Total adult blowfly flight-muscle mitochondrial protein was labelled in vivo with [14C]leucine. The labelled proteins were enumerated and characterized by their electrophoretic mobility using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autoradiography.

When different gel concentrations were used to resolve the maximum number of bands and their molecular weights, it was possible to observe at least 35 electrophoretic bands after staining and scanning; their molecular weights ranged between 11000 and 130000.

When mitochondria were labelled in the presence of cycloheximide, only 6–8 bands could be identified on gradient gels after electrophoresis and autoradiography. By comparison, controls (where cycloheximide was absent), which were run alongside the drug-treated mitochondria, revealed 17–20 radioactively labelled bands from densitométrie tracings. Whilst the molecular weights of these bands could be estimated, it was difficult to identify the precise nature and function of the proteins made in these mitochondria.

Mitochondria have been firmly established as organelles capable of autonomous synthesis of certain macromolecules including DNA, RNA and protein (Ashwell & Work, 1970). The mitochondrial nature of this synthesis has been characterized by experiments with whole cells in vivo, utilizing drugs to separate cytoplasmic from mitochondrial protein synthesis, since both nuclear and mitochondrial gene products are required for formation of functional mitochondria (Borst, 1972; Schatz & Mason, 1974).

At present it is possible to account for about 5–10% of the total mitochondrial protein as the contribution from the intrinsic mitochondrial protein-synthesizing system (Beattie, 1971; Ashour, Tribe & Whittaker, 1980a).

For lower eukaryotes, it has been demonstrated convincingly that the polypeptide gene products of mitochondrial DNA account for 3 subunits of cytochrome c oxidase, 1 subunit of cytochrome b, and protein necessary for the assembly of cytochrome c. Until recently, it was thought that mitochondrial DNA contributed 4 subunits to the ATPase complex, but according to Linnane (1980),2 of these may be artefacts. Furthermore, it seems that one ribosomal protein is translated on mitochondrial ribosomes. For details of mitochondrial gene products in lower eukaryotes see: Mahler, Perlman & Mehrotra, 1971; Sebald, Machleidt & Otto, 1973; Mason, Poyton, Wharton & Schatz, 1973; Tzagoloff, Akai & Rubin, 1974; Ross et al. 1974; Weiss & Ziganke, 1974; Poyton & Schatz, 1975; Cabral et al. 1976.

In the present work on a more advanced eukaryotic organism, we have adopted the approach in vivo of radioactively labelling proteins in the presence of inhibitors of cytoplasmic and mitochondrial protein synthesis, respectively. This approach has been primarily responsible for providing the most comprehensive picture of the inner membrane polypeptides synthesized both inside and outside the mitochondrion (Schatz & Mason, 1974). However, despite its conceptual simplicity, the inhibitor approach has a number of pitfalls that must be avoided. Some of these have already been alluded to in previous papers (Ashour et al. 1980a; Ashour, Tribe, Danks & Whittaker, 1980b), but special mention should be made here of the length of time of exposure to an inhibitor, as it is of critical importance. For example, if cells are exposed to chloramphenicol for a relatively long time, the synthesis of polypeptides on cytosolic ribosomes will eventually decline because they require ‘partner’ proteins from the mitochondrial ribosomal system. The end result will be an apparent blockage of those polypeptides synthesized outside the mitochondria by inhibitors of mitochondrial protein synthesis. The reverse, of course, would be true with long-term incubation using cycloheximide. In this study, therefore, exposure time to inhibitory drugs was never more than 12 h.

Since maximal protein synthesis shown by insect flight-muscle mitochondria takes place in parallel with development of the cristae, during the period just prior to eclosion (Birt, 1970), and during the first 4 days after eclosion (Tribe & Ashhurst, 1972; Tribe & Webb, 1979; Ashour et al. 1980a, b), the experiments reported here were carried out with 1 to 3-day-old flies.

