The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

Biochimica etBiophysicaActa, 781 (1984) 153-164

153

Elsevier BBA91314

T H E F O R M A T I O N OF A DEFECTIVE SMALL SUBUNIT O F T H E M I T O C H O N D R I A L R I B O S O M E S IN P E T I T E M U T A N T S OF S A C C H A R O M Y C E S CEREVISIAE KEWAL K. MAHESHWARI and SANGKOT MARZUKI Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia)

(Received September20th, 1983)

Key words: Varl protein," Ribosome assembly," Mitochondrial biogenesis; Protein synthesis; Petite mutant; (S. cerevisiae)

The involvement of mitochondriai protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of S a c c h a r o m y c e s cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes a n d / o r one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the varl and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the varl protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.

Introduction Although the bulk of the mitochondrial proteins are imported from the extra-mitochondrial cytoplasm, the development of functional mitochondria is dependent on the synthesis within the mitochondria of a small number of inner membrane proteins. The structure of the mitochondrial D N A which codes for these proteins is now well characterized. The control mechanism that regulates the expression of the genetic information encoded in the mitochondrial genome, however, is still poorly understood. In particular, very little is known about the assembly process involved in the 0167-4781/84/$03.00 © 1984 ElsevierSciencePublishers B.V.

formation of functional mitochondrial ribosomes which are responsible for the synthesis of the mitochondrially coded proteins, and the mechanism which regulates this assembly process. The focus of the present study is a polymorphic mitochondrial translation product ( M r in different strains of yeast varies between 40 000 and 44 000) [1] which might be an important component of the mitochondrial ribosomal assembly process. This protein, which has been shown to be associated with the small subunit of the mitochondrial ribosome [2,3], is the only protein component of the ribosomes which is synthesized in the mitochondria. Thus, while the two mitochondrial rRNAs

154

are coded for by the mitochondrial DNA (mtDNA) (see Re/. 4), the ribosomes are assembled almost exclusively from protein components which are synthesized in the extra mitochondrial cytoplasm. The varl protein, therefore, might play an important role in the regulation of the mitochondrial protein synthesis, as the only protein product of the organelle genetic system which can influence the assembly of the mitochondrial ribosomes. The involvement of the varl protein in the assembly of the mitochondrial ribosomes can be studied by investigating whether mitochondrial protein synthetic activity is essential for the assembly process. The most commonly employed approach to specifically inhibit the mitochondrial protein synthesis is to use an antibiotic, such as erythromycin or chloramphenicol, which do not affect the cytoplasmic protein synthesis [5]. However, this approach has two major drawbacks in that (a) it is difficult to ensure complete inhibition, and (b) the possibility of side-effects cannot be eliminated. For these reasons, we have investigated the involvement of the mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes, by analysing the extent to which this assembly process can proceed in petite strains, which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes a n d / o r one of the rRNA genes on the mtDNA. A group of three petite strains was used. Two of these strains retain only one of the rRNA genes: strain Y6 containing the 15-S rRNA gene and petite Y1.5 containing the 21-S rRNA gene. The third petite, strain Y8, has previously been described in a preliminary report [6] and contains both the 15-S and 21-S rRNA genes. Results presented in this communication show that mitochondrial protein synthesis is essential for the assembly of the 37-S small mitochondrial ribosomal subunit. Thus, although the 15-S rRNA was found to be synthesized in petite strains which retain the rRNA gene, no small ribosomal subunit could be detected in these strains. Instead, a novel ribonucleoparticle, sedimenting at 30 S, was observed. This ribonucleoparticle contains most of the proteins of the small ribosomal subunit.

