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Home , Ribosomal RNA

Proceedings of the National Academy of Sciences Vol. 68, No. 2, pp. 324-328, February 1971

Characterization of Mitochondrial from

H. MORIMOTO AND H. 0. HALVORSON Laboratory of Molecular and Department of Bacteriology, University of Wisconsin, Madison, Wis. 53706 Communicated by David E. Green, November 2, 1970

ABSTRACT A method for the preparation of mito- METHODS chondrial ribosomes from yeast is presented. Mitochon- and culturing conditions drial ribosomes differ from their cytoplasmic counterpart in composition, electrophoretic mobility of a A wild type homothallic diploid strain of Saccharomyces cere- number of their respective ribosomal , and ther- visiae (Y55) was grown with vigorous aeration at 30'C in a mal denaturation profiles. The mitochondrial and cyto- New Brunswick fermentor, with a synthetic medium contain- plasmic ribosomes have identical sedimentation co- efficients (80 S) and dissociate into 60S and 40S subunits. ing 2% galactose as the carbon source to derepress mito- These findings demonstrate that, in yeast, mitochondrial chondrial synthesis (H. B. Lukins et al., manuscript in prepa- ribosomes are distinct from cytoplasmic ribosomes. ration). When the optical density (600 nm) of the suspension reached 8 units, the cells were chilled by the addition of crushed ice and harvested by centrifugation in a Sharples Nuclear and cytoplasmic (1, 2) govern mitochondrial continuous-flow centrifuge. The cells (yield about 200 ml of functions in yeast. Only a few of the products of mito- well-packed cells) were used immediately for preparation of chondrial DNA have been identified (3, 4). The presence of a mitochondria. -synthesizing system in mitochondria, like that in and apparently different from the mammalian type Isolation of mitochondria present in the (5), is suggested by the following ob- Freshly harvested yeast cells were transformed into sphero- servations. Various , such as , plasts, and crude mitochondrial fractions were prepared, as inhibit in vivo and in vitro only the protein synthesis in the described by Lamb et al. (14). The mitochondrial suspension mitochondria of yeast (6). Mutants resistant to these anti- was layeredona 12-26%o linear Renografin gradient containing biotics show non-Mendelian or cytoplasmic inheritance (2). 0.6 M sorbitol and centrifuged as described by Schatz et al. fMet-tRNA, the chain-initiating tRNA in bacteria (7), has (15). Mitochondrial fractions between the densities of 1.2 and been detected in the mitochondria of yeast (8). Cytoplasmic 1.15 were pooled, pelleted by centrifugation, and washed ribosomal RNA fails to form complementary hybrids with twice to remove Renografin (which absorbed UV radiation mitochondrial DNA (9), and the ribosomal-like RNA ex- strongly). The final preparation (purified mitochondria) tracted from isolated mitochondrial particles hybridizes was free of contaminating protoplasts and nuclei. These readily only with mitochondrial DNA (10). Finally, the two freshly prepared mitochondria were then quick-frozen in high molecular weight of RNA from mitochondria Dry-Ice-alcohol and stored at -20'C. each have lower sedimentation values than the corresponding RNA from cytoplasmic ribosomes (10-12). Isolation of mitochondrial ribosomes In Neurospora crassa, mitochondrial ribosomes and their subunits have been isolated and characterized, and differences The purified mitochondria were thawed and carefully sus- pended by homogenization in TMK buffer (50 mM Tris. HCl between the mitochondrial and cytoplasmic ribosomal pro- (pH 7.4)-10 mM MgCl2-25 mM KCl) containing 10 mM di- teins have been detected chromatographically (13). This paper describes the isolation and physical characterization of ribo- thiothreitol and 0.5% Nonidet. To release the ribosomes, somal particles from yeast mitochondria. Their sedimentation purified mitochondria were disrupted by slowly passing the properties and their types of RNA and protein indicate that suspension through a French press at 8000 psi. The homog- enate was centrifuged in a Ti 50 rotor (Spinco L2-65B) at yeast mitochondrial ribosomes are distinct from those present in the cytoplasm. 30,000 X g for 30 min; the clear bright-yellow supernatant was centrifuged for 1.5 hr at 105,000 X g. The yellow pellet (crude mitochondrial ribosomes) was suspended by homoge- MATERIALS nization in TMK buffer and subjected to centrifugation in a Chemicals were obtained from the following: glusulase, from 10-40% linear sucrose gradient in a SW 25.1 (Spinco) rotor Endo Laboratories; Renografin (N,N'-diacetyl-3,5-diamino- at 23,000 rpm for 8 hr at 40C. The primary monosome peak 2,4,6-triiodobenzoate), from E. R. Squibb; galactose, from was pooled; the ribosomes were collected by centrifugation and Pfanstiel Laboratories, Nonidet (NP40), from Shell Oil; recentrifuged on a similar sucrose gradient. The pooled ribo- acrylamide, bis (N,N-methylene bisacrylamide) and tetra- somal material was then washed twice with buffer. The final methylethylenediamine (TEMED), from Eastman Kodak clear pellet (purified mitochondrial ribosomes) was quick- Co.; RNase-free sucrose from Schwarz BioResearch, Inc.; frozen and stored at -800C in a Revco freezer until further toluidine blue and amido black, from Aniline Dye Co. use. Identical procedures were used to isolate cytoplasmic 324 Downloaded by guest on September 23, 2021 Vol. 68, 1971 Mitochondrial Ribosomes from Yeast 325

