A Study of Mitochondrial Protein Synthesis in Intact HeLa Cells

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1 Eur. J. Biochem. 22 (1971) A Study of Mitochondrial Protein Synthesis in Intact HeLa Cells Agnese BREGA and Corrado BAGLIONI Department of Biology, Massachusetts Institute of Technology, Cambridge (Received March 29/July 16,1971) Mitochondrial protein synthesis has been studied in intact HeLa cells by utilizing specific inhibitors. We have observed that in the presence of pederine, an inhibitor of cytoplasmic protein synthesis in mammalian cells, protein synthesis continues for at least 1 h at approximately 0.501, of the rate of untreated cells. When control cells and cells treated with pederine are fractionated after an incubation with labeled amino acids into a mitochondrial and a supernatant fraction, it is found that incorporation into the mitochondrial fraction is higher in the presence of the inhibitor. Mitochondrial protein synthesis has been shown to take place on aggregates of mitochondrial ribosomes. The synthesis of mitochondrial proteins has been studied by incubating HeLa cells with labeled amino acids and either pederine or chloramphenicol, an inhibitor of mitochondrial protein synthesis. We have been able to establish that the structural protein of mitochondrial ribosomes is synthesized on cytoplasmic ribosomes and its synthesis is thus inhibited by pederine, but not by chloramphenicol. Mitochondrial proteins have been analyzed by acrylamide gel electrophoresis after fractionation of the mitochondria into inner and outer membranes. Proteins synthesized by mitochondria in the presence of pederine are located predominantly on the inner mitochondrial membranes. Only few protein species synthesized by mitochondria are resolved by gel electrophoresis. This finding is discussed in relationship to the information content of mitochondrial DNA. It has been clearly established that mitochondria of mammalian cells have the ability to synthesize protein (see [l] for a review). Ribosomes have been isolated from mitochondria that differ from cytoplasmic ribosomes [2-51; these 55s mitochondrial ribosomes form polyribosomes that are presumably the site of mitochondrial protein synthesis [3]. The proteins synthesized by mitochondria have however not yet been unequivocally identified [l]. Several studies suggest that only some structural proteins of the inner mitochondrial membrane are synthesized by mitochondria whereas proteins of the outer mitochondrial membrane and soluble proteins of the matrix and inner mitochondrial membrane are not [ In these studies mitochondrial proteins have been labeled by incubating isolated mitochondria, with radioactive amino acids. It is not clear whether protein synthesis carried out by isolated mitochondria reflects with fidelity that carried out in intact cells. Moreover, the mitochondrial structural proteins are difficult to characterize because of their insolubility and of their tendency to aggregate. We have studied mitochondrial protein synthesis in intact HeLa cells. The inhibitor of cytoplasmic protein synthesis pederine [ll-131 has been used to suppress protein synthesis carried out by cytoplasmic ribosomes ; in complementary experiments the inhibitor of mitochondrial protein synthesis chloram- phenicol has been used. The results obtained indicate that very few proteins are synthesized by mitochondria of mammalian cells. MATERIALS AND METHODS HeLa cells were cultured in Eagle's medium [14] in spinner bottles at a concentration between 1 and 5 x lo6 cells/ml. Before incubation the cells were concentrated and resuspended in the same medium at the cell density indicated for each experiment. Pederine was a kind gift of Prof. Mario Pavan (Istituto di Entomologia Agraria, Universita' di Pavia, Italy). Cell Fractionation for the Analysis of Mitochondrial Ribosomes At the end of an incubation the cells were centrifuged 5 min at 800 x g and resuspended in 0.5 to 1 ml of 0.01 M NaCl, 0.01 M Tris-HC1 ph 7.4, 1.5 mm MgCl,. After standing for 10 min at 0 "C the cells were homogenized by 15 strokes of a Dounce homogenizer. An equal volume of a solution containing 0.02M Tris-HC1 ph 7.4,0.02 M MgCl,, 0.1 M NaCl and 0.5 M sucrose was added to the homogenate when mitochondrial polyribosomes were analyzed. It has been shown previously [3] that under these conditions mitochondrial polyribosomes are stable. The nuclei 28.

