Biogenesis of mitochondria

ARCHIVES

OF

RIOCHEMISTRY

AND

RIOPHYSICS

Biogenesis 22. The Sensitivity

Deptcrtment

Between

H. DIXON, of

(1972)

of Mitochondria to Antibiotics;

of Rat Liver Mitochondria

Difference N. R. TOWERS,

361-369

161,

Biochemistry, Received

a Mammalian

System

G. M. KELLERMAN, Monash

November

University, 29, 1971;

accepted

and Yeast AND

Clayton, April

a Phylogenetic

Victoria

A. W. LINNANE 3168,

Australia

19, 1972

Protein synthesis in intact rat liver mitochondria is strongly inhibited by chloramphenicol, mikamycin, carbomycin, and spiramycin but is insensitive to erythromycin, lincomycin, and paromomycin. We have investigated methods of damaging the mitochondrial membrane to destroy any possible permeability barriers to these latter antibiotics. Criteria to demonstrate the access of antibiotics to the mitochondria have been based on the finding that paromomycin at 1000 rg/ml inhibits mitochondrial respiration when the mitochondrial membrane has been sufficiently damaged. Under conditions where membrane damage permits free access of paromomycin, neither this antibiotic nor erythromycin nor lincomycin has a specific inhibitory effect on protein synthesis. The possibility is discussed that the evolution of the ribosome from the bacterial ribosome through the yeast mitochondrial ribosome to the mammalian mitochondrial “miniribosome” may be expressed in the loss of certain antibiotic-binding proteins of the protein-synthesizing system.

particular we have reported that amino acid incorporation into protein by isolated mammalian mitochondria is insensitive to erythromycin, lincomycin, neomycin, and paromomycin (1, 2). Kroon and De Vries (4, 5) have suggested that there are no phylogenetic differences between the yeast and mammalian mitochondrial protein-synthesizing systems, on the basis of their experiments showing that protein synthesis by mammalian mitochondria is inhibited by low concentrations of erythromycin and lincomycin when permeability barriers are destroyed. However, substantial evidence for a phylogenetic difference between yeast and mammalian mitochondria has accumulated recently. Differences in the sizes of mitochondrial DNA (6, 7) mitochondrial ribosomal RNA (7, 8, 11, l2), and the mitochondrial ribosome itself (9-14) have all been reported. In all cases the

We have earlier reported that there are marked differences between the mitochondrial protein--synthesising systems of yeast and mammals (l-3). In particular, studies in this laboratory on the effect of antibiotics which inhibit, bacterial protein synthesis have highlightecl a number of phylogenetic similarities and differences among the protein-synthesizing systems of bacteria and of mitochondria from yeast and mammals. At low concentrations the 50 S-binding antibiotmikamycin, carboics chloramphenico!, mycin, spiramycin, erpthromycin, and lincomycin and the 30 S-binding antibiotics paromomycin and neomycin inhibit protein synthesis by both bacterial ribosomes and yeast mitochondria whereas the 30 S-binding antibiotics streptomycin and kanamycin are apparently effective only against bacterial ribosomes (1). The protein-synthesizing system of mammalian mitochondria is sensitive to a still smaller range of ant#ibiotics: in 361 Copyright All rights

@ 1972 by Academic I’ress, of reproduction in any form

Inc. reserved.

362

TOWERS

mammalian species is substantially smaller than its yeast counterpart. This communication is concerned with a reexamination of the sensitivity of rat liver mitochondrial functions to erythromycin, lincomycin, and paromomycin. The findings do not support the claims of Kroon and De Vries. Mitochondrial protein synthesis even in the damaged organelle is apparently insensitive to erythromycin and is inhibited only slightly by lincomycin, making an interpretation of the action of this antibiotic uncertain. The situation in regard to paromomycin is complicated; paromomycin has no effect on intact mitochondria but if the membrane is damaged, the drug functions at high concentrations as an inhibitor of mitochondrial respiration and it has some marginal effects on protein synthesis. MATERIALS