Materials and Methods

Yeast strains and growth conditions The respiratory competent (rho +) strain of the yeast S. cerevisiae used in this study were strain J69-1B a adel his and strain Y a ura[capl-r eryl-r olil-r anal-rparl-r]. Petite strains Y6, Y1-5 and Y8 were spontaneous isolates from strain Y [7]. Respiratory competent cells were grown aerobically in batch cultures at 28°C in 500 ml or in 11 litre medium containing 1% (v/v) ethanol, 1% ( w / v ) yeast extract, a salt mixture [8] and their auxotrophic requirements (100/~g adenine/ml and 50 /~g histidine/ml for strain J69-1B and 25 ~tg uracil/ml for strain Y). Respiratory-deficient petite cells were grown in glucose-limited chemostat cultures under catabolite-derepressed conditions as described previously [9]. The working volumes of the cultures were either 500 ml (in modified LKBBiotec polyferm fermentors) or 4 li.tre (in LKB Ultraferm fermentation system). The growth medium used contained 1% (w/v) yeast extract, 25 ~ g / m l uracil, a salt mixture [8] and 2% (w/v) glucose as a carbon source. The chemostat cultures were inoculated with logarithmically growing cells and then maintained at 28°C for approx. 45 h at a dilution rate of 0.05 h-~. Under these conditions, the steady-state glucose level of the cultures was maintained below that which induces catabolite repression [9]. Isolation of rnitochondria Mitochondria were isolated from spheroplasts formed by partial digestion of the cell wall with the zymolyase enzyme essentially as described previously [10]. Spheroplasts were resuspended in a 6.5 mM Tris-HC1 buffer (pH 7.4) containing 330 mM mannitol, 270 mM sorbitol and 10 mM EDTA and ruptured in a French pressure cell at 30-40 MPa. Disrupted spheroplasts were diluted with the above buffer and unbroken cells and cell debris were removed as previously described [10]. Mitochondria were then collected by centrifugation at 14000 x g for 10 min in Sorvall SS-34 rotor, and washed three times in the same buffer. The washed mitochondria were freed of EDTA by resuspending in a 6.5 mM Tris-HC1 buffer (pH 7.4) containing 330 mM mannitol, 270 mM sorbitol and 2 mM magnesium acetate, and collecting

155

the mitochondria by centrifugation at 14000 × g for 10 min. All the steps were performed at 0-4°C except for the incubations prior to spheroplast formation.

Isolation of mitochondrial ribosomes and ribosomal subunits Freshly prepared mitochondria were resuspended at a final concentration of 6 mg protein/ml in 10 mM Tris-HC1 buffer (pH 7.4), containing 10 mM magnesium acetate, 500 mM NH4CI and 1 mM dithiothreitol (buffer 1) and were solubilized by the addition of one-tenth volume of 10% Triton X-lO0. The mitochondrial lysate was clarified by centrifugation at 27 000 × g for 20 rain and layered directly on a 1.4 M sucrose pad in buffer 1. The pad represents 40% of the nominal tube volume. The ribosomes were collected by centrifugation at 250000 × g for 15 h at 4°C in a Beckman 70.1 Ti rotor. The gelatinous pellet was rinsed with buffer 2 (essentially the same as buffer 1 except that the concentration of NH4C1 was 50 mM instead of 500 raM) and resuspended by homogenization in the same buffer. The suspension was centrifuged at 12000 × g for 10 min to yield a low-speed pellet and supernatant. The pellet was resuspended in a small volume of buffer 2 and centrifuged as above. The two supernatant fractions were combined and further clarified by centrifugation at 27 000 × g for 10 min. This ribosomal preparation was snapfrozen in small aliquots in solid CO2/acetone and stored at -70°C. The concentration of ribosomes was estimated by determining its absorbance at 260 nm and multiplying the absorbance with a factor of 0.06 [11]. Mitochondrial ribosomal subunits were isolated and analysed on sucrose density gradient as described previously [6]. Isolation of cytoplasmic ribosomal subunits For the isolation of cytoplasmic ribosomes, spheroplasts were disrupted in 6.5 mM Tris-HC1 buffer (pH 7.4) containing 330 mM mannitol, 270 mM sorbitol and 0.6 mM EDTA. Postmitochondrial supernatant was collected and clarified by centrifugation at 30000 × g for 20 min. Ribosomes were then collected by centrifugation at 170000 × g for 2 h and resuspended in buffer 2. The suspension was clarified by centrifu-

gation at 27000 × g for 20 rain and ribosomes were again collected at 170 000 x g for 2 h. The resulting transparent ribosomal pellet was resuspended in a small volume of buffer 2 and stored at - 7 0 ° C in small aliquots. Cytoplasmic ribosomal subunits were isolated by resuspending the cytoplasmic ribosomal pellet in buffer I (2-4 A260 units/ml), and separating the two subunits in 15-30% (w/v) sucrose density gradients in buffer 1 [6].