2 416 Mitochondria1 Protein Synthesis Eur. J. Biochem. were then centrifuged 5 min at 1000 x g and discarded. The supernatant was centrifuged 10 min at I0000 xg; the pellet obtained was resuspended in 0.45ml of Tris-Mg-saline (0.01 M Tris-HC1 ph 7.4,O.Ol M MgCl, and 0.05 M NaC1) with a magnetic stirrer and 0.05 ml of loo/, Brij-58 and sodium deoxycholate added. The sample was then layered on /, (wlv) sucrose gradients in Tris-Mg-saline and centrifuged 15 h at rev./min at 4 "C. in a Spinco SW 27 rotor. The fractions collected were precipitated with 5O/, trichloroacetic acid and counted in a liquid scintillation counter. In the experiments on the synthesis of the structural protein of mitochondrial ribosomes, ethylenediaminetetracetic acid (EDTA) was added to the postnuclear supernatant to 30 mm concentration. The samples were then layered on step gradients made with 7.5 ml of 15O/, and 7.5 ml of 30 /, sucrose and centrifuged 40 rnin at rev./min. The pellets obtained were resuspended as indicated above, applied to /, sucrose gradients and centrifuged 20 h at rev./min. Isolation and Fractionation of Mitochondria The method devised by Parsons et al. [15] has been followed with minor modifications. The centrifuged cells (4 x lo7) were resuspended in 1 ml of 0.07 M sucrose, 0.21 M D(-)mannitol, 0.1 mm Na,-EDTA and 1 mm Tris-HC1 ph 7.2. The cells were then homogenized in a Potter homogenizer with a teflon pestle. The nuclei were spun 10 min at 900 x g and discarded. The supernatant was centrifuged 10 min at 9000 x g ; the pellet obtained was resuspended in 1 ml of the same medium and the low-speed centrifugation repeated. The supernatant was centrifuged 10min at 9750xg. The pellet obtained was resuspended in 2 ml of 20 mm phosphate buffer ph 7.2 containing 0.02O/, bovine serum albumin. After standing for 20 min the swollen mitochondria were centrifuged 20 min at xg and then resuspended in 2 ml of 20mM phosphate buffer. The swollen mitochondrial suspension was centrifuged 15 min at 2000 x g to obtain the inner mitochondrial membranes in the pellet. The outer mitochondrial membranes were obtained from the supernatant by a 20 min centrifugation at x g. The pellets containing mitochondrial membranes were resuspended in 0.1 ml of 20 mm phosphate buffer and 0.01 ml of loo/, SOdium dodecylsulphate added before analyzing the samples by gel electrophoresis. Acrylamide Gel Electrophoresis The procedure devised by Maize1 [l6] was followed. Gels 6 ~ 0.4 cm or 11 ~0.4 cm were used as indicated and 40 V were applied for 6 h. 20 to 50 pl of mitochondrial fractions preparations were applied to each gel after addition of 10 to 20 p1 of 0.1,Ilo bromo- phenol blue in 50 O/, sucrose. The gels were fractionated by using a gel crusher (Savant Instruments). The crushed gels fractions were left overnight in approximately 0.8ml of water; 10ml of a scintillant made up of an equal volume of methylcellosolve and toluene, containing 0.25O/, butyl-pbd (C.I.B.A.) were then added for counting. RESULTS Protein Synthesis in the Presence of Pederine The addition of pederine to a culture of cells causes almost complete inhibition of protein synthesis within a few minutes [12]. During this time cytoplasmic polyribosomes are converted to 80s ribosomes ; this effect of pederine is due to a selective inhibition of initiation of protein synthesis [13]. We have observed that in cells treated with pederine protein synthesis takes place at less than lolo of the rate of controll cells (Fig. 1). In order to show this residual rate of protein synthesis it has been necessary to use ten times more cells/ml and a concentration ten times higher of [3H]leucine than in control cells. Protein synthesis at this reduced rate can be observed after the inhibition by pederine has become established. The rate of protein synthesis is linear for approximately 1 h and declines markedly afterwards. For this reason in all the following experiments we have limited the incubation with pederine to 1 h and 15 min. Time (min ) Fig. 1. Protein synthesis in wntrol HeLa cells and in cells treated with pederine. The cells were transfered to Dulbecco's modified Eagle medium without leucine and incubated 15 min without addition (control) or with 20 ng/ml of pederine. The control had 2 x lo5 cells/ml and 1 pci of [3H]leucine was added after 15 min; 2 x lo6 cells/ml were incubated with pederine and 10pCi/ml of [3H]leucine were added after 15min. Aliquots of 0.2 ml were taken at the times indicated, treated with an equal volume of 1 N NaOH and precipitated with trichloroacetic acid for counting (see Methods). Solid line, control cells; broken line, cells incubated with pederine