Maintenance and injection of flies

Flies (1 to 3-day-old) in groups of 20–30 were injected with 1 μl water (control) or with a solution of inhibitors (cycloheximide, chloramphenicol and ethidium bromide) as indicated in figures and legends, by the method described before (Ashour et al. 1980a). One hour later, the same flies were each injected with a solution of 0·1 μCi L-[U-14C]leucine of high specific activity (342 mCi mmol−1; Radiochemical Centre, Amersham, Bucks). Flies were maintained in a clean 1 1. beaker before mitochondrial isolation. Ten hours later mitochondria were isolated by the method described by Chappell & Hansford (1972), using the bacterial proteinase, nagarse. The mitochondrial pellet was purified further by centrifugation in a sucrose gradient (0·9 M to 1·2 M-sucrose containing 5 mM-EDTA and 0·02 M-Tris at pH 7·4) for 1–2 h at 23000 rev./min in the Spinco SW 25-1 rotor. The orange-brown mitochondrial layer was visible as a band about half-way down the tube. The mitochondrial layer was recovered directly from the gradient by gentle aspiration using a Pasteur pipette. The mitochondrial fraction was diluted 4-fold with 0·25 M-sucrose and collected by centrifugation at 10000 g for 10 min. Mitochondrial protein was determined by the method of Lowry, Roseborough, Farr & Randall (1951); precautions being taken to add 0· 025% bromophenol blue to the standard protein solutions.

SDS/polyacrylamide slab gel electrophoresis of mitochondrial proteins

Electrophoretic resolution of mitochondrial protein subunits was based on the methods described by Studier (1973) using the discontinuous buffer system of Laemmli (1970), with slight modifications.

The final purified mitochondrial pellet was resuspended in a very small volume of sample buffer (0·05 M-Tris-HCl, pH 6·8, containing 2% sodium dodecylsulphate (SDS), 5% 2-mercaptoethanol, 0·5% glycerol, 0·1% bromophenol blue and 0·02 M-EDTA), to give a final concentration of 10–20 mg/ml mitochondrial protein.

For the determination of molecular weights, the running gel (7·5% or 12·5%) was poured and left for 1 h in the electrophoretic apparatus before adding the 5% acrylamide stacking gel. To obtain better band resolution 5% to 15% exponential gradients were used with no upper layer of stacking gel. These gradient gels were prepared by using a peristaltic pump.

After the gel solution had set (1 h), the comb and the bottom spacer were removed. Then the sandwiched gel was clamped to the electrophoresis apparatus and sealed at the upper chamber with 2% agar. The upper and lower buffer chambers were then filled with the running buffer solution (0·05 M-Tris-HCl, pH 8·3 containing 14·41 g glycine, 1 g SDS and 2·4 g sodium thioglycollate per 1). The mitochondrial protein samples or protein markers were loaded with a Hamilton micro syringe (loading was between 10 and 50 μl/well). In experiments to determine the molecular weights, the marker proteins were run in the same gel with the sample proteins. Samples were heated to 100 °C for 3 min prior to electrophoresis.

The gel was electrophoresed at constant amperage of 30 mA, until the blue tracing dye reached the bottom of the slab (about 3 h). After electrophoresis, the slab gel was removed from the glass sandwich and transferred to a tray for staining. The gels were fixed for 30 min in 50% TCA (trichloroacetic acid) followed by 20 min in staining solution (0·1% Coomassie brilliant blue in 40% TCA). The gels were destained overnight in a 7% acetic acid solution.

Autoradiography of gel slabs

For autoradiography, the slabs were dried first in a steam bath under vacuum. Dried gels (cooled to room temperature) were placed facing a Kodak X-ray 13×18 cm negative film and left for 3 weeks to display the profile of labelled protein on the gel. The exposures were developed with liquid X-ray developer for 5 min. The autoradiograms were scanned with a doublebeam recording microdensitometer (Mk HIC, Joyce Loebl & Co.).

Molecular weight determination

The following reference proteins were used as molecular weight markers: bovine serum albumin (68000), pepsin (34700), trypsin inhibitor (21 500), lysozyme (14500) and cytochrome c (12400). The molecular weight values were taken from Dunker & Rückert (1969).