Amino acid incorporation by isolated mitochondrial ribosomes The activity of mitochondrial ribosomes and ribosomal subunits was determined in a polyuridylic acid (poly(U)) directed translation system. The reaction mixture (100 #1) contained 50 mM Tris-HCl (pH 7.4), 20 mM magnesium acetate, 1 mM ATP, 5 mM phosphoenolpyruvate, 0.7 mg poly(U)/ml (ammonium salt), 0.015-0.25 mM L[2,4,6-3H]phenylalanine (spec. act. 40-160 #Ci/mmol), 0.015 mM each of other amino acids, 0.5-0.7 mg ribosomes/ml, and 40 #1 E. coli S-100 supernatant fraction (prepared from strain MRE 600 as described by Modolell [11] except that the S-100 supernatant was desalted by passing through a Sephadex G-25 column instead of dialysis). Chloramphenicol, cycloheximide or puromycin was added to the antibiotic control tubes to a final concentration of 0.5 mM. The reaction was started by incubating the tubes at 34°C and was terminated at intervals as indicated by the addition of 7% trichloroacetic acid containing 2 mg phenylalanine per ml (trichloroacetic acid/ phenylalanine). The tubes were kept at 4°C overnight and the precipitates were collected by centrifugation. Precipitates were washed with trichloroacetic acid/phenylalanine twice at room temperature and twice with incubation at 60°C for 10 rain each. The precipitates were solubilized in 0.25 ml of NCS (Amersham, IL), and the radioactivity incorporated was determined in a liquid scintillation counter. Estimation of total mitochondrial RNA contents RNA was extracted from washed mitochondria and estimated essentially as described by Ceriotti [12] using D-ribose as the reference pentose. mtRNA contents were calculated, and correction

156

was made for any contamination from cytoplasmic ribosomes. The extent of contamination was calculated from the guanine plus cytosine contents of the extracted RNA, assuming that the guanine plus cytosine content of mitochondrial RNA is 28% and that of cytoplasmic RNA is 47% [13]. Nucleotide analysis was performed on a small volume of alkaline hydrolysate of the RNA, adjusted to pH 4.5-5.0 with dilute H3PO 4. Any precipitates formed were removed by centrifugation and a sample containing 5-10 #g nucleotides was injected onto a reverse-phase C-18 HPLC column (Waters Assoc., MA, U.S.A.). The nucleotides were eluted by using a pH gradient of sodium phosphate (pH 5.0-6.5) at a flow rate of 3 ml/min, over 20 min. Absorbance was recorded at 254 nm.

Analysis of mitochondrial rRNA Mitochondrial RNA was extracted from twicewashed mitochondria [14] and analysed by electrophoresis on agarose-urea gels as described previously [21]. Polyacrylamide gel electrophoresis of ribosomal subunit proteins Ribosomal subunit proteins were analyzed by one- or two-dimensional gel electrophoresis. For one-dimensional gel analysis, ribosomal subunit proteins were precipitated with 5% trichloroacetic acid and electrophoresed on 11% polyacrylamide gel (24 x 16 x 15 cm) in the presence of SDS as described previously [15]. The ribosomal proteins were stained with Coomassie brilliant blue R-250. Two-dimensional polyacrylamide gel electrophoretic analysis of the ribosomal subunit proteins was essentially as described by Mets and Bogorad [16], except that the proteins were visualized by a silver base color staining procedure [17]. Protein cooncentration was estimated by the method of Gornall et al. [18] or Lowry et al. [19] using bovine serum albumin as standard. Results

