Molec. gen. Genet. 173, 159-170 (1979) © by Sprlnger-Verlag 1979

Biogenesis of Mitochondria 51 Biochemical Characterization of a Mitochondrial Mutation in Saccharomyces cerevisiae Affecting the Mitochondrial Ribosome by Conferring Resistance to Aminoglycoside Antibiotics Terence W. Spithill, Phillip Nagley, and Anthony W. Linnane* Department of Biochemistry,Monash University,Clayton, Victoria 3168, Australia

Summary. An examination of the effect of the aminoglycoside antibiotics paromomycin and neomycin on mitochondrial ribosome function in yeast has been made. Both antibiotics are potent inhibitors of protein synthesis in isolated mitochondria. With isolated mitochondrial ribosomes programmed with polyuridylic acid (poly U), the drugs are shown to inhibit polyphenylalanine synthesis at moderately high concentrations (above 100 gg/ml). At lower concentrations (about 10 gg/ml), paromomycin and neomycin cause a 2 3 fold stimulation in the extent of misreading of the UUU codons in poly U, over and above the significant level of misreading catalyzed by the ribosomes in the absence of drugs. Comparative studies have been made between a paromomycin sensitive strain D585-11C and a mutant strain 4810P carrying the parl-r mutation in mtDNA, which leads to high resistance to both paromomycin and neomycin in vivo. A high level of resistance to these antibiotics is observed in strain 4810P at the level of mitochondrial protein synthesis in vitro. Whilst the degree of resistance of isolated mitochondrial ribosomes from strain 4810P judged by the inhibition of polyphenylalanine synthesis by paromomycin and neomycin is not extensive, studies on misreading of the poly U message promoted by these drugs demonstrate convincingly the altered properties of mitochondrial ribosomes from the mutant strain 4810P. These ribosomes show resistance to the stimulation of misreading of the codon UUU brought about by paromomycin and neomycin in wild-type mitochondrial ribosomes. Although strain 4810P was originally isolated as being resistant to paromomycin, in all the in vitro amino acid incorporation systems tested here, the 4810P mitochondrial ribosomes show a higher degree of resistance to neomycin than to paromomycin. *

Author to whom requests for reprints should be addressed

It is concluded that the parl-r mutation in strain 4810P affects a component of the mitochondrial ribosome, possibly by altering the 15S rRNA or a protein of the small ribosomal subunit. The further elucidation of the functions in the ribosomes that are modified by the parl-r mutation was hampered by the inability of current preparations of yeast mitochondrial ribosomes to translate efficiently natural messenger RNAs from the several sources tested.

Introduction The activity of the yeast mitochondrial protein synthesis system is sensitive to several inhibitors of bacterial protein synthesis. These inhibitors include erythromycin, chloramphenicol and spiramycin (Lamb et al., 1968), which in bacteria affect the function of the large subunit of the ribosome (Vazquez, 1974), as well as the aminoglycoside antibiotics paromomycin and neomycin (Davey et al., 1970) which affect small ribosomal subunit functions (Tanaka, 1975). Several mutations in mitochondrial DNA (mtDNA) conferring resistance to these drugs have been identified in Saccharomyces cerevisiae by many laboratories (for review see Nagley et al., 1977). The determination of the physical map positions of these mutations on the yeast mitochondrial genome (Linnane and Nagley, 1978) has revealed that the mutations leading to erythromycin, chloramphenicol and spiramycin resistance lie very close to (perhaps within) the 21S rRNA gene on mtDNA, whilst the paromomycin resistance mutations map some 27 kilobase pairs away from the former group, being found in the region of the 15S rRNA gene. Biochemical studies on yeast mutants resistant to those drugs which inhibit functions of the large mitochondrial ribosomal subunit have shown these resis-

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T.W. Spithill et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast

tance mutations to be expressed at the level of mitochondrial protein synthesis (Linnane et al., 1968; Molloy et al., 1973; Trembath et al., 1973; Grivell et al., 1973). More detailed analyses demonstrated that the isolated mitochondrial ribosomes showed resistance to the inhibitory effects of these drugs on polypeptide chain elongation reactions (Grivell et al., 1971b, 1973; Spithill et al., 1978b). The biochemical analysis of yeast strains resistant to paromomycin has not been carried out at such a detailed level as for the drugs discussed above that are inhibitory to large subunit functions. The isolation of mutations in mtDNA conferring resistance to paromomycin has been reported by three laboratories (Thomas and Wilkie, 1968; Kleese et al., 1972; Kutzleb et al., 1973), and in one case cross-resistance in vivo to the closely related aminoglycoside neomycin, has been demonstrated (Kutzleb et al., 1973). Protein synthetic activity by mitochondria isolated from two paromomycin resistant yeast strains was found to show some resistance to inhibition by paromomycin (Wilkie, 1970; Kutzleb et al., 1973). These results suggested that the small subunit of the mitochondrial ribosome was modified by the mutations in mtDNA. However, no direct examination of the response to paromomycin of isolated mitochondrial ribosomes from normal or mutant strains has yet been reported. We considered that such a study, involving both paromomycin and neomycin, would be valuable in the light of the following observations. The aminoglycoside antibiotics have deleterious effects on a range of ribosomal functions in bacteria, including the stimulation of mistranslation of codons in mRNA both in vivo and in vitro (see Tanaka, 1975). This misreading is conveniently assayed in vitro using ribosomes programmed with a synthetic messenger such as polyuridylic acid (poly U) (Davies et al., 1964, 1965). It has been shown that different aminoglycosides, including paromomycin and neomycin, do not have identical effects on miscoding by bacterial ribosomes (Davies et al., 1965; Davies and Davis, 1968; cf. Tanaka, 1975). Therefore, a comparative study of paromomycin and neomycin in yeast would be useful for two main reasons. First, it would be possible to determine the action of these drugs on yeast mitochondrial ribosomes. Second, by analyzing a mutant strain isolated as being resistant to paromomycin, it becomes feasible to define the nature of the change in the mitochondrial ribosomes of the mutant. In this paper we describe the effects of paromomycin and neomycin on two yeast strains. D585-11C is a paromomycin sensitive strain from which was isolated a paromomycin resistant strain 4810P, carrying the parl-r mutation (Kleese et al., 1972). We demonstrate that strain 4810P is cross-resistant to neo-

mycin. A systematic comparison between strains D585-11C and 4810P is made at the level of cell growth, mitochondrial protein synthesis in vitro, and properties of the isolated mitochondrial ribosomes. Both paromomycin and neomycin at relatively low drug concentrations were found to stimulate mistranslation of poly U by mitochondrial ribosomes isolated from the parent strain D585-11C. However, the ribosomes from mutant strain 4810P (parl-r) showed a changed response to these drugs, and were strikingly resistant to these miscoding effects of neomycin. We also describe in detail the preparation of purified mitochondrial ribosomes that are highly active in poly U directed polyphenylalanine synthesis. Ribosomes prepared in this way have been used in recent studies on a number of drug resistant and temperature sensitive yeast mutants (Groot Obbink et al., 1977; Spithill et al., 1978a, b). We have found that neither this new isolation procedure, nor a previously reported procedure (Grivell et al., 1971 a), yields ribosomes which show significant response to added natural mRNA from bacterial or mitochondrial sources.