Subsequent to the electrophoretic and staining procedures, the relative mobilities of the protein bands in relation to the dye marker were calculated. The migration paths of the single bands of the calibration proteins and the sample were measured starting from the beginning of the gel. Then the molecular weights of the calibration proteins were plotted against the measured migration paths (relative mobility) on semilogarithmic paper and a straight calibration line was obtained. By knowing the migration path of the sample, the molecular weights of the polypeptides were read directly from the calibration line.

Electrophoretic patterns of the total mitochondrial protein subunits

When two concentrations (7·5 and 12·5%) of sodium dodecyl sulphate/polyacryla-mide slab gel are used, the proteins extracted from the flight-muscle mitochondria can be separated by electrophoresis into at least 35 well-defined bands with different intensities and widths, as shown in Fig. 1 A, B and Fig. 2. The 35 bands and their molecular weights were obtained from the 2 sets of gel concentrations stated above and not from any single gel concentration. However, it is possible to find some bands with the same molecular weight on both the gels used.

Fig. 1.

Coomassie brilliant blue-stained mitochondrial proteins separated by SDS/polyacrylamide slab gel electrophoresis; A, 7·5% gel concn; B, 12·5% gel concn; c, 5% to 15% gradient gel concn.

Fig. 1.

Coomassie brilliant blue-stained mitochondrial proteins separated by SDS/polyacrylamide slab gel electrophoresis; A, 7·5% gel concn; B, 12·5% gel concn; c, 5% to 15% gradient gel concn.

Fig. 2.

Densitometrie tracings of total flight-muscle mitochondrial proteins of newly emerged flies (1–3 days old) after electrophoresis in 7·5% (A) and 12·5% (B) SDS/polyacrylamide slab gels.

Fig. 2.

Densitometrie tracings of total flight-muscle mitochondrial proteins of newly emerged flies (1–3 days old) after electrophoresis in 7·5% (A) and 12·5% (B) SDS/polyacrylamide slab gels.

Fig. 2A, B shows typical densitogramms of the electrophoretic pattern, representing the unlabelled mitochondrial proteins after staining the gels with Coomassie brilliant blue. Provided that the experiments are repeated under exactly the same conditions, the molecular weights obtained from the gels are highly reproducible. Table 1 shows the molecular weights of the bands obtained from densitométrie scanning of the 2 sets of gel concentrations (7·5 and 12·5%) as calculated from Fig. 2. The molecular weights for these mitochondrial polypeptides range from about 11 500 to 130000. It should be noted that during a period of 10 h after labelling the mitochondrial protein profiles obtained by electrophoresis were stable.

Table 1.

The molecular weights of subunit polypeptide bands obtained from two sets of gel concentrations

The molecular weights of subunit polypeptide bands obtained from two sets of gel concentrations
The molecular weights of subunit polypeptide bands obtained from two sets of gel concentrations

Electrophoretic patterns of in vivo labelling of mitochondrial proteins under the influence of protein synthesis inhibitors analysed by autoradiography

Autoradiographic examples of the results obtained from several labelling experiments of control (untreated) and drug-treated flies with inhibitors of protein synthesis are shown in Tig. 3. In all these experiments only 5% to 15% gradient SDS/polyacrylamide gels were used. As regards the control, the autoradiogram of the flight-muscle mitochondrial proteins shows approximately 20 well-defined bands with molecular weights ranging between 11000 and 100000. This can also be seen from the Coomassie blue-stained gradient for untreated flies in Fig. 1 c. Some of these bands arc much ‘darker’ (more intense radioactivity) and thicker than the other bands, and will be referred to subsequently as the dominant bands. Fig. 4 A illustrates diagrammatically the dominant bands in the control ; they are numbered 1, 2, 5, 6, 7, 8 and 11. Three ‘bands’ (9, 10 and 12), with molecular weights of about 50000-35000 are relatively diffuse. Such diffuse bands have been reported in other mitochondrial systems (Yatscoff & Freeman, 1977; Young & Hunter, 1979). From the absorbance scanning profile of the original X-ray film (Fig. 5 A), however, it can be seen that each of these diffuse bands is in fact made up of 2 close but discrete bands (a and b). The densitometry scanning in Fig. 5 also serves to emphasize the variable level of the different bands, i.e. it illustrates the different labelling intensities and band-widths more clearly than the original auto-radiogram. The pattern is very reproducible and therefore the bands have been designated by number.