Analysis of rRNA species present in mitochondria of the petite mutant strains Detailed physical and genetic maps of the mitochondrial DNA retained in petite strains used in the present study have previously been de-

scribed [7, 20] The mitochondrial DNA of the petite strain Y6 is approx. 28 kb long and retains the 15-S rRNA gene (Fig. 1). The mtDNA of petite strain Y1.5 is approx. 27 kb long and retains the 21-S rRNA gene. The mtDNA of the petite strain Y8, which is approx. 27 kb long retains the genes for both the 15-S and 21-S rRNA, but has lost the oar1 and some tRNA genes (Fig. 1). To determine whether these petite strains can synthesize the mitochondrial rRNA, total RNA was extracted from isolated mitochondria and analysed by electrophoresis on 1.5% agarose gels containing 6 M urea [21]. Fig. 2 shows that both the 15-S and 21-S rRNAs are present in mitochondria of petite strain Y8. Mitochondria from petite strain Y6 and Y1.5 contain only the

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Y6 Fig. 1. Mitochondrial D N A retained by petite strains used in the present study. Petite strains Y6, Y1.5 and Y8 have previously been characterized by genetic and physical mapping of their mitochondrial genome for the retention of mitochondrial genetic loci, and for segments of mitochondrial D N A retained [7,20]. Data from the previous reports were used to construct the present map. 01il and oli2 are structural genes for ATPase subunits 6 and 9, respectively; oxil, 2 and 3 are structural genes for cytochrome oxidase subunits II, III and I; cyb is the structural gene for the cytochrome b apoprotein; eryl; capl and parl are antibiotic resistance loci for erythromycin, chloramphenicol and paromomycin, respectively. The heavy solid lines indicate the portions of the mitochondrial genome retained by the above petites.

157

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extracted with Triton X-100, and analysed on 15-30% (w/v) sucrose linear density gradient. No small subunit (37 S) was detected in petite Y8 (Fig. 3), which contained a significant amount of the large subunit (54 S). Instead a new particle with a sedimentation coefficient slightly lower than that of the small subunit was observed. Similarly, when petite strain Y6 (which retains the 15-S

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Fig. 2. Electrophoretic analysis of mitochondrial rRNA from petite strains. Mitochondrial RNA extracts from twice-washed mitochondria of wild-type strain J69-1B and petite strains Y8, Y1.5 and Y6 were electrophoresed in a 1.5% agarose slab gel containing 6 M urea. The gel was stained with ethidium bromide and examined under ultraviolet light. The staining patterns shown are as follows: Lane a, strain J69-1B; lane b, strain Y8; lane c, strain Y1.5; lande d, strain Y6. The positions in the gel of the 21-S and 15-S mitochondriai rRNAs, the contaminating large and small cytoplasmic rRNAs (Cyto L and Cyto S) and m t D N A are indicated. The positions of missing rRNA bands in the petite samples are indicated by arrows.

15-S rRNA and the 21-S rRNA, respectively. This observation is in agreement with previous reports that petites retaining one or more rRNA genes can synthesize the respective rRNA species [22,23]. Petite stratus retaining the gene for the 15-S rRNA lack the small mitochondrial ribosomal subunit

To determine whether the mitochondrial rRNAs in the petite strains can be assembled into mature ribosomal subunits, mitochondrial ribosomes were

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Fig. 3. Analysis of mitochondrial ribosomal subunits in petite strains Y8, Y1.5 and Y6. Cells were grown under catabolite-derepressed conditions in glucose-limited chemostat cultures. R i b o s o m e s were extracted from three-times-washed mitochondria (3 mg protein) and analyzed on 15-30% (w/v) linear sucrose density gradients as described in Materials and Methods. The absorbance of the gradient contents was recorded at 254 nm by their passage through a Uvicord I spectrophotometer. Traces of gradient profiles of strain J69-1B, strain Y8, strain Y1.5 and strain Y6 mitochondria are shown, as well as of strain Y8 mitochondria mixed with cytoplasmic ribosomes from strain J69-1B. The direction of sedimentation is from left to right. The sedimentation coefficient of the new 30-S particle's peak observed in petite strain Y8 and Y6 was determined by comparison with those of E. coli ribosomal subunits (30 S and 50 S) which were analyzed in parallel or mixed gradients. The vertical line indicates the position for 30 S. A small amount of contamination by the cytoplasmic ribosomes can be observed in all petite mitochondrial preparations.