AND

METHODS

Preparation of mitochondria. Rats (150-200 g body weight) were killed by decapitation and the livers removed and chilled in preparation medium SET34 (0.34 M sucrose, 1 mM EDTA, 2 mM Tris-HCl, pH 7.4, and 0.2oj, bovine serum albumin). The livers were homogenized in SET 34 (9 vol) in a motor-driven Potter-Elvehjem homogenizer. The cell debris, nuclei, and red blood cells were centrifuged from the homogenate at lOOOgM,, for 10 min. The mitochondria were collected by centrifuging at 1O,OOOg~~,, for 10 min, and washed twice by resuspending in SET 34 (9 ml per g liver) and centrifuging at 806Ogn., for 10 min. All centrifugations were in the Sorrall SS34 rotor and all procedures were carried out at 04°C. The mitochondria were resuspended at the concentrations and in the buffers given in the table legends before respiration or protein synthesis activity was measured. For some of the respiration studies suspensions of mitochondria were fragmented by four 15set periods of sonication using a MSE 60W Ultrasonic Power Unit. When mitochondria were to be subjected to osmotic stress two procedures were used. Method 1. Assays were carried out in a medium of moderately low osmotic strength (Medium B, see below). Method .z?. The swelling procedure published by Kroon and De Vries (4) was followed: this involved pretreating the mitochondria in 0.025 M sucrose for 15 min at 4°C and then carrying out the assays in an isotonic medium (Medium C). Incubation bugers. Three buffers were used to maintain the mitochondria during assays.

ET

AL.

Medium A is the standard buffer used in this laboratory for the assay of protein synthesis by mammalian mitochondria. It contains 50 mM Bicine-KOH buffer, pH 7.4, 50 mM sucrose, 50 mM KCl, 5 mM MgClQ, and 1 mM EDTA. Medium B is the buffer of lowest osmotic strength which we have found still reliably maintains normal levels of protein synthesis. Medium B contains 25 mM Bicine-KOH buffer, pH 7.4, 25 mM sucrose, 25 mM KCl, 2.5 mM MgC12, and 0.5 mM EDTA. Medium C is the buffer used for protein synthesis by Kroon and De Vries (4). It consists of 50 mM Tricine-KOH buffer pH 7.4,50 mM sucrose, 20 mM KCI, 30 mM NH&l, 5 mM ME;%, and 1 mm EDTA. ATP Generating System. The energy for protein synthesis was normally generated by an exogenous ATP-generating system, which consisted of 2.5 mM ATP, 12.5 mM phosphoenol pyruvate, 5 eu of pyruvate kinase (EC 2.7.1.40), and 10 pg oligomycin/ml. [W]Leucine incorporation assays. [14C]Leucine incorporation was assayed in the media and with the additions given in the tables; all incorporations were corrected for a zero time incorporation and all experimental tubes were in duplicate. The tabulated results are those of a single representative experiment except where indicated. The incubations were stopped by adding 7yo (w/v) trichloracetic acid (TCA) containing 1 mg/ml leucine. The precipitate was collected by centrifugation and digested for 60 min at 34°C in 1 M NaOH (1 ml) containing 1 mg/ml leucine. The protein was precipitated with 20% (w/v) TCA in 2 M HCl (0.5 ml) and collected by filtration on glass fiber discs (Whatman GF/C). The filters and precipitates were washed five times with 7% TCA containing leucine (5 ml), twice with acetone (5 ml), and then dried at 1lO’C for 15 min. The radioactivity of the collected protein was determined by liquid scintillation counting in a toluene-based scintillator. Respiratory measurements. These were made polarographically at 30°C using a Clark oxygen electrode. Mitochondrial suspensions were diluted to about 0.5 mg protein/ml in the appropriate buffer (2 ml) plus the additions specified in the table legends. Tabulated results are those of single representative experiments. Protein assays. Mitochondrial protein was measured using the biuret procedure (15). Antibiotic solutions. All antibiotic concentrations refer to the concentration of the active material. The antibiotics (except mikamycin) were dissolved in water and the pH adjusted to 7.4. Mikamycin was dissolved in a small amount of methanol, which was then diluted with water.