3 V01.22, No.3,1971 A. BREGA and C. BAGLIONI 417 Table 1. Labeling of the mitochondrial fraction of wntrol HeLa cells incubated with pederine or chlorampheniwl The experimental conditions for the incubation are those described under the legends of Figs.4,5 and ml cultures containing 2 x lo6 cells/ml were incubated with 0.1 mci of [3H]leucine for 1 h; pederine (20 ng/ml) was added 15 min before leucine and chloramphenicol 5 min before leucine. The cells were fractionated following the procedure described by Parsons et al. [15]. The 9000xg supernatant is the supernatant obtained after spinning down the mitochondria. The 1st and 2nd wash are obtained by resuspending and resedimentmg the pellet containing mitochondria as described under Methods Fraction Control lo-. x Radioactivity of Pederine Chloramphenicol total countslmin per 4 x 10 cells 9000 x g supernatant st wash supernatant nd wash supernatant Total supernatants and washes Mitochondria1 fraction (15.0 /,) (41.12O/,) (15.56O/,) The distribution of labeled protein between the fraction of the homogenate sedimenting at 9000 x g and the supernatant was then determined. Cultures of HeLa cells were incubated with [3H]leucine and with pederine or chloramphenical. The cells were then homogenized and the postnuclear supernatant fractionated (see Methods). The pellet sedimenting at 9000 x g contains mitochondria, endoplasmic reticulum and other cosedimenting cellular organelles. This pellet (called henceforth mitochondrial fraction ) was washed and sedimented repeatedly to free it from contaminating cytoplasmic proteins, following the procedure of Parsons et al for the isolation of mitochondria. Table 1 shows that a larger percentage of labeled cell protein was associated with the mitochondrial fraction from cells that were incubated with pederine, than from control cells or from cells incubated with chloramphenicol. No significant difference in the labeling of the mitochondrial fraction of cells incubated with chloramphenicol was observed relative to control cells. The labeled protein found in the mitochondrial fraction in the presence of pederine equals approximately 0.5O/,, of the labeled protein found in the same fraction in control cells or in cells incubated in the presence of chloramphenicol (Table 1). If this drug inhibits the synthesis of only those proteins that are synthesized in the presence of pederine, this might explain the absence of any significant difference in the labeling of the mitochondrial fraction between control cells and cells incubated with chloramphenicol. We have determined the site of protein synthesis resistant to pederine inhibition, by analyzing the mitochondrial fraction of cells incubated with pede- I I I d I Fig. 2. The cellular site of pederine-resistant protein synthesis. Two 10 ml cultures containing 2.4 x lo6 HeLa cells/ml were incubated 15 min in Dulbecco s modified Eagle medium with 20nglml of pederine. To one culture 120pg/ml of chloramphenicol were added during the last 5 min. To both cultures were added 50 VCi of [3H]leucine and the incubation continued for 15 min. The cells were collected by centrifugation, washed once with Earle s saline and homogenized as described under Methods. The mitochondrial fraction was isolated by differential centrifugation (see Methods) and resuspended in 0.4 ml of Tris-Mg-saline solution with the aid of small magnetic stirrer; 50 pl of loo/, Brij-deoxycholate solution were added and the samples were applied to /, (w/v) sucrose gradients. These were centrifuged 15 h at rev./min in a Spinco SW-27 rotor. The fractions were collected and counted as described under Methods. Solid line, cells incubated with pederine only; broken line, cells incubated with pederine and chloramphenicol rine and [3H]leucine on sucrose gradients (Fig.2). This analysis showed the presence of labeled peptide chains associated with components sedimenting between 55s and 1805 ; addition of chloramphenicol before [3H]leucine prevented the labeling of these peptide chains (Fig.2). These data confirm that protein synthesis in the presence of pederine occurs predominantly on mitochondrial polyribosomes since mitochondrial protein synthesis only is inhibited by chloramphenicol The structures sedimenting faster than 555 have been previously identified with mitochondrial polyribosomes, on the basis of their association with nascent (puromycin-sensitive) chains [3]. The Site of Synthesis of Proteins of Mitochondria1 Ribosomes We proposed to investigate the products of mitochondrial protein synthesis by analyzing the proteins present in the mitochondrial fraction obtained from cells incubated with pederine and from control cells. Since mitochondrial ribosomes of HeLa cells have recently been described and characterized [3-51 we studied first the synthesis of the ribosomal proteins contained in mitochondrial ribosomes.

4 418 Mitochondrial Protein Synthesis Em. J. Biochem I i, 0.2 I z 0 Lo N P Fig.3. Labeling of mitochondria1 ribosomal proteins in the presence of pederine and chloramphenicol. The illustration shows the results of three separate experiments; in two of these the labeling of mitochondrial ribosomal proteins in the presence of pederine or chloramphenicol was studied. In the third experiment the RNA of mitochondria was specifically labeled to identify mitochondrial ribosomal subunits. Proteins were labeled in two 20 ml cultures containing 2 x lo6 cells/ml; one was incubated 15 min with 20 ng/ml of pederine and then with 10 pci/ml of [3H]leucine for 1 h; the other for 5 min with 120 pg/ml of chloramphenicol and then with 10 pci/ml of [3H]leucine for 1 h. The cells were homogenized and the mitochondrial fraction isolated as described under Methods after adding 30 mm EDTA to the postnuclear supernatant. The mitochondrial fraction was resuspended and treated with detergent as indicated under the legend of Fig.2; the samples were then applied to /, sucrose gradients and centrifuged 20 h at rev./min in a Spinco SW-27 rotor. Mitochondrial RNA was labeled by incubating a 16.5 ml culture containing 2 x los cells/ml first for 25 min with 0.04 pg/ml of actinomycin D and then for 155 min with 3 pci/ml of [3H]uridine. The cells were homogenized and the postnuclear supernatant prepared as described under Methods. The postnuclear supernatant was treated with 30 mm EDTA and applied to a step gradient containing 7.5ml of 3O0/, sucrose and 7.5 ml of 15O/, sucrose in Tris-Mg-saline. The gradients were centrifuged 40 min at rev./min and the pellets obtained resuspended in 0.45 ml of Tris-Mg-saline and 50 pl of loo/, Brij-58 and deoxycholate. These samples were applied to sucrose gradients and centrifuged as indicated above; the absorbance at 260nm (solid line) was recorded with a Gilford recording spectrophotometer and the fractions collected were counted as indicated in Methods. 0-0, leucine label in the presence of leucine label in the presence of chloramphenicol; A----.A, uridine label Cultures of HeLa cells were incubated with [3H]- leucine in the presence of pederine or chloramphenicol and control cultures were incubated with [3H]uridine under conditions in which mitochondrial ribosomal RNA is specifically labelled [3,4]. The cells were then homogenized and EDTA added to 30 mm concentration. The treatment with this chelating agent releases all the small cytoplasmic ribosomal subunits and half of the large subunits from the membranes of the endoplasmic reticulum [21]. In control experi- ments in which mitochondrial ribosomes were specifically labeled in their RNA we observed that this treatment with EDTA had no effect on the recovery of mitochondrial ribosomes. The mitochondrial fraction was then sedimented through a step gradient of 15 and 30 /, sucrose. The pellet obtained was then resuspended and a mixture of the detergents deoxycholate and Brij -58 added to solubilize mitochondrial membranes. The samples were then applied to sucrose gradients to separate mitochondrial ribosomes and ribosomal subunits. It has previously been