158 r R N A gene) was analysed, the 37-S subunit was also found to be absent, and the new particle was again observed (Fig. 3). The absence of the 54-S subunit in this strain was due to the fact that petite strain Y6 did not contain the 21-S r R N A gene. The difference between the sedimentation coefficient of the new particle and that of the small ribosomal subunit is significant. Thus while the mitochondrial and the cytoplasmic small ribosomal subunits (37 S and 40 S, respectively) run as a single peak when the cytoplasmic ribosome is used as an internal standard (data not shown), the new particle in petite strain Y8 is clearly separated f r o m the cytoplasmic small subunit (Fig. 3). The sedimentation coefficient of the new particle was estimated to be about 30 S, using the subunits from the yeast cytoplasmic ribosome (40 S and 60 S) and E. coli ribosome (30 S and 50 S), run in parallel gradients, as standards. The absence of mitochondrial protein synthesis does not appear to have any effect on the asembly of the large subunit (54 S), since this subunit is present in petite Y8. Furthermore, the petite strain which retains the gene for the 21-S r R N A (Y1.5) contains the large mitochondrial ribosomal subunit which is indistinguishable from the large subunit in the wild-type strain on the basis of its sedimentation coefficient (Fig. 3). Neither the 30-S particle or the mitochondrial ribosomal subunits (37 S and 54 S) were observed when a petite strain lacking both the 15-S and 21-S r R N A genes was analysed (data not shown), indicating that the accumulation of the 30-S particle occurs only in petite strains that retain the 15-S r R N A gene. Is the petite 30-S particle related to the 37-S small subunit? The concurrent appearance of the new 30-S particle with the loss of the 37-S ribosomal subunit in petites which retain the 15-S r R N A gene suggests that the particle sedimenting at 30 S is related to the small mitochondrial ribosomal subunit. For instance, this 30-S particle m a y be a p r o d u c t of defective assembly of the small subunit (37 S) or a precursor to the small subunit which is partially assembled in the absence of mitochondrial protein synthesis. To investigate these possibilities, the 30-S particle and the small and large ribosomal

subunits were isolated. Initially, these subunits were analysed by polyacrylamide gel electrophoresis in the presence of SDS (Fig. 4). However,

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Fig. 4. Analysis of mitochondrial ribosomal subunit proteins by polyacrylamide gel electrophoresis in the presence of SDS. Purified mitochondrial ribosomal subunits (37 S and 54 S) from strain J69-1B, the 30-S ribonucleoparticle from petite strain Y8 and cytoplasmic subunits from strain J69-1B were isolated as described in Materials and Methods. About 0.2 mg protein of each ribosomal subunit and 0.2 mg protein of mitocondria. labeled with [ 35S]sulphate in vivo for mitochondrial translation products [15], were precipitated with 5% trichloroacetic acid, solubilized and electrophoresed in an 11% polyacrylamide gel in the presence of SDS [15]. Labelled mitochondrial protein bands were detected by autoradiography and ribosomal subunit proteins were visualized by Coomassie blue staining. Displayed are: (a) mitochondrial translation products from strain J69-1B; (b) 40-S small cytoplasmic ribosomal subunit from strain J691B; (c) 37-S small mitochondrial ribosomal subunit from strain J69-1B; (d) 30-S particle from petite strain Y8; (e) 54-S large mitochondrial ribosomal subunit from straifi J69-1B; (f) 60-S large cytoplasmic ribosomal subunit from strain J69-1B.

159 although the gel pattern suggests similarities between the 30-S particle and the 37-S subunit, no firm conclusion can be made from this analysis due to the limited resolution of the one-dimensional gel system. In order to increase the resolution, the 30-S particle was analysed by two-dimensional gel electrophoresis. In the first dimension, proteins were separated on the basis of their net charge in a low concentration of acrylamide (4%), and in the presence of a high concentration of urea (8 M) to prevent the proteins from aggregating. After separtion in the first dimension the proteins were then electrophoresed in the presence of SDS into a polyacrylamide slab gel to separate further the proteins on a molecular weight basis. It is apparent from Fig. 5 that the two-dimensional gel protein pattern of the 30-S particle obtained from the petite strain Y8 (Fig. 5c) is