ribosomes from the 105,000 X g pellet prepared from glucose- somes (1.92). The adsorption spectra of purified cytoplasmic grown cells. and mitochondrial ribosomes are identical and typical of ribosomal particles. The absorption maxima and minima for Ribosomal proteins both are 268 and 242 nm. Pure mitochondrial and cytoplasmic ribosomes were washed twice with 0.5 M NH4Cl-0.1 M MgCl2-TK buffer. Ribosomal Sedimentation properties of mitochondrial ribosomes proteins were prepared from both preparations and subjected Sedimentation of the mitochondrial ribosomal fraction on a to acrylamide gel electrophoresis according to the method of linear sucrose gradient yields a major monosome peak at ap- Gesteland and Staehelin (16) after dialysis of ribosomal pro- proximately 80 S (Fig. 1). The absorbance at the top of the teins against 4 M urea and TMK buffer containing 10 mM tube is due to dithiothreitol. Preliminary experiments indi- mercaptoethanol. cated that the monosomes readily dissociate in a medium low in magnesium. Ribosomal RNA (rRNA) The sedimentation values of mitochondrial and cytoplasmic Purified cytoplasmic and mitochondrial ribosomes were ribosomes were determined by analytical ultracentrifugation. treated with LiCl (17). The rRNA fraction was extracted Schlieren photographs show one predominant species for each. twice with phenol and several times with ether, and the rRNA A mixture of the two types of ribosomes does not resolve into was precipitated with 70% ethanol. distinct sedimenting species. From these data the corrected Mitochondrial RNA s20,w value of each was calculated as 80 S. Subunits of mitochondrial ribosomes were formed by sus- Mitochondrial RNA was extracted with 2% sodium dodecyl pending the ribosomes according to the method of Schmitt sulfate in TM buffer (no KCl) and deproteinized by phenol. (21) in TM buffer containing 0.3 M KCl. The sedimentation This RNA was electrophoresed according to the method of values calculated for these subunits were 60 and 40 S. Peacock and Dingman (18). Ribosomal RNA was removed from the acrylamide gels by electrophoresis and analyzed for Thermal transitions of cytoplasmic and base composition. mitochondrial ribosomes If the structure or composition of the two types of ribosomes Chemical determinations differed, one might expect this to be reflected in their thermal Protein and RNA content of the ribosomal fractions were transitions. In TK buffer containing 5 mM MgCl2, both determined by the method of Lowry et al. (19) and by the purified preparations showed a biphasic change in orcinol (20) method, respectively. Bovine serum albumin and hyperchromicity with increasing temperature. The hyper- commercial yeast RNA were used as standards. chromic