shown that most of the mitochondrial ribosomes are found dissociated into subunits under conditions similar to those used in the present experiment [3,4]. Two peaks identified with the large and the small mitochondrial ribosomal subunit were separated (Fig. 3) ; mitochondrial ribosomal RNA was associated with these peaks and it has previously been shown that each peak contains a distinct species of RNA [3,4]. Labeling of the protein present in each peak was observed when HeLa cells were incubated with [3H]- leucine in the presence of chloramphenicol, but not when the cells were incubated in the presence of pederine. The resolution of the small mitochondrial ribosomal subunit from other slowly-sedimenting components was rather poor. These might be large aggregates formed when membrane proteins solubilized by the detergent, sediment into the sucrose gradient which does not contain detergent. These results indicate that most of the mitochondrial ribosomal proteins of HeLa cells are synthesized on cytoplasmic ribosomes; their synthesis is thus inhibited by pederine but not by chloramphenicol. We cannot rule out that some of these proteins are synthesized on mitochondrial ribosomes, since the sensitivity of our method of analysis is not sufficiently high. It has been previously shown that the proteins of mitochondrial ribosomes of Neurospora are synthesized on cytoplasmic ribosomes [21,22] ; their synthesis is inhibited by cycloheximide but not by chloramphenicol. Our present data confirm in HeLa cells work of other laboratories carried out in Neurospora [2 1,221. The Products of Mitochondrial Protein Synthesis Inner and outer mitochondrial membranes have been prepared from cells incubated with pederine or chloramphenicol and from control cells following the procedure described by Parsons et al. [15]. In all cases the cells were incubated for 1 h with leucine. The inner and outer mitochondrial membrane preparations were analyzed by acrylamide gel electrophoresis after solubilization with detergent according to Maize1 [16]. Mitochondrial proteins have been previously examined by this method of analysis ; a complex pattern has been observed with several incompletely separated peaks [6,24].

5 Vo1.22, No.3,1971 A. BREGA and C. BAGLIONI _ c 6 c r UI _ r al I 92 U - al n N Fig.4. Acrylamide gel electrophoresis of the protein of (A) obtained were resuspended in 0.1 ml of 20 mm phosphate inner and (B) outer mitochondria1 membranes of HeLa cells buffer ph 7.2; 10 p1 of sodium dodecylsulphate were Zabeled for 1 h. A 20 ml culture containing 2 x lo6 cells/ml added before electrophoresis and 40 p1 of the clear solution was incubated for 1 h in Dulbecco s modified Eagle s medium used for each gel. The 6 x 0.4 em gels contained 7.50/0 minus leucine with 0.1 mci of [3H]leucine. The cells were washed once with cold Earle s saline and inner and outer acrylamide and 0.2% bis-acrylamide and were run and fractionated for counting according to the method of mitochondrial membranes prepared according to the method of Parsons et al. [15], as described under Methods. The pellets Maize1 [16] as described under Methods. Eight drops per fraction were collected I I I I I I Fig.5. Acrylamide gel electrophoresis of the protein of the inner legend of Fig.4; 120 pg/ml of chloramphenicol were added mitochondrial membrane of HeLa cells labeled for 1 h in the 5 min before [3H]leucine. The mitochondrial membranes presence of chloramphenicol. A 20 ml culture containing were prepared as described in Fig.4 and 50 pl of this prepara- 2 x lo6 cells/ml was incubated as described above under the tion were analyzed on the gel Fig.4--7 show the resolution of labeled proteins obtained by acrylamide gel electrophoresis. The pattern of labeled proteins from the mitochondrial membrane fractions of control cells is rather complex (Fig.4). This is true for both the inner and the outer membrane preparation, although somewhat more distinct peaks appear to be present in the latter. The patterns of labeled proteins obtained from the mitochondrial membrane fractions of cells treated with chloramphenicol are very similar to those of control cells. We have been unable to detect any relevant difference between these and the patterns obtained from the corresponding fractions of control cells. Fig. 5 shows the pattern obtained from the inner membrane preparation of cells treated with chloramphenicol. In contrast, the patterns of labeled proteins obtained from the mitochondrial fraction of cells treated