Fig. 3.

Autoradiograph of the 5% to 15% gradient slab gel electrophoresis of mitochondrial proteins. Flies 3-day-old were injected with inhibitors; cycloheximide, 5 μg/fly (B) ; ethidium bromide, 10 μg/fly (c); or chloramphenicol, 250 μg/fly (D); 1 h before labelling with [14C]leucine. Control flies (A) were injected with water 1 h before label injection.

Fig. 3.

Autoradiograph of the 5% to 15% gradient slab gel electrophoresis of mitochondrial proteins. Flies 3-day-old were injected with inhibitors; cycloheximide, 5 μg/fly (B) ; ethidium bromide, 10 μg/fly (c); or chloramphenicol, 250 μg/fly (D); 1 h before labelling with [14C]leucine. Control flies (A) were injected with water 1 h before label injection.

Fig. 4.

A diagrammatic illustration of the autoradiograph presented in Fig. 3, showing mitochondrial protein subunits from control flies (A), or from flies treated with 5 μg cycloheximide (CH) (B), 10 μg ethidium bromide (EthBr) (c), and 250 μg chloramphenicol (CAP) (D).

Fig. 4.

A diagrammatic illustration of the autoradiograph presented in Fig. 3, showing mitochondrial protein subunits from control flies (A), or from flies treated with 5 μg cycloheximide (CH) (B), 10 μg ethidium bromide (EthBr) (c), and 250 μg chloramphenicol (CAP) (D).

Fig. 5.

Densitométrie scanning profile of the original X-ray film presented in Fig. 3, showing the mitochondrial protein subunits of untreated flies (A), or flies treated with 250 μg chloramphenicol/fly (D), 10 μg ethidium bromide/fly (c), or 5 μg cycloheximide /fly (B).

Fig. 5.

Densitométrie scanning profile of the original X-ray film presented in Fig. 3, showing the mitochondrial protein subunits of untreated flies (A), or flies treated with 250 μg chloramphenicol/fly (D), 10 μg ethidium bromide/fly (c), or 5 μg cycloheximide /fly (B).

When the electrophoretic pattern (Figs. 3B and 4B) and the densitométrie scanning of the exposed X-ray film (Fig. 5B) of flies treated with 5μg cycloheximide are examined, a drastic reduction is seen in both the number of bands and in the size of the densitométrie peaks. Only 6–8 bands can be identified from the above figures. A comparison of the mitochondrially synthesized proteins with total mitochondrial proteins (control experiments, slot A) shows that they represent quantitatively only minor components of this organelle. Furthermore, the proteins synthesized by mitochondria (i.e. cycloheximide-insensitive proteins), fall into a group with lower molecular weights (55000 or less) than most of the proteins synthesized on cytosolic ribosomes.

The sensitivity of the labelling of the mitochondrial proteins to chloramphenicol was also examined. Figs. 3D, 4D and 5D show the mitochondrial polypeptide patterns of flies after injection with 250 μg chloramphenicol. Of the many polypeptides resolved in the absence of antibiotics, only a relatively small number (if any) are decreased or eliminated by labelling in the presence of chloramphenicol, since mitochondrially synthesized proteins only constitute about 5–10% of total mitochondrial proteins (see Ashour et al. 1980a).