shifts and Tm values were: mitochondrial ribosomes RESULTS 87% and 52.5°C and cytoplasmic ribosomes 55% and 58°C As mitochondrial ribosomes are purified the RNA: protein (inset Fig. 2). The changes in hyperchromicity can be more ratio rises appreciably and approaches that of purified cyto- readily seen if one compares the differential changes in ab- plasmic ribosomes. The initial mitochondrial fractions con- sorbance with temperature (Fig. 3). Mitochondrial ribosomes tained 19 ug of RNA per mg of proteins. The first crude ribo- have maximal hyperchromatic change at 49.5°C, cytoplasmic qomal pellet was composed of 120 ,ug/ml protein. Purified ribosomes at 60.5°C. Lesser changes in hyperchromicity ribosomes contained 900 ug/ml protein-equivalent to 47% occur for mitochrondrial ribosomes at 58.5°C and for cyto- RNA, in contrast to 50% for cytoplasmic ribosomes. The plasmic ribosomes at 50.50C. A2a/Amo ratio of purified mitochondrial ribosomes is only Species of RNA in mitochondrial ribosomes slightly less (1.88) than that observed with cytoplasmic ribo- Table 1 summarizes the base composition of RNA extracted from pure cytoplasmic and mitochrondrial ribosomes. The G+C content of total cytoplasmic rRNA is 52.6%, in agree- ment with previous observations that both 28S and 18S rRNA from yeast contain approximately equal proportions of each . 3.0 E c nucleotide (9). In sharp contrast, total mitochondrial rRNA, 0 as well as the ribosomal subunits, contain approximately and I 2.0 equal proportions of purine pyrimidine bases but have considerably lower G+C contents (30.2-33.3%). These CD z findings provide, for the first time, proof that the bulk of the a) mitochondrial RNA previously isolated (11, 12) is ribosomal. o 1.0 I n In addition to differences in base composition, rRNA from pure mitochondrial and cytoplasmic ribosomes differ with respect to migration in acrylamide gels (Fig. 3). Mitochondrial o 10 20 30 rRNA species migrate more slowly than their cytoplasmic FRACTION NUMBER rRNA counterparts in 2.5% acrylamide gels. FIG. 1. Centrifugation of mitochondrial ribosomes (mono- Comparison of ribosomal proteins somes) on sucrose gradients. Crude mitochondrial ribosomes were layered on a 10-40% linear sucrose gradient and centri- Cytoplasmi9 and mitochondrial ribosomes differ in their RNA fuged at 23,000 rpm for 8 hr in a Spinco SW 25.1 rotor at 4°C. species (as well as in their physical properties). To further 30-drop fractions were collected by siphoning from the bottom of compare these particles, we extracted the predominant pro- the tube. tein species present in cytoplasmic and mitochondrial mono- Downloaded by guest on September 23, 2021 326 : Morimoto and Halvorson Proc. Nat. Acad. Sci. USA

