 0.6 68 113

y -Globulin I H chain

\

Myoglobin

4.0 I I I I

0 0.2 0.4 0.6 0.8 1.0 Relative mobil ity

Fig.4. Calibration of the loo / , plyacrylamide gel using protein standards. 20 p1 (8 pg) of each protein was applied to separate gels, and following electrophoresis the mobility of each protein was measured relative to the leading edge of the marker dye

electrophoresis. Table 2 shows the results obtained by the washing procedure following labelling of mito- chondrial proteins in vivo for 60 rnin by [14C]leucine in the presence of cycloheximide. Although some radio activity is lost by the washing procedure, the spe- cific activity of the soluble proteins is considerably lower than that of the insoluble proteins.

Gel Electrophoresis of Mitochondrial Membrane Proteins following Double Labelling in vivo

Approximately 1 g wet weight of mycelium was incubated in 10 ml of growth medium. Double label- ling was carried out as described, except that 1 mCi

Em. J. Biochem. 40 (1973)

G. Turner 205

O------- 0.6 -

.

500

c .- E . u)

000 f B - %

> " m

c ._ ._ c

j00 2 &!

1

Fraction no.

Fig.5. Electrophoresis of labelled mitochondrial-membrane proteins. Mycelium was labelled with r3H]leucine for 30 min prior to the addition of cycloheximide, followed by 60-min labelling with [14C]leucine in the presence of cycloheximide (100 pg/ml). (----) Ratio of 14C/3H

[3H]leucine and 50 pCi [Wlleucine were used in each experiment. Mitochondria1 membrane proteins were prepared as described and subjected to electro- phoresis. Following electrophoresis, duplicate gels were either stained for protein, or sectioned and counted for 14C and 3H. The calibration of the gels for molecular weight is shown in Fig.4, and electro- phoresis of labelled mitochondrial proteins in Fig. 5. To ensure that no artifacts were caused by the order of the double labelling, labelling was carried out in the reverse manner, i.e. [14C]leucine without cyclo- heximide, [3H]leucine in the presence of cyclohexi- mide. The pattern of labelling remained the same (Fig. 6).

Since non-mitochondria1 proteins are labelled in the presence of cycloheximide (Fig. 3), mitochondrial- ly synthesized proteins are most clearly distinguished by examination of the l4Cl3H ratio (or vice versa), and this ratio has been plotted on the traces.

At least four components can be distinguished, with molecular weights of 13000, 18000, 27000 and

Molecular weight 10 20 30 40 50

1500

Fraction no.

Fig. 6. Electrophoresis of labelled mitochondrial-membrane proteins. Mycelium was labelled with [14C]leucine for 30 min prior to the addition of cycloheximide, followed by 60-min labelling with [3H]leucine in the presence of cycloheximide (100 pg/ml). (----) Ratio of 3H/14C

40000. I n addition, "high-ratio" material can be seen at the top end of the gels, representing high- molecular-weight material, though this has not been clearly resolved. It is possible that this consists of aggregates of the other components. Three of the components reproducibly coincide with certain dye- stained bands, though it is possible that these are masking the bands responsible for the labelling observed. None of the major components of the mitochondrial membrane proteins appear to be of mitochondrial origin, as far as their synthesis is concerned.

DISCUSSION

Before examining mitochondrial components, it is essential to obtain as pure a fraction as possible. Density gradient centrifugation of crude mitochon- drial fractions of Aspergillus nidulans resulted in the formation of a band containing most of the cyto- chrome oxidase of the crude fraction, and electro-

Em. J. Biochem. 40 (1973)

206 G. Turner: Mitochondrial-Membrane Components in Aspergillus

phoresis of membrane proteins derived from this preparation gave reproducible results for the protein bands and labelling pattern.

Electrophoresis of mitochondrial proteins has been carried out by a variety of methods in the past [ll, 20,211 though, in the experience of the author, methods involving phenol/urea/acetic acid and phe- nol/formic acid often result in incomplete dissolution of the relatively insoluble mitochondrial membrane proteins, leaving much material a t the top of the gels following electrophoresis.

The use of a double-labelling technique, which has also been used by other workers [4], is essential when i t is clear that the inhibitor used does not give 1000/, inhibition of cytoplasmic protein synthesis. Using a single-labelling technique, major cytoplasmi- cally-synthesized protein components could give small peaks of radioactivity as a result of the leak past the site of inhibition.

The variety of methods used makes comparisons difficult between results obtained by different workers with different organisms. Electrophoresis of membrane proteins on dodecylsulphate-polyacryl- amide gels has been carried out by several workers with Neurospora crassa [4], Saccharomyces cerevisiae [12] and rat liver [ll]. It appeared that both Neuro- spora and Saccharomyces coded for approximately four or five components over the molecular weight range of 12000-50000. Although the molecular weights of the components of these organisms do not exactly coincide, i t is interesting to note that similar labelling patterns are obtained with these two organisms as with Aspergillus in this present work. However, Coote and Work [ll] studied mitochondrial components of mammalian origin, following labelling in vitro and in vivo, and showed the presence of a t least 10 components over a similar molecular weight range. It seems unlikely that mammalian mito- chondria (molecular weight DNA = lox 106 [22]) would be able to code for more mitochondrial proteins than yeast (molecular weight DNA = 50 x lo6 [23]) or Neurospora (molecular weight DNA = 40x 106 [24]). It is, of course, possible that some of the pro- teins synthesized on the mitochondrial ribosome might be encoded by nuclear DNA, though this has not yet been demonstrated. It is also possible that there may be minor products of mitochondrial pro- tein synthesis which remain undetected by the meth- ods used with these microorganisms.

It is more difficult to determine the function of mitochondrially synthesized proteins, since the harsh treatments required for dissolution of membrane proteins prior to electrophoresis are not favourable for studying physiological behaviour of the components thus separated. Another approach to this problem

is to examine the membrane proteins of mitochondrial mutants, and to relate the results to the biochemical lesions of such mutants. Extrachromosomally in- herited, oligomycin-resistant [9,10] and cold-sensitive mutants [25] have now been isolated in Aspergitlus nidulans, and a combination of biochemical and genetical studies on these and other mutants may well improve our understanding of the function of the few mitochondrially Synthesized proteins which seem to be so essential for the proper functioning of the mitochondrion.

I wish to thank Miss S. Maliphant for her technical assistance, and Dr M. J. Tanner for his advice on the electro- phoresis. This investigation was supported by a Science Re- search Council grant.

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G. Turner, Department of Bacteriology, The Medical School, University Walk, Bristol, Great Britain BS8 1TD

Eur. J. Biochem. 40 (1973)