Materials and Methods 1. Yeast Strains The strains of Saccharomyces cerevislae used in the main part of this work are D585-11C a lysl [rho + parl-s], and 4810P a lysl [rho + parl-r] kindly provided by R.A. Kleese. Saccharomyces earlsbergensis (N.C.Y.C. 74S) was used for some experiments on the properties of mitochondrial ribosomes.

2. Media and Growth Conditions a) For Determination of Cellular Growth Rate. Cells were grown in liquid media in 50 ml capacity Erlenmeyer Flasks fitted with a glass side-arm. The flasks contained YEPE medium (10 ml) consisting of 1% Difco yeast extract, 2% Bacto peptone, 2% ethanol. The antibiotics were added to the media after autoclaving and cooling to room temperature. Cells were inoculated at about 106 cells/ml and the cultures were shaken aerobically at 28 °. Growth was measured in a Klett-Summerson colorimeter (using the sidearm) and generation times were determined during the logarithmic phase of growth. b) For Biochemical Studies. Large scale cultures were grown at 28 ° under forced aeration in fermentors containing 11 1 of the following medium: Difco yeast extract (1%), a salts mixture (Wallace et al., 1968) supplemented with lysine (50 ~tg/ml), and ethanol (1%) as carbon source. Cells were harvested in the late logarithmic growth phase (2-3 mg dry weight cells/ml).

3. Preparation of Mitochondria Mitochondria were prepared by the method of Cobon et al. (1974), which involves conversion of cells to spheroplasts with snail gut

T.W. Spithili et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast enzyme, and rupture of spheroplasts by passage through a French pressure cell at 0 ° C. The following modifications to the procedure of Cobon et al. (1974) were made here. Spheroplasts were ruptured in buffer A (0.6 M sorbitol, 10 m M EDTA, 15 m M Tris, pH 7.2). Crude mitochondria were collected at 12,000 x g~v for 12 min, resuspended in buffer A and centrifuged at 1,000 x g~ for 10 min. Mitochondria were again collected at 12,000 x g~ for 12 rain and washed twice more in buffer A. The final mitochondrial pellet was resuspended in a small volume of a modified buffer A which contained 0.6 m M E D T A . This three-times washed mitochondrial fraction contained about 36 gg R N A / m g mitochondrial protein and was devoid of detectable contamination with cell sap ribosomal RNA.

4. Amino Acid hTcorporation by Isolated Mitochondria Protein synthesis in isolated mitochondria at 28 ° was measured as described by Lamb et aI. (1968) as modified by Groot Obbink et al. (1977).

5. Preparation of Purified Mitochondrial Ribosomes Ribosomes were always isolated from fleshly prepared (i.e. not frozen-thawed) mitochondria. Purified mitochondria were incubated with puromycin in order to discharge nascent polypeptides from mitochondrial ribosomes (Grivell et al., 197i a). Mitochondria were suspended at a concentration of 5 mg protein/ml in medium containing 150 m M KCI, 10 m M KH2PO~, 1 0 m M Mg acetate, 2 m M ATP, 5 m M 2-oxoglutaric acid, bovine serum albumin (2 mg/ml), and adjusted to a final p H of 6.7 by addmg Tris base (approx. 20 m M is usually required). After incubation of the mitochondrial suspension for 10 rain at 30 °, puromycin was added to a final concentration of 50 ~tg/ml and the incubation was continued for a further 3 min. The suspension was then rapidly chilled to 4 ° following the addition of an equal volume of the modified buffer A and the mitochondria were collected by centrifugation at 14,000xgav for 5 rain. All subsequent steps were performed at 0-4 °. Mitochondria were suspended at 3 m g protein/ml m T M N 500 buffer (10 m M Tris-HCl, pH 7.5 at 0 °, 10 m M Mg acetate, 500 m M NH4CI, 6 m M 2-mercaptoethanol) and lysed by rapid addition of 1/40th volume of 20% (w/v) Triton X-100. The lysate was centrifuged at 19,000x g~v for 20 min, and the supernatant was layered directly over a pad of 1.2 M sucrose containing 0.2% Triton X-100, 10 m M Tris-HC1, p H 7.5 at 0 °, 10 m M Mg Acetate, 1 5 0 m M NH4C1 and 6 m M 2-mercaptoethanol. The pad represented 0.4 of the nominal tube volume. Mitochondrial ribosomes were collected by centrifugation of the lysate at 190,000 x ga~ for 14 h in the 8 × 25 ml rotor of an MSE Superspeed 65 preparative ultracentrifuge. The upper layer and pad were removed by suction and the walls of the tube drmd with tissue paper. The colorless, transparent, gelatinous pellet was rinsed with T M N 50 buffer ( 1 0 m M Tris-HC1, p H 7 . 5 at 0 °, 1 0 r a M Mg acetate, 5 0 m M NH4CI, 6 m M 2-mercaptoethanol) and suspended with homogenization in the same buffer. The suspension was centrifuged at 8,500 x g~ for 10 min to yield a low speed pellet and a supernatant of suspended purified ribosomes. The pellet was re-extracted with a small volume of T M N 50 buffer and centrifuged again. The combined supernatants contained 8 34% of the total mitochondrial R N A in the form of purified ribosomes with A260:A2s o and A26o :Aza5 ratios of > 2.0 and > 1.7, respectively, and contained 40 gg R N A per Az6 o unit of ribosomes. The ribosomes could be used immediately in amino acid incorporation systems directed by poly U (see section 6 below), or

161

could be frozen in liquid nitrogen, stored at - 7 0 ° and thawed once with no appreciable loss in activity.