TABLE 1. Base composition of ribosomral RNA some preparations depend on the purity of the mitochondrial fractions from which they were derived and the separation of Molar percent ratio ribosomes from other mitochondrial components. Source Species C A U G G+C The bulk of the RNA isolated from yeast mitochondria has s values characteristic of rRNA (25 S and 15 S). Assuming Cytoplasmic Total 26.0 22.6 24.7 26.6 52.6 these to be rRNA, we adopted the following criteria to moni- 18S 26.5 23.5 25.0 25.0 51.5 tor the isolation of "mitochondrial ribosomes": These 28S 25.9 24.1 22.1 27.8 53.7 ribosomes should contain RNA species analogous in (i) size Mitochondrial Total 12.5 36.1 34.5 17.7 30.2 and (ii) base composition to the major RNA species in mito- Small 12.9 33.3 36.4 17.4 30.3 chondria, and (iii) support bacterial-like protein synthesis. Large 16.1 32.6 34.0 17.2 33.3 In the present paper the first two of these criteria have been met for the isolation of ribosomes from yeast mitochondria. The ribosomal RNA subunits were separated by gel electro- Some of these data have been reported elsewhere (23). Orga- phoresis. The RNA, either total or extracted from the gels, was nelle ribosomes differ from cytoplasmic ribosomes in number of precipitated with alcohol, dissolved in 0.05 M ammonium acetate buffer (pH 5.5) containing 10-3 M EDTA, and then dialyzed (Table 2). Our findings that mitochondrial and against 0.05 M ammonium acetate buffer for 5 hr to remove cytoplasmic ribosomes of yeast sediment at 80 S is an agree- Mg++ ions and EDTA. RNA was reprecipitated with alcohol, ment with the value reported by Schmitt (21) and Vignais dissolved with NH4OAc buffer, and digested with T, and T2 et al. (24). In low concentrations of Mg++, both ribosomes RNase for 3 hr at 30'C. The digest was lyophilized overnight to dissociate into 60S and 40S subunits. Schmitt (21) reported remove NH4OAc. The RNA digest was then dissolved in 0.1 M that in high KCl concentrations, 6(S subunits were trans- phosphate buffer, pH 3.5, and the relative amount of each base formed into 50S particles. There are conflicting reports on the (with a standard error of ± 3%) was determined by measuring size of ribosomes from Neurospora mitochondria (13, 25). the area under each nucleotide peak eluted at 70'C by a linear ribosomes from Chlamydomonas (26) and tobacco 0.1-0.3 M in a gradient of potassium phosphate, pH 3.5, Picker (27) are 70 S. nucleotide analyzer. The size of ribosomal RNA is as yet unclear. In sucrose gradients and ultracentrifugation studies, the mito- somes and compared them by polyacrylamide gel electro- chondrial rRNA of yeast and Neurospora appear bacterial- phoresis (Fig. 4). Eighteen major bands were observed in like in size (Table 2). However, under various conditions of each preparation, which is probably a minimal estimate if we acrylamide gel electrophoresis, mitochondrial rRNA from remember the findings on bacterial ribosomes (22). Although yeast migrates more slowly than cytoplasmic rRNA. These most of the bands from both ribosomal preparations have differences could reflect conformational rather than molecular similar migration rates, six of the major mitochondrial pro- weight differences between the RNA species. Various factors, tein bands appear to have unique positions in which there are including the buffer used for RNA isolation and the ionic no counterpart bands from cytoplasmic ribosomal proteins. strength of the buffer used during electrophoresis are known to affect the rate of RNA migration. Differences have likewise DISCUSSION AND CONCLUSIONS been reported in the base composition, molecular weight, and Based on differences in the sensitivity to antibiotics of the thermal denaturation of cytoplasmic and mitochondrial rRNA protein-synthesizing activity, as well as the composition and in Aspergillus nidulans (29). properties of bulk mitochondrial RNA, it is probable that Ribosomes from mitochondria and cytoplasm clearly differ mitochondria from yeast contain ribosomes distinct from in a number of properties. First, in Neurospora (13) and in those in the cytoplasm. Isolation, and thereby characteriza- yeast (this paper), mitochondrial and cytoplasmic ribosomes tion, of mitochondrial ribosomes is complicated by the fact have different patterns of ribosomal proteins. Second, in that these ribosomes comprise a very small portion (less than yeast, mitochondrial rRNA has a lower G+C content than 5%) of the total in the . The characteristics of these ribo- does cytoplasmic rRNA. Finally, the melting profiles of the