ANTIBIOTIC

ACTION

IN

The final concentration of 2% methanol in the assay medium had no effect on protein synthesis. Bacterial contamination. All glassware used in the preparation of mitochondria was autoclaved before use and solutions were either autoelaved or filtered through an 0.22~ MiIlipore filter. Surgical instruments were kept in 707$ ethanol and the incision area of the animals was bathed in 70% ethanol before removal of the liver. Assays for total viable bacteria were conducted on a blank incubation by direct scoring of colonies on nutrient agar plates. Routinely less than 100 bacteria per incubation were recorded, and no experiment with more than 1000 bacteria per incubation has been presented. Materials. All reagents used were of Analar or equivalent grade. Bicine, Tricine, Trizma base, and pyruvate kinase were obtained from Sigma Chemical Co., and [U-%]L-leucine from the Radiochemical Centre, Amersham, England. Antibiotics were obtained from the following comand paromomycin sulpanies ; chloramphenicol fate, Parke Davis (Aust); mikamycin, Calbiothem; carbomycin, Pfizer (Aust); erythromycin glucoheptonate, E. Lilly & Co.; lincomycin HCl, Upjohn & Co.; oligomycin, Sigma Chemical Co. RESULTS

In Table I is shown the effect of seven antibiotics-chloramphenicol, mikamycin, carbomycin, spiramycin, erythromycin, lincomycin, and paromomycin-on amino acid incorporation by yeast and rat liver mitochondria. Protein synthesis in these experiments was supported by an exogenous energy source of phosphoenol pyruvate, pyruvate kinase, ATP, and oligomycin. The use of this energy source, rather than an endogenous system dependent on oxidative phosphorylation, ensures that only the reactions of protein synthesis are being studied and possible secondary in.hibition of protein synthesis consequent on effects of the antibiotics on mitochondrial ATP-generation are avoided. All seven antibiotics at low concentrations strongly inhibited the yeast system. However, while four of the compounds were also potent inhibitors of the mammalian system, erythromycin., lincomycin, and paromomytin were without significant effect even at 250 @g/ml, as we have earlier reported (l-3). It is of interest that two macrolides, carbomycin and spiramycin, inhibit the mammalian system while a third macrolide erythromycin does not.

DAMAGED

363

MITOCHONDRIA TABLE

EFFECT OF INCORPORATION

Antibiotic

I

ANTIBIOTICS BY INTACT

Concentration WmC

ON [WILEWINE MITOCHONDRIA~ Percentage inhibition of [‘“Cl-Leucine incorporation Yeast

Chloramphenicol Mikamycin Carbomycin Spiramycin Erythromycin

Lincomycin

Paromumycin

10 3 10 30 1 5 250 1 5 250 1 5 500

73 83 78 75 74 74 74 40 60 88 37 72 -

Liver 81 82 62 67 0 0 5 0 0 12 0 0 4

a The rat liver mitochondrial pellet was resuspended in Medium A to give 5-10 mg protein/ml and incubations contained liver mitochondria (0.5-l mg), 10 mM potassium phosphate buffer, 0.04 mM [‘K!]leucine (12.5 &i/pmole), the ATPgenerating system detailed in Materials and Methods, 25 rg of an amino acid mixture (16), and the specified antibiotic in a total volume of 0.5 ml of medium A. Incubations were at 34°C for 30 min. Yeast mitochondria were prepared from strain L410 and [‘*C]leucine incorporation assayed under the conditions of Lamb, Clark-Walker, and Linnane (17). Incorporation rates were 9 pmoles/ mg protein/30 min and 50 pmoles/mg protein/30 min for rat liver and yeast mitochondria, respectively.

Kroon and De Vries (4) have similarly reported that the mammalian system is inhibited by carbomycin; they have, however, reported that erythromycin (50 fig/ml) and lincomycin (100 pg/ml) inhibit the system 64 % and 42 %, respectively, when the membrane permeability is destroyed. As their amino acid-incorporating system depends on the surviving mitochondrial oxidative phosphorylation for energy the question arises as to whether these inhibitions are due to interactions of the antibiotics with the mitochondrial protein-synthesizing system per se, or with the energy-supplying processes. We were unable to confirm that the mitochondrial protein synthesis became sensitive to

364

TOWERS

ET AL.

erythromycin and lincomycin after membrane damage (see later): in an attempt to resolve this disagreement we initially investigated in some detail the interactions between ATP availability, antibiotic penetration into the mitochondria, and membrane damage. To this end we have used the observation that under appropriate conditions high concentrations (1000 pg/ml) of paromomycin inhibit mitochondrial respiration.