6. Amino Acid Incorporation by Isolated Mitochondrial Ribosomes a) Poly U Directed Polyphenylalanine Synthesis. The system used for mitochondrial ribosomes is based on that described by Modolell (1971) for E. coli ribosomes, but with some modifications so as to obtain optimal conditions for translation of poly U message by yeast mitochondrial ribosomes (Spithill, 1977; cf. Grivell et aI., 1971 a). The incubation mixture (50 gl) was assembled at 0 °, and contained 50 m M Tris-HCI, pH 7.8 at 34 °, 25 m M Mg acetate, 30 m M NH,C1, 2 m M dithiothreitol, 1 m M ATP, 5 m M phosphoenol pyruvate, 0.02 m M GTP, pyruvate kinase (30 gg/ml), 0.02 m M L-[U-14C]-phenylalanine (final specific activity 40-60 mCi/mmol), poly U (0.66 rag/m1), E. coli S100 supernatant fraction (20 gl) prepared from strain M R E 600 as described by ModolelI (1971), and mitochondrial ribosomes prepared as in section 5 above (about 0.01 0.02 mg on an R N A basis, estimated from the A26o). Where antibiotics were included, these were added to the incubation mixture as aqueous solutions, after the addition of all other components. The amino acid polymerization reaction was then started by transfer of the reaction tubes to a 34 ° water bath. After 15 rain at 34 °, the reaction was terminated by the addition of 2 mI of ice cold TCA-phe solution (5% trichloracetic acid containing 1 m g phenylalanine/ml). The tubes were kept at 0 ° for at least 30 rain, then heated for 90 ° for 20 rain, chilled again then filtered onto glass fibre discs ( W h a t m a n GF/C 25 m m diameter). The discs were washed three times with 3 ml portions of cold TCA-phe, then placed in glass vials and dried at I00 ° for 60 rain. Radioactivity on the discs was determined after addition of a toluene based scintillation mixture. Incorporation was expressed either in terms of dpm, or pmol p¢C]-phenylalanine incorporated/rag R N A / I 5 min.

b) Misreading of Poly U Messenger. The mistranslation of the poly U messenger was determined by measuring the incorporation of a mixture of 14C- or all-amino acids in the presence and absence of excess (15 raM) unlabelled phenylalanine (cf. Davies and Davis, 1968). The incubation mixture was that utilized in section 6 a above, with the following mo&fications : either 7.5 ~tCi/ml [U-tgC]-protein hydrolysate (57 nrCi/matom C) or 150 gCi/ml [3Hi-amino acid mixture was used as the source of radioactivity; a complete unlabelled amino acid mixture (0.015 m M each amino acid) was included, and the a m o u n t of E. eoli S100 supernatant fraction added was reduced to 14 ~1. Incubations were performed at 34 °, terminated after 15 rain, and the radioactivity incorporated was determined as described above (section 6a) except that the T C A washing solution contained each unlabelled amino acid at 1 mg/ml In experiments incorporating 3H-amino acids, the washed glass fibre discs were placed in glass vials, NCS (0.5 ml) was added, the vial was capped and the radioactive material was extracted from the discs by incubation overnight at 45 ° . The radioactivity incorporated was determined after addition of a toluene based scintillation mixture.

c) Natural mRNA Directed Protein Synthesis. The incubation mixture (50 ~L1)contained 50 m M Tris-HC1, pH 7.8 at 34 °, 8 m M Mg acetate, 60 m M NH4C1 , 2 m M dithiothreitol, 1 m M ATP, 5 m M phosphoenol pyruvate, 0.02 m M GTP, pyruvate kinase (30 gg/ml), a complete unlabelled amino acid mixture (0.03 m M each amino acid), 14 ~tl E. coli S100 supernatant fraction (Modelell, 1971), E. coh initiation factors (0.6 mg/ml) (Iwasaki et al., 1968), 6 ~tCi/ml [U-I4C] protein hydrolysate (57 m C i / m a t o m C), together with the appropriate m R N A and ribosomes. Reactions were carried out at 34 ° for 15 min, an the acid-insoluble radioactivity incorporated was determined as described in section 6a above.

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7. Other Analytical Procedures Protein was estimated by the methods of Gornall et al. (1949) or Lowry et al. (1951) using bovine serum albumin as the reference protein. RNA was estimated chemically by the orcinol method of Ceriotti (1955) using xylose as the reference pentose.

8. Chemicals Chemicals were obtained from the following sources : poly U, ATP, GTP, dithiothreitol, Triton X-100, pyruvate kinase, puromycin, and 2-mercaptoethanoi from Sigma Chemical Co., St. Louis, Mo. ; bovine serum albumin from Commonwealth Serum Laboratories, Melbourne, Vic.; sucrose (RNAase-free) from Schwarz-Mann, Orangeburg, N.Y. ; Nuclear Chicago Solubilizer (NCS) from Amersham-Searle, Illinois; L-[U-14C]-phenylalanine (code CFB 70), L[U-l~C]-protein hydrolysate (code CFB 25), and [3H]-amino acid mixture (code TRK 440) from the Radiochemical Centre, Amersham, Bucks.; paromomycin sulphate from Parke-Davis and Co., Sydney, N.S.W.; neomycin B sulphate, Upjohn Co., Michigan. All other chemicals were of analytical grade or of the highest purity available.

Results

1. Effect of Paromomycin and Neomycin on Cell Growth P a r o m o m y c i n (and n e o m y c i n ) selectively inhibit the g r o w t h o f S. cerevisiae on n o n - f e r m e n t a b l e c a r b o n sources ( D a v e y et al., 1970; K u t z l e b et al., 1973). The p a r o m o m y c i n resistant m u t a n t strain 4810P was isolated f r o m D585-11C (Kleese et al., 1972) a n d was resistant to 2 m g p a r o m o m y c i n / m l in a g a r plates. In the p r e s e n t study the g r o w t h rates o f strains D58511C a n d 4810P in liquid m e d i a c o n t a i n i n g e t h a n o l as c a r b o n source are c o m p a r e d in the presence of v a r i o u s c o n c e n t r a t i o n s o f p a r o m o m y c i n or n e o m y c i n (Table 1). The parI-r m u t a t i o n in strain 4810P confers

Table 1. Effect of paromomycin and neomycin on the growth of strains D585-11C and 4810P" I5o for drug (gg/ml) u