6 420 Mitochondria1 Protein Synthesis Em. J. Biochem. Fig.6. Acrylamide gel electrophoresis of the protein of the (A) inner and (B) outer mitochondrial membranes of HeLa cells incubated for 1 h in the presence of pederine. A 20 ml culture containing 2 x lo6 cells/ml was incubated as described above were collected under the legend of Fig.4; 20 ng/ml of pederine were added 15 min before [3H]leucine. The mitochondrial membranes were prepared as described in Fig.4 and 50 pl of each sample were analyzed in each gel. Twelve drops per fraction Fig. 7. Acrylamide gel electrophoresis of the total mitochondrial mitochondria was resuspended in 0.1 ml of phosphate buffer proteins of HeLa cells incubated for 1 h with [3H]leucine. and dissolved with 10 p1 of dodecylsulphate (see Methods). The cells were incubated and the mitochondria isolated as 40 p1 was analyzed in a 11 ~ 0.4 cm gel. The gels were run described in Fig.4; the final separation of the inner and outer until the bromophenol blue (arrow) migrated approximately mitochondrial membranes was omitted. The pellet containing two thirds of the way toward the bottom of the gel. Ten drops per fraction were collected 80 with pederine are relatively simple, with only few peaks (Fig. 6). The same peaks are present in the inner and outer mitochondrial membrane preparations ; the inner membrane appear to contain, however, a larger amount of the labeled protein. Because of the very small amount of material yielded by the procedure used we could not estimate the contamination of outer membranes by inner membranes or vice versa. Even assuming that some cross-contamination occurred, the results clearly suggest that most of the proteins labeled in the presence of pederine are localized in the inner mitochondrial membrane. Only two clearly distinct peaks have been observed in the gels of protein synthesized in the presence of pederine (peaks 1 and 2, Fig.5). The other peaks are rather broad and not well defined. It should be pointed out that the specific activity of the proteins obtained from the mitochondrial fraction of cells incubated with pederine is very low. In order to ensure the reliability of radioactivity measurement it was necessary to compromise at the expense of resolution ; thus fewer fractions were collected from these gels than from the gels of control cells (Fig.4 and 5). We do not know whether each peak represents a different protein

7 Vo1.22, S0.3, 1971 A. BREGA and C. BAGLIOXI 421 species or whether several proteins are present within each peak. This method of analysis separates proteins on the basis of their molecular weight [16]; therefore it is possible that several proteins of similar size migrate together. The failure to observe any major difference between the gel patterns of control cells and those of chloramphenicol treated cells may be explained by the relatively small contribution of the actual products of mitochondrial protein synthesis to the pattern of labeled proteins. If the proteins synthesized in the presence of pederine are the only products of mitochondrial protein synthesis, we may not be able to observe any clearcut difference between control and chloramphenicol treated cells, due to the very high background of products of cytoplasmic protein synthesis. It has recently been proposed [25] that small molecular weight proteins (called miniproteins ) are among the constituents of mitochondrial and other biological membranes. We have analyzed our mitochondrial fraction preparation for the presence of small molecular weight proteins and have been unable to confirm their existence (Fig.7). For this analysis mitochondria were not fractionated into inner and outer membranes, but analyzed directly. Longer gels were also used (1 1 em) and precautions were taken to avoid possible loss of any protein by running the gels till the tracking dye migrated approximately 7 em [25a]. We have been unable to observe the presence of any labeled