similar to that of the 37-S mitochondrial ribosomal subunit (Fig. 5a). With the exception of 3 - 5 proteins (including the varl protein), the 30-S particle appears to contain most of the proteins of the small subunit. In addition, however, 3 - 4 proteins which are not c o m p o n e n t s of the small subunit were also observed in the 30-S particle preparation. These proteins might be contaminants which co-purify with the 30-S particle. The possibility cannot be excluded, however, that some of the proteins might be involved in the assembly of the small subunit but are associated only transiently with this subunit at certain stages of its assembly. The two-dimensional pattern of the 30-S particle proteins has no resemblance to that of the larlge mitochondrial ribosomal subunit (Fig. 5b) or those of the cytoplasmic subunits (Fig. 5e and g). Furthermore, when a mixture of the 30-S particle and the 37-S subunit was analysed by

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Fig. 5. Analysis of mitochondrial ribosomal subunit proteins by two-dimensional polyacrylamide gel electrophoresis. Purified ribosomal subunits from sucrose gradients were separated by two-dimensional gel electrophoresis as described in Materials and Methods. Separation in the first dimension was in 4~ acrylamide and in the presence of 8 M urea (from left to right), and in the second dimension in 11% polyacrylamide containing 0.1% SDS (from top to bottom) [16]. Proteins were visualized by silver-based staining [17]. Molecular weights were determined by running standard proteins (bovine serum albumin, ovalbumln, aldolase, chymotrypsinogen and cytochrome c) in a parallel track in the second dimension. The varl protein is indicated by an arrow ( *- ) and was identified by comparison with the mobility of varl protein. A mitochondrial sample, labelled in vivo with [35S]sulphate for mitochondrial translation products, was used for this purpose. The absence of the varl protein is indicated by an open circle (O) around its inferred position. Displayed are: (a) small ribosomal subunit (37 S) from strain J69-1B; (b) large ribosomal subunits (54 S) from strain J69-1B; (c) 30-S ribonucleoparticle from petite strain Y8; (d) large ribosomal subunit (54 S) from petite strain Y8; (e) small cytoplasmic ribosomal subunit (40 S) from strain J69-1B; (f) 30-S ribonucleoparticle mixed with small ribosomal subunit (as above a and c); (g) large cytoplasmic ribosomal subunit (60-S) from strain J69-1B' (h) RNAases A + T1 (blank).

160

two-dimensional gel electrophoresis (Fig. 5f), all the 30-S particle proteins co-migrated with the small subunit proteins, with the exception of the 30-S particle proteins, which are not found in the 37-S small subunit. When the two-dimensional protein patterns of the 54-S subunit from petite strain Y8 (Fig. 5d) and from wild-type strain J69-1B (Fig. 5b) are compared, they appear to be identical. This finding supports the conclusion of the earlier section that mitochondrial protein synthetic activity is not required for the assembly of the large ribosomal subunit.

Quantitation of the 30-S ribonucleoparticle and the 54-S subunit in petite mitochondria When several gradient profiles of mitochondrial ribosomal subunits from the petite strains were examined and quantitated, two interesting phenomena became apparent. First, it was found that the amount of the 30-S ribonucleoparticle in petite strain Y8 is always slightly lower than the amount of the small subunit in strain J69-1B. Thus the amount of the 30-S particle in strain Y8 was found to be about 0.18 A260/mg mitochondrial protein, which is about 80% of the amount of the small subunit (37 S) present in mitochondria of a wildtype strain (Table I). The amount of 30-S particle in petite strain Y6 is lower than that of petite Y8 and varied from 0.12 to 0.16 A260/mg mitochondrial protein (Table I).