Paromomycin Neomycin

D585-11C

4810P

Degree of resistance of 4810P

100 200

6,000 6,000

60 30

" Yeast cells were grown in YEPE medium containing various concentrations of drugs, as described in Materials and Methods 2a. The division times of both strains m YEPE medium in the absence of drugs was 5.9 h b I50 signifies the concentration of drug which leads to halfmaximal inhibition of the rate of growth. The Iso values for the inhibition by paromomycin and neomycin of the growth of strains D585-11C and 4810P in YEPD medium (as YEPE, but containing glucose (2%) instead of ethanol) were of the order of 6,000 gg/ml

a 60-fold e n h a n c e m e n t o f resistance to p a r o m o m y c i n a n d a high level (30-fold) o f cross-resistance to neom y c i n r e s e m b l i n g the p a r o m o m y c i n resistant strain described by K u t z l e b et al. (1973). The action o f b o t h p a r o m o m y c i n a n d n e o m y c i n on m i t o c h o n d r i a l ribosomes is c o n s i d e r e d in m o r e detail in the following sections.

2. In vitro Antibiotic Sensitivity of Mitochondrial Protein Synthesis The effect o f p a r o m o m y c i n on the p o l y m e r i z a t i o n o f a m i n o acids c a t a l y z e d by m i t o c h o n d r i a isolated f r o m strains D585-11C a n d 4810P is shown in Fig. 1A. M i t o c h o n d r i a f r o m strain D585-11C s h o w 50% i n h i b i t i o n o f p r o t e i n synthesis at a b o u t 0.3 gg p a r o m o m y c i n / m l . By c o n t r a s t 4810P m i t o c h o n d r i a show a high level o f resistance in vitro to p a r o m o m y cin: 50% i n h i b i t i o n occurs at a b o u t 100 gg/ml, and even at 1,000 g g / m l p r o t e i n synthesis is p r o c e e d i n g at a b o u t 30% o f the u n i n h i b i t e d rate. Based on the p a r o m o m y c i n c o n c e n t r a t i o n s at which 50% i n h i b i t i o n occurs, m i t o c h o n d r i a f r o m strain 4810P show a 300fold level of resistance c o m p a r e d to m i t o c h o n d r i a f r o m strain D585-11C. T h e cross-resistance o f strain 4810P to n e o m y c i n in vivo is reflected in a high level of resistance to n e o m y c i n o f m i t o c h o n d r i a l p r o t e i n synthesis in vitro (Fig. 1 B). The m i t o c h o n d r i a f r o m the sensitive strain D585-11C are m o r e sensitive to n e o m y c i n t h a n to p a r o m o m y c i n : 50% i n h i b i t i o n occurs at a b o u t 0.1 jag n e o m y c i n / m l . The 4810P m i t o c h o n d r i a are n o t m a r k edly i n h i b i t e d by n e o m y c i n b e l o w 10 gg/ml, a n d 50% i n h i b i t i o n is seen at a b o u t 40 gg/ml, r e p r e s e n t i n g an 400-fold level of resistance to n e o m y c i n c o m p a r e d with the m i t o c h o n d r i a f r o m strain D585-11C. The isolated m i t o c h o n d r i a f r o m strain 4810P are slightly m o r e sensitive to n e o m y c i n t h a n to p a r o m o m y c i n , with 50% i n h i b i t i o n o f p r o t e i n synthesis o b s e r v e d at 40 gg n e o m y c i n / m l as c o m p a r e d with 100 gg p a r o momycin/ml. The o b s e r v a t i o n t h a t the in vivo resistance of strain 4810P to b o t h p a r o m o m y c i n a n d n e o m y c i n is a c c o m p a n i e d b y very high levels o f resistance to these drugs o f p r o t e i n synthesis in isolated m i t o c h o n d r i a s u p p o r t s the view t h a t the parl-r m u t a t i o n directly affects the m i t o c h o n d r i a l r i b o s o m e s , as has been suggested for o t h e r p a r o m o m y c i n resistant m u t a n t s by W i l k i e (1970) a n d K u t z l e b et al. (1973).

3. Effect of Paromomycin and Neomycin on Isolated Mitochondrial Ribosomes In o r d e r to study the precise i n h i b i t o r y m o d e o f a c t i o n o f p a r o m o m y c i n a n d n e o m y c i n on m i t o c h o n d r i a l ri-

T.W. Spithill et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast

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Fig. 1A and B. Effect of paromomycin (A) and neomycin (B) on amino acid incorporation by isolated mitochondria of strains D585-11C (open symbols) and 4810P (filled symbols). The incorporation activities of mitochondria incubated in the presence of different concentrations of the drugs are expressed as a percentage of the activity of control samples incubated in the absence of paromomycin and neomycin. These control activities were 4838 and 4307 dpm l*C-leucine incorporated/mg protein/20 min, for strains D585-11C and 4810P. respectively

bosome function, it would be necessary to establish in vitro systems for analyzing the individual steps of the protein synthesis cycle (including initiation, elongation, codon recognition and termination) since both paromomycin and neomycin are known to affect each of these steps in the translation process in bacteria (Tanaka, 1975). The first system that we have used is to test the effects of paromomycin and neomycin on polypeptide chain elongation catalyzed by mitochondrial ribosomes engaged in the poly U directed synthesis of polyphenylalanine. Secondly, we have been able to determine the effect of paromomycin and neomycin on the codon recognition process catalyzed by mitochondrial ribosomes as studied with a homopolymer such as poly U, since the incorporation of any amino acid other than phenylalanine directed by this messenger represents misreading of the UUU codons in this template (cf. Davies et al., 1964, 1965). In this section we describe the effects of the aminoglycosides on poly U directed amino acid incorporation catalyzed by mitochondrial ribosomes isolated from strains D585-11C and 4810P. a) Poly U Directed Polyphenylalanine Synthesis When mitochondrial ribosomes were isolated from strains D585-11C and 4810P respectively, as described in Materials and Methods, section 5, the yields of ribosomes obtained with independent ribosome preparations from each strain were similar (representing 12 19% of total mitochondrial RNA). Moreover, the specific catalytic activities exhibited by these different ribosome preparations were comparable, being of the order of 1,000 pmol ~¢C-phenylalanine incor-