resulted in complete loss of respiratory control; under these conditions greater than 70 % inhibition of respiration by 1000 pg/ml paromomycin was obtained. Less severe damage, achieved by a hypotonic treatment of the mitochondria which preserved some respiratory control, resulted in an intermediate degree of sensitivity of respiration to paromomycin. These results are consistent with there being a permeability barrier to paromomycin in intact mitochondria which is abolished by fragmentation and partially E$ect of Paromomycin on Mitochtmdrial overcome by mild swelling procedures. At Functions concentrations up to 2000 pg/ml, erythromycin and lincomycin did not inhibit resRespiration of intact mitochondria (with high respiratory control ratios) was insensi- piration of intact mitochondria and intive to paromomycin at low concentrations hibited by no more than 20 % even after but was about 40% inhibited by paromo- fragmentation by sonication. Mitochondria fragmented by sonication mycin at 2000 pg/ml (Table II). The degree of inhibition by paromomycin could be in- show no detectable amino acid incorporation creasedif the mitochondria were fragmented into protein even with an external energyby 60-set sonication, a treatment which TABLE TABLE EFFECT

Antibiotic

OF

II

ANTIBIOTICS ON RESPIRATION LIVER MITOCHONDRIA~

Gg/ml)

Intact mi~;$on-

--__-~ pmoles

Fragmented mitochondria Oz/mg

Swollen mitochondria (method 1) protein/hr

2.1 9.1 Respiratory control ratio None 4.2 1.0 Percentage inhibition of respiration Paromomycin 23 500 0 74 1000 6 81 2000 40 Erythromycin 0 15 2000 Lincomycin 2000

0

18

BY

III

INHIBITION OF MITOCHONDRIAL P~RO~UOMYCIN IN THE ATP-GENERATING

Additions

Intact mitochondria

RICSPIRATION PRKSICNCE OF SYSTEMS

BY

Swollen mitochondria Method Method 1 2

Respiratory

6.6 1.7

18 42 59 -

a Mitochondrial pellets were resuspended in SET 34 (10-15 mg/ml) and diluted to 0.5 mg/ml in Medium A (intact mitochondria) 9~ Medium B (swollen mitochondria). Fragmented mitochondria were prepared by sonication as described in Materials and Methods and diluted tc 0.5 mg/ml in Medium A. Respiration was meaaur :d polarographically at 30°C in the same buffers with the following additions: 5 rnM potassium phosphate buffer, pH 7.4, 5 m&f succinate, and 0.5 mM ADP.

control ratio 3.8 2.1 Percentage inhibition by paromomycin 9 28 None (state 4) 10 26 ADP (0.5 mM) (state 3) 7 8 ATP-generating system 28 DNP (0.1 mM) 15 DNP + ATP-generating 12 29 system None

1.3 65 56 20 58 48

a Intact and mitochondria swollen by Method 1 were treated as in Table II. Mitochondria swollen by Method 2 were pretreated in 0.025 M sucrose at 4°C for 15 min and diluted in Medium C. Respiration wm assayed in Medium A, B, or C, respectively, with the addition of 10 mM potassium phosphate buffer, pH 7.4, and 5 mM succinate. The paromomycin concentration in all cases was 1000 rg/ml. The ATP-generating system is described in Materials and Methods. The state 3 respiration rates were 5.7, 3.6, and 1.3 pmoles O,/mg protein/hr for intact mitochondria, Method 1 and Method 2 swollen mitochondria, respectively.

ANTIBIOTIC

ACTION

IN

generating system; milder treatments to alter t.he permeability barrier are required for [14C]leucine incorporation studies to be possible. Two swelling procedures, Methods 1 and 2, were chosen for the investigations into prot,ein synthesis; they are detailed in Materials and Methods. They represent a compromise on t.he degree of mitochondrial damage which still permits protein synthesis but at the same time allows penetration of Using mitochondria treated paromomycin. in this way:, the effect of paromomycin on respiration under the conditions used in the protein synt,hesis assays by different authors was examined (Table III). Paromomycin inhibited both state 3 and state 4 respiration in swollen mit,ochondria. The addition of the ATP-generating system, however, almost abolished the inhibition of respiration by indicating some reversal of paromomycin, the permeability change induced by swelling. As shown in Table III, this “ATP effect” could be successfully reversed by the addition of 2,4-dinitrophenol (DNP). DNP itself does not inhibit mitochondrial prot.cin synthesis (Table IV) ; indeed with intact mitochondria DNP caused a Zfold stimulation of [14C]leucine incorporation, consistent with the removal of a permeability barrier. With mitochondria swollen by Method 1 t,he same stimulation was seen; however, where the permeability barrier had already been destroyed by the harsher hypotonic pretreatment (Method 2), DNP had no stimulatory effect and the net rate of incorporation was only 30% of the control incorporation. Having established conditions in which paromomycin has access to the mitochondrial matrix, and shown that these conditions were suitable for assaying protein synthetic activity, the effect of paromomycin on protein synthesis was investigated (Table IV). In no case did the presence of paromomycin lead to any inhibition of protein synthesis; indeed there was a stimulation of incorporation by paromomycin in the experiments with mitochondria swollen by Method 2. The possibility that this stimulatory effect is the consequence of in v&o misreading of the genetic code promoted by the high concentrations of paromomycin, an aminoglycoside effect well known in bacteria