In the last group of experiments (Figs. 3c, 4c and 5c) the effect of the inhibitor, ethidium bromide, on the mitochondrial protein profile can be seen. Surprisingly, 10 μg ethidium bromide per fly showed a more drastic effect on the total mitochondrial protein than chloramphenicol. After treatment with ethidium bromide, bands 3, 12 b, 13 and 14 can no longer be identified, while bands 5, 6 and to some extent 7 show decreased peak sizes (Fig. 5 c). This might be due to side effects of ethidium bromide on some other cellular function (nuclear transcription and cytosolic protein synthesis), rather than a direct effect on mitochondria (see Ashour et al. 1980a, b).

Studies in vitro with mitochondria isolated from insect flight muscle show that they incorporate amino acids only into the water-insoluble fraction of the membrane proteins (Bronsert & Neupert, 1966; Williams, 1972). This finding is also supported by Roodyn and his co-workers in their investigation in vitro of mammalian mitochondria (Roodyn & Wilkie, 1968). With the advent of methods for the electrophoretic separation of subunits from mitochondrial proteins, Sebald, Bûcher, Olbrick & Kaudewitz (1968) examined the insoluble mitochondrially synthesized protein of yeast and reported that 20 bands could be observed after using phenol medium. They also found that proteins synthesized in vitro were identical to those produced in vivo. After electrophoretic separation of insoluble mitochondrial proteins labelled in vitro, bands can be distinguished by autoradiographic technique coincident with the protein bands formed by insoluble mitochondrial protein synthesized in vivo. Later, when mitochondria from other organisms were investigated by Sebald and his colleagues using the same technique, it was found that the band pattern obtained was almost identical to that found for the mitochondrial membrane proteins of yeast; notably, rat liver mitochondria (Neupert, Brdizka & Sebald, 1968) and locust flight-muscle mitochondria (Kleinow, Sebland, Neupert & Bûcher, 1970). Kleinbw and his colleagues, however, found that a few of these bands were labelled by incorporation of amino acids in vivo. They therefore concluded that the intrinsic system is restricted not only to the insoluble fraction of mitochondrial proteins, but to a quantitatively minor part of this fraction.

The more advanced technique introduced later, of using in vivo labelling with amino acids of very high specific activity in the presence of cycloheximide, has given a much clearer view of the products of mitochondrial protein synthesis in many different organisms. This method, which has been used in our study, has made it possible to visualize at least 6–8 polypeptide species that fulfil the criteria of mitochondrial translation products, as well as the overall profiles of mitochondrial polypeptides labelled in the absence of antibiotics. The number of bands obtained here after labelling in vivo of blowfly flight-muscle mitochondria in the presence of cycloheximide, however, is far less than the number obtained with some of the other systems investigated. In yeast, for example, Douglas & Butow (1976) have reported that at least 20 bands are present. Nevertheless the majority of the 20 bands found in yeast were extremely minor components relative to the dominant 8 or 9 bands. Labelling in vitro of intact plant mitochondria has also demonstrated up to 20 components (Forde, Oliver & Leaver, 1978). It is not known, however, to what extent this system faithfully reproduces in vivo conditions.

From the results reported in this work, it seems that the mitochondrial translation products of blowfly flight muscle represent one of the smallest translation systems known. But even though it appears that the number of distinct polypeptides synthesized within mitochondria is generally low, there is no agreement about the precise number. For example, Costantino & Attardi (1975), using sodium dodecyl sulphate disc gel electrophoresis, gave evidence that 10 proteins are synthesized in the mitochondria of HeLa cells. Jeffreys & Craig (1976a), working on the same type of cell but using sodium dodecyl sulphate slab gel electrophoresis and autoradiography, detected 8 predominant bands, one of which was thought to consist of 2 components.