porated/mg RNA/15min for several preparations from each strain. These results indicate that the parl-r mutation is not deleterious to the function of the mitochonderial ribosome in strain 4810P. The effect of paromomycin on poly U directed polyphenylalanine synthesis catalyzed by mitochondrial ribosomes isolated from strains D585-11C and 4810P is shown in Fig. 2A. Consider first the behaviour of D585-11C mitochondrial ribosomes, where it can be noted that amino acid incorporation programmed with poly U is relatively insensitive to inhibition by paromomycin. Thus, at the paromomycin concentration (0.3 gg/ml) sufficient to inhibit amino acid incorporation activity by 50% in D58511C mitochondria (Fig. 1 A), polyphenylalanine synthesis is inhibited by less than 20% with mitochondrial ribosomes from this strain (Fig. 2A). These isolated ribosomes are only significantly affected at quite high paromomycin concentrations, with 50% inhibition being observed at about 500 gg/ml. The general response of mitochondrial ribosomes from strain 4810P to inhibition by paromomycin is found to be similar to that of D585-11C ribosomes over the concentration range tested (0.1 3,000gg/ml), although at concentrations of paromomycin between 1 and 100 ~tg/ml the 4810P ribosomes show less inhibition of incorporation than for the D585-11C ribosomes. This slight resistance of 4810P ribosomes at moderate levels of paromomycin is seen much more clearly when neomycin is used in this system. As shown in Fig. 2B, neomycin is a more potent inhibitor than paromomycin of polyphenylalanine synthesis catalyzed by wild-type (D585-11C) mitochondrial ribosomes, with 50% inhibition of activity

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T.W. Spithi11 et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast

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(t-l") Fig. 2A and B. Effect of paromomycin (A) and neomycin (B) on poly U directed polyphenylalanine synthesis catalyzed by isolated mitochondrial ribosomes of strains D585-11C (open symbols) and 4810P (filled symbols). Each point represents the mean of duplicate determinations on ribosome preparations from each strain. The activities are expressed as a percentage of the activities of control ribosomes incubated with poly U in the absence of drugs. These control activities (expressed as pmol 14C-phenylalanine incorporated/mg RNA/15 min) were for strains D585-11C and 4810P, 1323 and 1658, respectively

being observed at 100 gg/ml. As was found with paromomycin, the effects of neomycin on poly U directed amino acid incorporation take place at higher drug concentrations than those required for inhibition of amino acid incorporation by mitochondria from strain D585-11C (cf. Fig. 1B). The mitochondrial ribosomes from the mutant (4810P) show a significantly higher resistance to the inhibitory effect of low neomycin levels over the concentration range 1 100 gg/ ml, than was seen with paromomycin. Above 100 gg/ ml, however, significant inhibition of polyphenylalanine synthesis on 4810P ribosomes is observed, with 50% inhibition being obtained at about 200 gg/ml. Finally, it is evident from Fig. 2B that mitochondrial ribosomes from both strains exhibit a similar sensitivity to neomycin concentrations above 300 gg/ml. b) Misreading of the Poly U Messenger

(i) Natural Misreading in the Absence of Added Drugs. Normally, poly U codes for poly phenylalanine (codons UUU) but depending on the environmental conditions (such as Mg 2÷ concentration), bacterial ribosomes programmed with poly U catalyze the incorporation of other amino acids such as leucine, isoleucine, serine and tyrosine (cf. Davies et al., 1965). The extent of this natural misereading is normally low, of the order of 1-5% of the rate of incorporation of phenylalanine, but can approach the rate of incorporation of phenylalanine itself at sufficiently high Mg 2÷ concentrations (see e.g. So et al., 1964). The overall extent of misreading of the poly U messenger by ribosomes can be determined by employing a mixture of radioactive amino acids as a source of label in

the incubation mixture. By adding a large excess of unlabelled phenylalanine to the incubation system to suppress completely the correct incorporation of radioactive phenylalanine, the extent of incorporation of other amino acids represents mistranslation of the UUU codons in poly U. Using this procedure, the natural misreading of the poly U messenger catalyzed by mitochondrial ribosomes was examined. Incubations were performed under the conditions previously determined to be optimal (Spithill, 1977; see Materials and Methods, section 6a) for the correct translation of the poly U messenger by mitochondrial ribosomes (involving inter alia 25 mM Mg acetate and 30 mM NH,C1) but employing a mixture of 1~C- or 3H-amino acids as the source of radioactivity. The poly U directed incorporation of radioactive amino acids catalyzed by mitochondrial ribosomes is hown in Table 2. Mitochondrial ribosomes isolated from the wild-type strain D585-11C catalyze a high rate of incorporation of mixed 14C-amino acids which is dependent on the addition of the poly U messenger. In the presence of excess (15 mM) unlabelled phenylalanine, which completely suppresses the incorporation of 14Cphenylalanine under these conditions (Spithill et al., 1978a), a high rate of incorporation of a4C-amino acids was observed to an extent of 38% of radioactivity incorporated in the absence of unlabelled phenylalanine (Table 2). This residual misreading incorporation was strongly suppressed by the addition of excess (15 raM) unlabelled leucine (Table 2), indicating that the misreading catalyzed by mitochondrial ribosomes is due almost exclusively to the incorporation of 14Cleucine present in the radioactive amino acid mixture.

T.W. Spithill et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast Table 2. Poly U directed amino acid incorporation catalyzed by isolated mitochondrial ribosomes a Ribosome source

Incubation b mixture

Incorporation ° (dpm incorporated/ tube/15 min)

D585-11C

Complete -poly U + Phe + Phe + Leu

3,414 149 1,294 99

Complete - poly U +Phe + Phe + Leu

52,687 2,785 7,305 0

4810P

Misreading d (%)

27

" The mitochondrial ribosome preparations isolated from strains D585-11C and 4810P were those employed in Fig. 3. Incorporation of amino acids was determined under the conditions described in Materials and Methods, section 6b, using a 14C-amino acid mixture (D585-11C) or 3H-amino acid mixture (4810P) as the source of radioactivity. Each tube contained 0.02 mg (D585-11C) or 0.014 mg (4810P) of ribosomes (estimated as RNA) b Phe signifies addition of 15 mM unlabelled phenylalanine; Leu signifies addition of 15 mM unlabelled leucine ° The figures are corrected for zero-time incorporations of 152 dpm (D585-11C) and 3,240 dpm (4810P) respectively d The extent of misreading is expressed as the mole percentage of leucine incorporated, out of the total amino acid incorporation (phenylalanine+leucine). This parameter is calculated from the observed percentage of total radioactivity which is incorporated in the presence of excess unlabelled phenylalanine (38% and 14%, for D585-11C and 4810P, respectively) taking into account the relative specific activities of phenylalanine and leucine in the mixture of radioactive amino acids added to the ribosomes. For both the 14C- and 3H-amino acid mixtures used there is 1.7 times as much radioactivity in leucine as there is in phenylalanine (see Radiochemical Centre catalogue for specifications of CFB25 and TRK440, the catalogue numbers of the preparations used). Since unlabelled amino acids were also included in the reaction mixture each at 0.015 mM, representing a large molar excess over the mass of the relevant amino acids added in the radioactive preparations, the ratio of the specific activities of leucine and phenylalanine is close to 1.7