TABLE 2. Comparison of ribosomes from various organisms

Yeast (21, 24) Neurospora (13, 25) Chlamydomonas (26) Cyto- Mito- Cyto- Mito- Cyto- Chloro- Component plasmic chondrial plasmic chondrial plasmic plast E. coli Ribosomes 80 80 77, 81 73, 81 80 68 70 Large subunit* 60 50, 60 60 50 47 34 50 Small subunit* 40 30, 38-40 37 37 31 29 30 rRNA Large RNA* 28 22-25 28 25 25.1 22.4 23 Small RNA* 18 15 18 19 17.6 16.7 16 % G+C 52.6 30.2 49.3-51.3 34-36 52.4 sensitivityt Cyc CAP Cyc CAP CAP

* S20,w- t Cyc = cycloheximide; CAP = chloramphenicol. Downloaded by guest on September 23, 2021 Vol. 68, 1971 Mitochondrial Ribosomes from Yeast 327

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-JI w a: C-, z 0 5 4 -I _ i < 0 TEMPERATURE m n 0Cn x co C-) 0~~~~~~~~~~~~~- -. 4 w l F- - -J SPLIT a 3 w GE. FIG. 4. 2

RELATIVE MIGRATION 0 FIG. 3. FIG. 3. Separation of mitochondrial and cytoplasmic rRNA I by gel electrophoresis. Approximately 25 ,g of each RNA was 2(0 30 40 50 60 70 electrophoresed in 2.5% acrylamide gels prepared in quartz TEMPERATURE tubes (18). The gels were directly scanned at 260 nm in a Cary 15 spectrophotometer. FIG. 2. Thermal transitions of cytoplasmic and mitochondrial FIG. 4. Polyacrylamide gel electrophoresis of mitochondrial ribosomes. Purified mitochondrial (0) and cytoplasmic (0) and cytoplasmic ribosomal proteins. Dialyzed LiCl-split ribosomes (A260 = 0.50) were placed in a 3-ml cuvette in a Beck- proteins of ribosomes were electrophoresed (16) for 5 hr. The man DU spectrophotometer with a Gilford attachment. Change gels were stained for 1 hr with amido black in 10% acetic , in A260 was followed as the temperature was raised 1°C per min. destained, and photographed. Differences n band patterns are A thermistor was placed in the cuvette to measure the tempera- indicated by the arrows. ture accurately. The differential change of absorbance with temperature was calculated as (A T,-ATJ )/AT. ribosomes and factors, it is now possible to determine directly the molecular nature of the antibiotic mutants. Such experi- two ribosome are different. The conformational preparations ments are in progress. changes accompanying thermal denaturation of ribosomes reflect both protein-RNA interactions and RNA composition This investigation was supported by grants from The National (28). Science Foundation (GB 6993X), U.S. Public Health Service Although the sedimentation characteristics of mitochondrial research grant (AI-01459), National Institute of Health Training ribosomes differ from those from bacteria, both have similar Grant (GM-01874), and a National Institute of Health Fellow- ship (CA-18,203). One of us (H. 0. H.) is the recipient of a stability toward Mg++. In general, organelle ribosomes re- National Institutes of Health Research Career Professorship. quire a higher Mg++ concentration for their stability than their cytoplasmic counterpart (13, 25, 27). 50S subunits from 1. Ephrussi, B., Nucleocytoplasmic Relations in Microorga- nism (Clarendon Press, Oxford, 1953). Neurospora mitochondrial ribosomes (13) are sensitive to 2. Wilkie, D., G. W. Saunders, and A. W. Linnane, Genet. chloroamphenicol; this is similar to the behavior of 50S Res., 10, 105 (1967). ribosomal subunits from bacteria. Both Neurospora (13) and 3. Wintersberger, E., and G. Viehhauser, Nature, 220, 699 yeast cytoplasmic cell-free protein-synthesizing systems are (1968). 4. M. and C. Mol. Gen. inhibited whereas the mitochondrial sys- Fukuhara, H., Faures, Genin, Genet., by cycloheximide, 104, 264 (1969). tems are sensitive to chloroamphenicol. 5. Bretthauer, R. K., L. Marcus, J. Chaloupka, and H. 0. Studies indicate that fMet-tRNA, some of the T-factors, Halvorson, Biochemistry, 2, 1079 (1963); So, A. G., and E. W. Mg++ requirements, and antibiotic sensitivities are common Davie, Biochemistry, 2, 132 (1963); Halvorson, H. O., and C. to both the bacterial and mitochondrial protein-synthesizing Heredia, Aspects of Yeast , ed. A. K. Mills (Blackwell Sci. Pubs, Oxford, 1968), p. 107. systems. Various antibiotic-resistant mutants that show 6. Huang, M., D. R. Briggs, D. G. Clark-Walker, and A. W. cytoplasmic inheritance have been reported in yeast (2). With Linnane, Biochim. Biophys. Acta, 114, 434 (1966). the present improvements in the isolation of mitochondrial 7. Adams, J. M., and M. R. Capecchi, Proc. Nat. Acad. Sci. Downloaded by guest on September 23, 2021 328 Genetics: Morimoto and Halvorson Proc. Nat. Acad. Sci. USA

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