The products of flight-muscle mitochondrial protein synthesis demonstrated in this study are, however, generally similar in ‘size’ to those reported in several other systems ranging from mammalian cell lines to yeast and other fungi (Schatz & Mason, 1974). The band (number 6 in Fig. 4) with an estimated molecular weight of 55000 is somewhat larger than is commonly found, although components of almost similar size have been reported ; for example, in rat liver (58 000) (Burke & Beattie, 1974), in Tetrahymena (60000) (Young & Hunter, 1979); and in the fungus Botryodiplodia theobromae, where a much larger one is found (85000) (Bramble & Handschin, 1976). In the latter case, however, no evidence was presented that the bands were actually sensitive to chloramphenicol. The significance of some of the apparent differences in molecular weight and the size of some of the individual proteins are difficult to assess. Some mitochondrial translation products are known to run anomalously under some gel conditions and therefore these differences may be apparent rather than real (Yatscoff & Freeman, 1977; Groot, Van Harten-Loosbrook & Kreike, 1978).

Yatscoff & Freeman (1977) and Yatscoff, Aujume & Freeman (1978) have characterized the mitochondrial proteins, labelled in vivo in the presence of cycloheximide with [35S]methionine or pH]leucine for 1–2 h, in whole Chinese hamster ovary cells and other mammalian cell lines. Ten to 13 distinct radioactive bands separated by electrophoresis were observed, and these were stable (i.e. always found in the same position on the gel) during a 2 h post-labelling period.

In agreement with the results of others (Lederman & Attardi, 1973; Jeffreys & Craig, 1975, 1976b), the components obtained from blowfly flight-muscle mitochondria do not apparently turn over rapidly and there is no change in the number of bands with different times of labelling, at least not within a period of 10 h. However, contrasting observations have been reported from experiments with fungi and with mammalian cells, suggesting that cytosolic proteins are required for mitochondrial protein synthesis (Tzagoloff, Rubin & Sierra, 1973; Schatz & Mason, 1974; Wallace, Williams & Freeman, 1975; Poyton & Kavanagh, 1976).

Previously, only the molecular weight ranges or apparent molecular weights of the mitochondrial polypeptide bands have been determined because of poor resolution (Coote & Work, 1971 ; Costantino & Attardi, 1975; Jeffreys & Craig, 1976a). By the more refined in vivo approach used in this study molecular weight determination was more accurate. However, the precise nature and function of the proteins synthesized in blowfly flight-muscle mitochondria is still not known. Components of the complexes of cytochrome c oxidase (Mason & Schatz, 1973; Rubin & Tzagoloff, 1973; Sebald, Machleidt & Otto, 1973, 1974), cytochrome b (Weiss, 1976) and ATPase (Tzagoloff & Meager, 1972) are synthesized in the mitochondria of yeast and Neurospora. Further, the synthesis of cytochrome c oxidase, cytochrome b, and an uncoupler binding protein in mammalian cells is inhibited by chloramphenicol (Kroon & Janson, 1968; Firkin & Linnane, 1969; Fettes, Halder & Freeman, 1972; Schatz & Mason, 1974; Fisher, 1976). Subunits of mammalian cytochrome c oxidase (Downer, Robinson & Capaldi, 1976; Bûcher & Penniall, 1976; Phan & Mahler, 1976), cytochrome b (Ohnishi, 1966) and the uncoupler binding protein (Hatefi, Hanstein, Galante & Stiggall, 1975) have molecular weights, to some extent, in the range reported here and could correspond to some of the observed bands.

Obviously the question of aggregation must be considered, particularly where wide and diffuse bands are obtained. These bands were frequently observed in samples that were solubilized in 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol and brought to 100 °C for 3 min. Such conditions should have prevented aggregation, so that by this criterion at least, these bands appear to represent proteins in a disaggregated state.

In this discussion, the importance of cycloheximide in elucidating the polypeptide subunits synthesized within mitochondria has been emphasized because it is agreed that this is a fundamental approach to this field of study. As far as the effects of ethidium bromide and chloramphenicol are concerned, considerable discussion has already been published (Ashour et al. 1980a, b) and will not be reported again here. Whilst it is clear that both drugs, especially ethidium bromide, have a direct effect on some of the electrophoretic bands by apparently complementing the action of cycloheximide, they do not permit any further identification of the polypeptide subunits involved or their site of synthesis.

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