The level of natural misreading on a m o l a r basis can be calculated to be 27% (see legend to Table 2) which is quite high, p r o b a b l y resulting f r o m the relatively high concentration of M g 2+ (25 m M ) employed in the incubation mixture. M i t o c h o n d r i a l ribosomes isolated f r o m the mutant 4810P catalyze a high rate poly U dependent i n c o r p o r a t i o n of ~H-amino acids (Table 2). This inc o r p o r a t i o n p r e d o m i n a n t l y represents the correct translation of the UUU c o d o n s in poly U as phenylalanine, since in the presence of excess unlabelled phenylalanine the rate of i n c o r p o r a t i o n of 3H-amino acids is suppressed to just 14% of the uninhibited rate. This low level o f misreading is completely suppressed

165

by excess unlabelled leucine, which again indicates that the mistranslation catalyzed by the mitochondrial ribosomes is represented only by the incorporation of 3H-leucine. The extent o f misreading of UUU on a m o l a r basis is calculated to be 9% (Table 2). It should be noted that this significant difference in the level of natural misreading catalyzed by mitochondrial ribosomes isolated f r o m strains D585-11C and 4810P has been observed in two independent ribosome preparations f r o m each strain, the levels of misreading observed being 27% and 47% for strain D585-11C and 9% and 14% for strain 4810P. Thus, mitochondrial ribosomes f r o m the p a r o m o m y c i n resistant m u t a n t 4810P translate the poly U messenger with higher fidelity than ribosomes f r o m the wild-type strain D585-11C. A n o t h e r wild-type strain (L410) has been shown to have an extent of natural misreading on mitochondrial ribosomes of 44% under the same conditions as used here (Spithill et al., 1978 a).

(ii) Effect of Paromomycbl. The effect of p a r o m o m y cin on the mistranslation of the UUU codons in poly U catalyzed by mitochondrial ribosomes isolated f r o m strains D585-11C and 4810P was examined. As shown in Fig. 3 A for mitochondrial ribosomes f r o m both strains there is a biphasic response to p a r o m o mycin. At low p a r o m o m y c i n levels misreading is stimulated, whereas at higher drug concentrations amino acid incorporation is inhibited. The p a r o m o m y c i n concentrations at which this second, inhibitory, phase is observed correspond to those concentrations at which polyphenylanine synthesis is inhibited by the drug (Fig. 2A). It can be seen in Fig. 3 A that the stimulation of misreading with ribosomes f r o m the m u t a n t 4810P is displaced to significantly higher p a r o m o m y c i n levels than in ribosomes f r o m strain D585-11C. In the concentration range 0.3-30 ~g/ml, p a r o m o m y c i n stimulates the misreading of poly U catalyzed by wildtype ribosomes about 2-fold; half-maximal stimulation is observed at a b o u t 1 pg p a r o m o m y c i n / m l . With mitochondrial ribosomes f r o m the mutant, however, only a very low degree of stimulation of misreading is observed at 1 pg p a r o m o m y c i n / m l ; half-maximal stimulation is obtained at a m u c h higher concentration (12 lag/ml), although the overall stimulation of mistranslation is higher (2.7-fold) than for D585-11C. At p a r o m o m y c i n levels above 500 pg/ml, the misreading activity catalyzed by both ribosome preparations is inhibited to a similar extent and almost all incorporation is blocked above 1,000 pg/ml. The 4810P mitochondrial ribosomes are thus seen to be altered in their miscoding properties in response to p a r o m o m y cin as c o m p a r e d to ribosomes f r o m the parent strain D585-11C.

166

T.W. Spithill et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast 300

A

B

I- 200 > lU < ,-I

o

I.Z 100

o

U

\\

V

0 0"01

I 0 1

I

I

1

10

I 100

! 1000

0.01

I

I

I

0 "1

1

10

I 100

AV I 1000

[o.uo] (.0 Ira') Fig. 3. Effect of paromomycin (A) and neomycin (B) on misreading of poly U catalyzed by isolated mitochondrial ribosomes of strains D585-11C (open symbols) and 4810P (filled symbols). Misreading represents the incorporation of radioactivity from a labelled amino acid mixture measured in the presence of excess unlabelled phenylalanine (Materials and Methods 6b; see also Table 2), which results mainly from incorporation of labelled lencine (see Table 2 and text). Each point represents the mean of duplicate determinations obtained with ribosome preparations from each strain. The misreading activities in this Figure are expressed as a percentage of the activities of control ribosomes incubated in the absence of drugs. These control misreading activities (expressed as dpm 14C- or 3H-amino acids incorporated/tube/I5 min in the presence of unlabelled phenylalanine) were for D585-11C (14C) and 4810P (3H), 1294 and 7305, respectively

(iii) Effect of Neomycin. The effect of neomycin on the misreading of the poly U messenger catalyzed by mitochondrial ribosomes isolated from strains D585-11C and 4810P is shown in Fig. 3 B. The overall effect of neomycin on the misreading catalyzed by wild-type mitochondrial ribosomes (D585-11C) is similar to that shown by paromomycin, in that there is biphasic response involving first a stimulation of misreading, followed by the second phase of a general inhibition of incorporation effected by the drug (cf. Davies and Davis, 1968). However, neomycin promotes misreading more efficiently than does paromomycin with ribosomes from strain D585-11C. Halfmaximal stimulation is observed at about 0.4 gg neomycin/ml. Nevertheless, the overall stimulation of activity is the same for both drugs (2-fold). In contrast, mitochondrial ribosomes from the mutant 4810P show a markedly altered response to the effects of neomycin. Over the entire concentration range 0.1-60 gg/ml, no stimulation of misreading by neomycin is observed with the mutant ribosomes. Indeed, neomycin is seen to inhibit the natural misreading by about 40%, an effect that is not seen with mitochondrial ribosomes from the wild-type strain. At neomycin concentrations above 60 lag/ml, the general inhibition of translation is observed with both ribosome preparations and all incorporation is blocked at about 1,000 gg/ml.