The significance of the above observation is not known, but one possibility is that it might simply be due to the fact that the mitochondria from petite strains are more fragile than those from the wild-type strains, resulting in the loss of the 30-S particle during the isolation of mitochondria due to the high concentration of EDTA in the buffer used. Alternatively, it is also possible that the 30-S particle is more sensitive to degradation by RNAases or proteinases. It was found that, when mitochondria from petite strain Y8 were stored for 5 - 7 days at - 2 0 ° C , the amount of the 30-S particle observed in the sucrose gradient profiles was reduced by 50%. Storage for a longer period can lead to a complete loss of the 30-S particle. In the present study, fresh petite mitochondria were always used for the analysis of the 30-S particle. Close examination of ribosomal profiles of the large mitochondrial ribosomal subunit in both petite strains Y8 and Y1.5 indicates that these strains contain significantly larger amounts of the 54-S subunit as compared to the wild-type strain used (Table I). The increase in the amount of the large subunit in petite strains Y8 and Y1.5 was reflected in the RNA contents of their mitochondria (Table II). Thus, while the mitochondria isolated from the wild-type strain contained about 36 /zg R N A / m g protein, those of the petite strains Y8 and Y1.5 contained 68 and 60 #g R N A / m g protein, respectively. TABLE II ESTIMATION OF RNA IN M I T O C H O N D R I A ISOLATED F R O M WILD-TYPE A N D PETITE STRAINS

TABLE I Q U A N T I T A T I O N OF M I T O C H O N D R I A L RIBOSOMAL SUBUNITS FROM PETITE STRAINS Results are expressed as the mean + S.D. of between three and seven independent determinations for strains J69-1B, Y1.5 and Y8. Results for strain Y6 are of two independent determinations. Strain

J69-1 B Y6 Y1.5 Y8

Amounts of mitochondrial ribosomal subunits (A 26o/3 mg mitochondrial protein) Small subunit or 30-S ribonucleoparticle

Large subunit

0.22 _+0.02 0.12, 0.16 0.18 + 0.02

0.40 + 0.02 0.51 + 0.02 0.49 + 0.02

Mitochondria were isolated from cells grown under catabolitederepressed conditions. The RNA contents of the mitochondria were determined as described in Materials and Methods. The degree of contamination by cytoplasmic RNA was calculated from the guanine plus cytosine (G + C) content of the RNA assuming that the G + C content of mitochondrial RNA is 28% and that of cytoplasmic RNA is 47% [13]. Strain

J69-1B Y6 Y1.5 Y8

%G + C

29 33 31 31

RNA content of mitochondria ( # g R N A / m g mitochondrial protein) Total

Corrected for cytoplasmic contamination

39.8 48.8 67.8 77.3

36.4 43.4 60.0 68.0

161

The 30-S ribonucleoparticle from petite Y8 is defective in poly(U) translation Petite strain Y8 has no in vivo a n d in vitro m i t o c h o n d r i a l p r o t e i n synthetic activity, p r e s u m a b l y b e c a u s e of the d e l e t i o n of some of the t R N A genes (Fig. 1). In o r d e r to d e t e r m i n e w h e t h e r the 30-S particle has a n y biological activity, the activity of the r i b o s o m a l p r e p a r a t i o n from petite Y8 in p o l y ( U ) t r a n s l a t i o n was d e t e r m i n e d . A n E. coli S-100 s u p e r n a t a n t fraction was used as a source of t R N A s a n d o t h e r catalytic factors, A s shown in Fig. 6, the m i t o c h o n d r i a l r i b o s o m a l p r e p a r a t i o n f r o m strain Y8 i n c o r p o r a t e d [ 3 H ] p h e n y l a l a n i n e i n t o trichloroacetic a c i d - p r e c i p i t a b l e m a t e r i a l at a

m u c h lower rate (770 d p m / m g r i b o s o m e s p e r min) as c o m p a r e d to that f r o m the w i l d - t y p e strain J69-1B (7670 d p m / m g r i b o s o m e s p e r rain). It is not clear at present w h e t h e r the low rate of [ 3 H ] p h e n y l a l a n i n e i n c o r p o r a t i o n observed is d u e to the activity of the Y8 r i b o s o m e s or to some c o n t a m i n a t i o n f r o m c y t o p l a s m i c ribosomes. It is also not k n o w n f r o m the a b o v e o b s e r v a t i o n w h e t h e r the a p p a r e n t i n a b i l i t y of the petite ribos o m a l p r e p a r a t i o n to translate p o l y ( U ) is due to a defect in a small s u b u n i t only or whether the 54-S s u b u n i t is also inactive. To investigate this, the 30-S particle a n d the large s u b u n i t (54 S) f r o m p e t i t e strain Y8 were r e c o n s t i t u t e d with the large