DAMAGED

MITOCHONDRIA

365

TABLE EFFECT OF LWCINP:

IV

DNP

AND PAROMOMYCIN ON INCORPORATION r3~ SW~LLF:E MITOCHONDRIAa

[‘“Cl-

pmoles/mg protein/30 min Untreated mitochondria

Additions

None DNP (0.1 mM) DNP (0.1 mM) paromomycin &ml)

8.0 15.5 lfi.2

+

Swollen mitochondria Method Method 1 2 8.2 16.0 15.8

5.2 5.3 7.4

(250

0 [“Cl Leucine incorporation into protein was assayed as outlined in Table 1 with the ATPgenerat,ing system being added last. Untreated mitochondria were resuspended in Medium -4 and the incubation was carried out in the same medium (0.5-1.0 mg protein). Mitochondria swollen by Method 1 were incubated in Medium B (0.5-1.0 mg prot.ein) and mitochondria swollen by Method 2 were incubated in Medium C (l-l.5 mg protein).

(lS), has been considered. However, this is probably not the case as the incorporation rates of three amino acids with widely differing codons (leueine, lysine, and phenylalanine) all showed a stimulation of the same magnitude over a wide range of paromomycin concentrations. Another possibility is that the stimulation in incorporation results from the cationic properties of paromomycin in some way stabilizing the protein-synthcsizing machinery; a similar phenomenon has been shown for other polycat,ions in bacterial systems (19). Whatever the explanation may be, there is certainly no evidence for the direct inhibition of mitochondrial protein synthesis by paromomycin. In damaged mitochondria paromomycin inhibits respiration and, therefore, when protein synthesis is measured under conditions where energy is provided by mitochondrial oxidative phosphorylation, paromomycin is found to inhibit [‘4C]leucine incorporation by about 20 % at 50 pg/ml and by 50 % and more at concentrations of 250 fig/ml and higher. However, it is clear from the data presented above that this inhibition is a secondary effect of the inhibition of mito chondrial respiratory phenomena.

366

TOWERS

ET

Effects of Erythromycin and Lincomycin on Protein Synthesis of Swollen Mitochundria

has been measured after a variety of other experimental procedures designed to destroy any relevant permeability barriers. These have included preswelling in the presence of high (500 pg/ml) concentrations of antibiotic; mild sonication procedures; digitonin treatments similar to those reported to remove the outer membrane from the mitochondrion (20), and variations to both the degree of hypotonicity and duration of treatment in the two basic hypotonic swelling procedures described. About 100 experiments have been carried out and in all cases the results have been essentially similar; we have statistically analyzed these results and they indicate no significant inhibition of mitochondrial protein synthesis by either antibiotic at 50 Mg/ml, a 20% inhibition at 250 pg/ml, and even at massive concentrations of 1000 pg antibiotic/ ml the mean inhibition is only about 20 % with erythromycin and 40 % with lincomycin. We conclude that protein synthesis by the mammalian mitochondrial ribosome is essentially insensitive to these antibiotics and that their insensitivity is a feature of the ribosome

These two antibiotics were without significant effects on respiration (Table II), and no other specific assay was discovered to demonstrate their access to the mitochondrial matrix; it was, therefore, assumed that the conditions under which paromomycin was demonstrated to have access would also be sufficient to allow access of erythromycin and lincomycin. Under Method 2 swelling conditions the control level of [14C]leucine incorporation was greatly reduced; however, Table V shows that the antibiotics chloramphenicol, mikamycin, and carbomycin still yielded the normal degrees of inhibition in mitochondria swollen by either Method 1 or 2. It is apparent from the data presented in Table V that neither swelling treatment induced any significant sensitivity to erythromycin or lincomycin at 50 pg/ml, i.e., at concentrations 50-fold those resulting in extensive inhibition of yeast mitochondrial protein synthesis. The sensitivity of mitochondrial protein synthesis to erythromycin and lincomycin TABLE EFFECT

OF ANTIBIOTICS

Antibiotic bdml)

ON

[14C]-L~~~~~~

AL.