It is possible that the 4810P ribosomes have acquired such a high degree of resistance to the stimulation of misreading by neomycin, that the neomycin concentration where this stimulation would occur now falls within the range where the general inhibition of translation is brought about by neomycin (> 100 gg/ml). Thus one can observe only a very slight stimulation of misreading by neomycin of 4810P ribosomes at 100 gg/ml (Fig. 3B). These results demonstrate that the parl-r mutation in strain 4810P is directly involved in determining the response of mitochondrial ribosomes to neomycin.

4. Further Studies on Amino Acid Incorporation by Isolated Mitochondrial Ribosomes " Attempts at Programming Using Natural Messengers For a more detailed study of the effects of paromomycin and neomycin on mitochondrial polypeptide chain initiation, elongation and termination in vitro, under conditions which most closely reflect the processes occurring in vivo, it would be necessary to establish conditions under which isolated mitochondrial ribosomes would efficiently and faithfully translate natural messenger RNAs. Unfortunately, we have not found a sufficiently high stimulation of our mitochondrial ribosome preparations by natural messengers

T.W. Spithill et al.: Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast

from mitochondrial, bacterial or viral sources, that would permit more detailed analyses of the mitochondrial ribosomes from the parent D585-11C or from the mutant 4810P. We summarize here our results concerning the response to added natural m R N A of ribosomes prepared by our method (Materials and Methods 5a), and by that of Grivell et al. (1971a). The latter method was chosen for comparison because ribosomes prepared by this procedure were used by Grivell and Reijnders (1974) in studies involving translation of phage Ms2 R N A by mitochondrial ribosomes from S. carlsbergensis. For the purposes of comparison we prepared mitochondrial ribosomes from S. carlsbergensis, both by our method, and by the method of Grivell et al. (1971a). The activities of co-factors and m R N A used in the assay systems were tested with ribosomes prepared from E. coli strain M R E 600 (Modelell, 1971). The results we obtained (Table 3) can be summarized to state that mitochondrial ribosomes from S. carlsbergensis were found to show little or no activity in translation of phage MS2 R N A (or other natural mRNAs), whilst being highly active in the synthesis of polyphenylalanine directed by poly U. These low catalytic activities of mitochondrial ribosomes are presently inadequate for studying the detailed properties of mitochondrial ribosomes engaged in the translation of natural messengers. It is possible that the detergent and high salt concentrations that are used in both our method and that of Grivell et al. (1971 a) in order to free mitochondrial ribosomes from the membranes to which they are tightly bound in vivo (Plummer et al., 1973 ; Spithill et al., 1978 b) may have removed some key factors from the ribosomes that are necessary for translation of natural mRNAs, particularly proteins involved in initiation. It is evident from Table 3 that the E. coli initiation factors are unable to restore the functions missing from yeast mitochondrial ribosomes while containing factors required by E. coli ribosomes. We have prepared from yeast mitochondria, using the method of Iwasaki et al. (1968), an extract that may be expected to contain mitochondrial initiation factors. When these were substituted for the E. coli initiation factors they were found not to stimulate either E. coli ribosomes or mitochondrial ribosomes in the translation of MS2 R N A (data not shown). It is of interest that Grivell and Reijnders (1974) suggest that mitochondrial ribosomes may be unable to recognize initiation sites on MS2 RNA. However, we are unable to explain why, in contrast to Grivell and Reijnders (1974), we did not observe any stimulation by MS2 R N A of amino acid incorporation catalyzed by yeast mitochondrial ribosomes prepared by our method, or by the method of Grivell et al.

167

Table 3. Activities of different mitochondrial ribosome preparations and E. coli ribosomes in natural m R N A directed protein synthesis" m R N A source b (mg/ml)

None MS2 (0.56) E. coli (2.48) Mitochondria

Ribosome source c E. coli

M-1

346 (318) 25,740 (23,680) 15,878 (14,608) 2,551 (2,347)

40 0 319 97

M-2

(114) (0) (910) (276)

32 18 181 94

(148) (84) (838) (435)

(0.50) poly U (0.66)

23,450

(3,451)

6,042 (2,730)

2,870 (2,124)

a Activity in natural m R N A directed protein synthesis was measured as described in Materials and Methods 6c. Each tube contained the indicated amount of an m R N A source (see footnote b, below) and the following amounts of ribosomes (see footnote c, below): E. coil (0.04 mg), M-1 (0.013 rag), M-2 (0.008 mg). Natural m R N A directed amino acid incorporation activity is expressed as dpm 14C-amino acids/tube/15 min, or as indicated in parentheses, pmo114C-amino acids incorporated/mg RNA/15 min. The corrections for zero time incubations were 136, 182, and 128 dpm for E. coli, M-1 and M-2 ribosomes, respectively. Poly U directed polyphenylalanine synthesis was determined as described in Materials and Methods 6a. In this case the activity is expressed as dpm 14C-phenylalanine incorporated/tube/15 min, or as indicated in prarentheses, pmol J*C-phenylalanine incorporated/rag RNA/15 rain. In the absence of poly U from this system, the amount of radioactivity incorporated was 225, 69 and 33 dpm/ tube/15 min for ribosomes from E. coli, M-1 and M-2 respectively. b The sources of m R N A were as follows. MS2 RNA was from bacteriophage MS2 prepared according to Kolakofsky (1971). E. coli RNA was extracted from E. coli MRE600, and sedimented through a sucrose gradient to collect the material sedimenting more slowly than the 16S r R N A ; this R N A preparation thus contains a population of m R N A s in addition to tRNAs. The mitochondrial R N A was a preparation of total R N A isolated from purified mitochondria of S. cerevisiae strain L410 ° E. coli ribosomes were isolated from strain MRE600 by the alumina grinding procedure (Modelell, 1971), and were washed in 1 M NH¢C1 to render them responsive to added initiation factors. MitochondriaI ribosomes were isolated from S. carlsbergensis N.C.Y.C. 74S either by the method described in this paper (Materials and Methods, section 5) and designated M-I, or by the method of Grivell et al. (1971 a) and designated M-2

(1971 a), even using the same strain of yeast as used by Grivell and colleagues. This situation has prevented further study of the functional alterations in mitochondrial ribosomes from strain 4810P.