protein that migrates faster than the tracking dye. The present results confirm those obtained in Neurospora and in other organisms by several authors (see [ 11) : a large number of proteins that are localized in mitochondria are synthesized on cytoplasmic ribosomes and are presumably coded for by nuclear genes. DISCUSSIOK The present investigation on mitochondrial protein synthesis in intact cells confirms previous results of studies on protein synthesis in isolated mitochondria [6-101 :most of the products of mitochondrial protein synthesis appear to be constituents of the inner mitochondrial membrane. We cannot rule out however t,hat some products of mitochondrial protein synthesis are soluble proteins which are lost during the fractionation of inner and outer mitochondrial membranes. A small amount of labeled protein was consistently found in the supernatant of pederine treated cells aft.er spinning down mitochondrial membranes (see Methods). These proteins are too diluted, and their specific activity too low, to be analyzed by gel electrophoresis. It is conceivable however that these proteins may be released from mitochondrial membranes because of fragmentation during their fractionation. The observations made with intact cells have provided some addit.iona1 information on mitochon- drial protein synthesis. We have observed that mitochondria have a limited autonomy in protein synthesis ; inhibition of cytoplasmic protein synthesis by pederine leads to the arrest also of mitochondrial protein synthesis after about I h. We suggest that continued synthesis of cytoplasmic proteins or possibly of other macromolecules is essential for mitochondrial protein synthesis. The proteins of mitochondrial ribosomes are synthesized on cytoplasmic ribosomes, since their synthesis is sensitive to pederine but not to chloramphenicol. Similar observations have been made in Neurospora [22,23] and it seems likely that this will be the general rule. The amount of information carried by mitochondrial DNA is rather limited; a DNA molecule of a mammalian cell can code only for approximately 30 proteins of molecular weight (see [I] for a detailed discussion). It is thus not surprising that proteins of mitochondrial ribosomes are specified by nuclear genes and are synthesized on cytoplasmic ribosomes. We have been unable to confirm the existence of miniproteins [25] in mitochondrial membranes. Either such proteins do not contain leucine and thus do not become labeled in our incubation, or they are not synthesized in a 1 h incubation, or they do not exist. We favor the last alternative and believe that the method used to show the existence of miniproteins, staining acrylamide gels with coomassie blue, is less reliable than our incorporation studies, unless it can be excluded that non-protein material is stained by the dye used. The most puzzling aspect of mitochondrial protein synthesis is the relatively small number and quantity of proteins synthesized by mitochondria. It is somewhat surprising to see a complex system, like that necessary for protein synthesis. to be kept in operation for the synthesis of only a few proteins. It seems non-economical for a cell to have a completely separate system for mitochondrial protein synthesis, with its own trnas [26], ribosomes [3--51, activating enzymes [27], initiation [28] and supernatant factors [29], when proteins synthesized on cytoplasmic ribosomes can apparently enter easily into mitochondria. It is however possible that mitochondrial protein synthesis is necessary for a small number of proteins that are highly insoluble and have to be synthesized at or near the site that they will occupy. This work was supported by Grant GB of the Nat,ional Srience Foundation, Washington, D.C. Note Added in Proof. Bfter this manuscript had been submitted for publication J. B. Galper and J. E. Darnell reported in J. Mol. Biol. 57 (1971) 363 an analysis by gel electrophoresis of the protein synthesized by mitochondrial ribosomes in intact HeLa cells. These authors have used cycloheximide to inhibit cytoplasmic protein synthesis and have obtained results that are in close agreement with the ones that we report.

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