V

INCORPORATION

Swollen

Untreated mitochondria

Method pmoles/mg

14.9 f Percentage Erythromycin 50 250 1000 Lincomycin 50 250 1000 Carbomycin 30 Mikamycin 10 Chloramphenicol 30 0 The results given for erythromycin duplicate (mean i standard deviation). the legend to Table IV.

1.8 of control

102 f 12 87 3~ 24 90 f 10 94 f 83 f 54 f

10 10 17

DY SWOLLI’N

mitochondria

1

protein/30

15.8 f incorporation

MITOCHONDRIA~

0.5

Method min. 4.1

+

1.2

104 i 89 f 85 f

7 2 6

98 i 87 f 70 f

10 15 11

93 f 76 f 49 f

2 5 6

102 f 88 f 70 f

12 7 9

12

18

15

18

9

20

19

12

11

and lincomycin Mitochondria

2

are those from at least three experiments done in were resuspended and incubated as specified in

ANTIBIOTIC

ACTION

IN DAMAGED TABLE

EFFECT

OF ANTIBIOTICS

Antibiotic Cidml) None Erythromycin 50 250 1000 Lincomycin 50 250 Chloramphenicol 30

ON [WI-LEUCINE ENERGY DERIVED

Untreated mitochondria

83 f 68 f

6 12

18

BY DAMAGED MITOCHONDRIA PHOSPHORYLATION~

Swollen mitochondria

15.9 f 3.1 Percentage of control 15 11 3

367

VI

INCORPORATION FROM OXIDATIVE

93 f 86 f 73 f

MITOCHONDRIA

EMPLOYING

Digitonin-treated mitochondria

pmoles/mg protein/60 min. 3.6 f 0.6 incorporation

5.9 f

1.8

95 f 6 106 f 13 88 zk 10

90 f 87 f

8 15

83 f 72 f

4 6

-

loo f 72 f

15

10 3 -

a The result,s are those from three experiments done in duplicate (mean f standard deviation). Mitochondria were swollen or digitonin-treated as described by Kroon and De Vries (4) and were also assayed by the method of these authors.

and not simply the result of a permeability barrier. E$ects of Erythromycin and Lincomycin on Mitochondrial Protein Synthesis Dependent on EndogenousEnergy Generation

specific effects on the mitochondrial ribosome. DISCUSSION

This work has attempted to determine whether the difference in the spectrum of To ensure that the difference between the antibiotic sensitivity of the protein-syntheresults repor ted above and those reported by sizing systems of yeast and rat liver mitoKroon and De Vries is not simply due to the chondria relates to a fundamental difference different assay conditions, the methods and in the systems of the two organisms, as we conditions of these authors were duplicated have previously reported (1, 2) or whether it as closely as the published details allowed results merely from a selective permeability (4). Our results, using mitochondria sub- barrier preventing accessof some antibiotics to the protein-synthesizing system (4). Exjected to both the hypotonic and digitonin treatments described (4) are shown in Table perimentally it is difficult to distinguish VI. In contrast to the data of Kroon and between genuine insensitivity to the antibiotics in question and apparent insensitivity De Vries, in our hands both treatments always led to extensive damage of the mito- resulting from the lack of accessof the antichondria resulting in the reduction of the biotics to the mitochondrial ribosome. Assay [14C]leucine incorporation to 25 % and 40 % conditions are required in which antibiotic accessis assured, and the data presented on of the control incorporation, respectively. Under these conditions no significant in- the effects of paromomycin on mitochondrial crease in the susceptibility of the mitochon- respiration clearly demonstrate the numdrial protein synthesis to erythromycin or erous interacting factors which must be lincomycin was observed. In six experiments considered. As there is no independent assay using a similar but milder hypotonic swelling available to show that erythromycin and lincomycin can reach their potential site of procedure, in which the incorporating ability action we have used the conditions shown was not reduced by more than 20 %, inhibitions by these two antibiotics were similar to adequate to permit accessof paromomycin to those given in Table VI. Hence, it appears study these other antibiotics. Under these that erythromycin and lincomycin have no conditions we could find no clear evidence