Discussion

The biochemical studies reported here on the nature of the resistance of strain 4810P to paromomycin and neomycin have centred on the activity of the isolated mitochondrial ribosomes in polypeptide synthesis, and particular attention has been paid to the misreading of the UUU codons in the model m R N A ,

168

T.W. Spithillet al. : MitochondrialRibosomesand AminoglycosideResistancein Yeast

poly U. It was found that mitochondrial ribosomes from the paromomycin resistant mutant strain translate poly U with higher fidelity than do wild-type mitochondrial ribosomes. It thus appears that the parl-r mutation in strain 4810P affects mitochondrial ribosome structure in the mutant in such a way that the natural mistranslation of mRNAs is diminished. This behaviour of mitochondrial ribosomes is analogous to that shown by ribosomes from streptomycin resistant bacteria which also translate poly U with higher fidelity than do wild-type ribosomes (Anderson et al., 1965; Ozaki et al., 1969). It was also established here that both paromomycin and neomycin at relatively low concentrations act on wild-type mitochondrial ribosomes (such as those from strain D585-11C) so as to enhance the natural level of misreading of the poly U message by about 2-fold. At higher concentrations these drugs lead to a general inhibition of amino acid polymerization by mitochondrial ribosomes. The similarity of the action of these drugs on yeast mitochondrial ribosomes is in contrast to their differential effects on E. coli ribosomes (Davies et al., 1965; Davies and Davis, 1968). Examination of the effects of these drugs on the misreading of poly U by mitochondrial ribosomes from strain 4810P revealed that ribosomes from the mutant were highly resistant to the misreading-inducing effect of the low neomycin levels, and showed significant changes in the miscoding response promoted by low paromomycin concentrations. However, at higher drug concentrations, no resistance to paromomycin or neomycin was observed and complete inhibition of incorporation occurred. Such a result indicates that the parl-r mutation directly influences the response of the mitochondrial ribosomes in strain 4810P to the specific misreading-inducing effects of paromomycin as well as of neomycin. In this respect, the behaviour of the mitochondrial ribosomes resembles that shown by ribosomes isolated from bacterial mutants resistant to streptomycin, neomycin or neamine which are also resistant to the misreading-inducing properties of these particular aminoglycosides (Davies et al., 1964; Van Knippenberg et al., 1965; Apirion and Schlessinger, 1968; Bollen et al., 1975). The evidence presented here is consistent with the view that a major effect of paromomycin and neomycin on yeast cells (at concentrations which selectively block growth on non-fermentable substrates) may be to promote misreading on mitochondrial ribosomes, and thus lead to the synthesis of non-functional mitochondrial proteins. In this view the in vivo resistance of strains 4810P to these aminoglycosides could be explained by the parl-r mutation in the mtDNA of 4810P leading to some change in the mitochondrial ribosomes which renders these ribosomes less sensi-

tive to the miscoding effects of these drugs. This view may however represent an over-simplification, as it does not immediately explain the behaviour of the isolated mitochondria of 4810P, in that they show a very high level of resistance to inhibition of amino acid incorporation by paromomycin and neomycin (Fig. 1). The experiments on ribosomes reported here do not eliminate the further possibilities that the aminoglycosides block some other mitochondrial ribosomal function that does not play a role in poly U directed translation. Such additional functions may include polypeptide chain initiation, certain eleongation steps, termination, or ribosome recycling (cf. Tanaka, 1975). Studies on mitochondrial ribosomes programmed with natural mRNA potentially provide information concerning these points, but as discussed in the results section 4, it has not yet been possible to obtain conditions for the efficient translation of natural mRNAs by isolated yeast mitochondrial ribosomes, in which these initiation and termination steps play a role. A further important question that is a yet not definitively answered is which particular component of the mitochondrial ribosome (i.e. a particular ribosomal protein, or rRNA molecule) is modified directly as a result of the parI-r mutation. Whilst no direct molecular evidence is yet available, some data from physical mapping studies of yeast mtDNA may have some bearing on this question. Using physical and genetic mapping procedures (Linnane and Nagley, 1978), the parl determinant and the cistron for the 15S rRNA component of the small ribosomal subunit have been physically mapped to the same region of mtDNA, in the segment 63 65 units on the physical map of the S. cerevisiae mitochondrial genome (Choo et al., 1977; Nagley et al., 1977). This segment of mtDNA is about 1,500 base pairs (1 x 106 dalton) in length. These results indicate that the parl-r mutation could reside within the structural gene for the 15S rRNA molecule of the small ribosomal subunit. However, these mapping data do not rigorously prove that the parl locus lies within the sequences coding for 15S rRNA. A fine structure map would have to be constructed using new sets of petite mutants retaining or losing genetic loci and defined mtDNA segments in this region of the genome, as has recently been carried out to determine the relationship of the eryl, tsrl, co and capl loci to the 21S rRNA gene (Atchison et al., 1978). It is also possible that the parl-r mutation resides within the structural gene of a mitochondrially-coded ribosomal protein of the small subunit. At least one protein of the small subunit of the mitochondrial ribosome in Neurospora is a mitochondrial translation product (and presumptively a mitochondrial gene product)

T.W. Spithill et al. : Mitochondrial Ribosomes and Aminoglycoside Resistance in Yeast ( L a m b o w i t z et al., 1976) a n d a s i m i l a r s i t u a t i o n c o u l d e x i s t i n y e a s t ( G r o o t , 1974), a l t h o u g h it h a s b e e n e s t a b l i s h e d t h a t a l m o s t all o f t h e p r o t e i n s o f y e a s t mitochondrial ribosomes are synthesized on cytosolic r i b o s o m e s ( D a v e y et al., 1969) a n d t h e r e f o r e p r e s u m a b l y s p e c i f i e d b y g e n e s in n u c l e a r D N A . Acknowledgements. This work was supported by a grant from the Australian Research Grants Committee (D2 75/15789). We thank Mr. R. Maxwell and Mr. K.K. Maheswari for their skilled assistance with various aspects of this work.

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Communicated by W. Gajewski Received November 5, 1978 / January 20, 1979

Biogenesis of mitochondria 51: biochemical characterization of a mitochondrial mutation in Saccharomyces cerevisiae affecting the mitochondrial ribosome by conferring resistance to aminoglycoside antibiotics.

Molec. gen. Genet. 173, 159-170 (1979) © by Sprlnger-Verlag 1979 Biogenesis of Mitochondria 51 Biochemical Characterization of a Mitochondrial Mutati...
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