368

TOWERS

for the specific inhibition of mammalian mitochondrial protein synthesis by paromomycin, crythromycin, or lincomycin; these antibiotics have only small effects on this system at concentrations two to three orders of magnitude greater than t,hose effective against yeast mitochondria and bacteria. Recent work with yeast mitochondria suggests that t’he mitochondrial ribosome is functionally closely associatedwith the inner mitochondrial membrane (21, 22), so that the erratic results and minor effects of t.hese three antibiot’ics may represent nonspecific interactions among the antibiotics, the membrane elements, and the ribosomes under varying degreesof membrane damage. At first sight the different responseof the liver mitochondrion to t’he three macrolides is unexpected; however similar responsesare seen in the bacterial ribosome (23-25) and in yeast mitochondria (26). Thus, in the formation of the first peptide bond in a bacterial cell-free system, erythromycin has no effect, spiramycin is a mild inhibitor, while carbomycin strongly inhibits the reaction (23-25). Also Trembath et al. (26), in recent genetic and protein synthesis studies on yeast mitochondria have obtained mutants which establish a complete independence of action of the three macrolides; thus, we have isolated mutants with mitochondrial protein synthesis resistant to erythromycin alone, to spiramycin alone, to both spiramycin and carbomycin, or to all three compounds. This difference in detailed action of the antibiotics suggests the possibility that there are differences in the ribosomal proteins with which they react. The lack of sensitivity of mammalian mitochondrial protein synthesis to the antibiotics discussed in this work thus appears to be a true manifestation of a phylogenetic difference between the mitochondrial ribosomes of rat liver and yeast. This explanation receives additional support from a variety of findings. The mammalian mitochondrion appears to have undergone a considerable loss of potential genetic information during the course of evolution; its DNA has a contour length of 5 ,J in contrast to a 26-p length of DNA present in mitochondria of yeast (6, 7). This loss of information is reflected in the much reduced

ET AL.

size of the mammalian mitochondrial ribosomesand its individual rRNA’s. Thus yeast (13, 14) and Neurospora (27) have been shown to have a “70 S” mitochondrial ribosome but the mitochondrial ribosome of higher animals (9-12, 2S), including the invertebrates (29), appears to be exceptionally small (55-60 S), the so-called miniribosome. The reduced size of the ribosomes is unlikely to be solely due to the smaller rRNA’s and thus it follows that the prot’ein complement is also reduced, either in the direction of fewer proteins or of smaller protein molecules. Of these two alternatives the first is more probable and we suggest that’ in demonstrating the insensitivity of the mammalian protein-synthesizing system to inhibition by some antibiotics we may be identifying in a functional sensethose protcins which are absent from the mammalian mitochondrial ribosome, although present in the yeast mitochondrial ribosome. It is not possible to distinguish phenotypically between the presence of a structurally altered “resistant” protein and the absence of the protein which normally carries the antibiotic-binding site, as both will assay as insensitive. Therefore, the nature of the phylogenetic differences in the mitochondrial ribosomes of yeast and liver will be finally established only when functional ribosomes are isolated from both speciesand the proteins of each identified and compared. However, if the different antibiotic sensitivity of yeast and liver mitochondrial protein synthesis is indeed a manifestation of the different sizes of the mitochondrial ribosomes, then we may suggestthat in any speciesthe spectrum of antibiotic sensitivity may be determined by the classof ribosome found in its mitochondria; the larger “70 S” ribosomes may show a sensitivity spectrum similar to that of yeast, while the sensitivity spectrum of the mammalian mitochondrial protein-synthesizing system may be the archetype for the “miniribosome.” The actual role of the mitochondrial ribosome appears to be limited to the synthesis of a small number of membrane associated proteins; it is possible that in the mammalian mitochondrial ribosome we have the smallest protein-synthetic apparatus that can remain functional, and

ANTIBIOTIC

ACTION

IN DAMAGED

that the reduced number of antibiotic inhibitions to which it is susceptible is correlated with the reduction of its functional complexity to a corresponding minimum. REFERENCES 1. FIRKIN, F. C., AND LINNANE, A. W. (1969) Fed.

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