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Fig. 6. The ability of ribosomal preparation from mitochondria of petite strain Y8 to translate poly(U). The biological activity of mitochondrial ribosomal preparation from petite strain Y8 was measured by its ability to translate poly(U) using E. coli S-100 supernatant as a source of tRNA and other factors needed. The assay was performed as described in Materials and Methods. The incorporation of [3H]phenylalanine is sensitive to inhibition by puromycin, and is dependent on the presence of added ATP and on S-100 supernatant. Plotted are the incorporations of [3H]phenylalanine in the presence of: (a) 74-S ribosome preparation from strain J69-1B, (b) ribosomal preparation from petite strain Y8, (c) no ribosomes.

Fig. 7. The ability of petite mitochondriai ribosomal subunits to support poly(U) translation in reconstituted ribosomes, q~ze

activity of mitochondrial ribosomal subunits in petite strain Y8 (30-S ribonucleoparticle and 54-S large subunit) was measured by their ability to translate poly(U) in a reconstituted ribosome. The assay was performed as described in Materials and Methods. Incorporation of [3H]phenylalanine is sensitive to inhibition by puromycin, and is dependent on the addition of ATP and S-100 supernatant. Plotted are the incorporations of [3H]phenylalanine by: (a) 74-S ribosome preparation from strain J69-1B, (b) 37-S plus 54-S subunits from strain J69-1B, (c) 54-S subunits from strain Y8 plus 37-S subunit from strain J69-B, (d) 30-S particle from Y8 plus 54-S subunit from strain J69-1B, (e) 37-S subunit alone from strain J69-1B, (f) 54-S subunit alone from strain J69-1B.

162 subunit (54 S) and the small subunit (37 S) from the wild-type strain J69-1B, respectively. When the 30-S particle from petite Y8 was reconstituted with the large subunit from strain J69-1B, no significant incorporation of [3H]phenylalanine was observed (Fig. 7), indicating that the 30-S particle is functionally defective. In contrast, a significant amount of [3H]phenylalanine incorporation was observed when the 54-S subunit from strain Y8 was reconstituted with the 37-S subunit from strain J69-1B. The rate of incorporation was similar to that of ribosomes reconstituted from subunits isolated from the wild-type strain J69-1B, but slightly less than that of the mitochondrial ribosomal preparation. This result further shows that the assembly of a functional large mitochondrial ribosomal subunit does not require any product of the mitochondrial protein synthesis. Discussion

Assembly of the 15-S rRNA into a 30-S ribonucleoparticle in the absence of mitochondrial protein synthesis In this paper evidence is presented which shows that petite deletion mutants of S. cerevisiae which retain the gene for the 15-S rRNA do not contain a small mitochondrial ribosomal subunit. Instead, a novel ribonucleoparticle which has a sedimentation coefficient of 30-S in sucrose density gradients was observed. These results contradict an earlier report [24] which claimed that petite ,mutants retaining the 15-S rRNA gene contain a normal small ribosomal subunit, and that no mitochondrial translation product is required for the assembly of this subunit. However, in the absence of the large subunit and any other internal markers in sucrose gradients used to analyse the petite mitochondrial extracts in the previous study [24], it is difficult to ascertain whether the subunit observed was indeed the normal small subunit (37-S) and not the 30-S ribonucleoparticle reported in this communication. A similar investigation into the role of the mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes has been reported in which mitochondrial protein synthesis was inhibited by growing a wild-type strain of yeast in

the presence of erythromycin, at a concentration which could be shown to inhibit mitochondrial protein synthesis almost completely [25]. It was shown that under this growth condition no small mitochondrial ribosomal subunit can be observed. Results presented in the present communication are in agreement with those of the above study in so far as both studies indicate that a product of mitochondrial protein synthesis, presumably varl, is needed for the assembly of the small ribosomal subunit. However, the 30-S particle has not been reported previously. The reason for the failure to detect the 30-S particle in erythromycin-grown cells in the previous study [25] is probably due to the effects of catabolite repression on the yeast cells used in this study. We have carried out a similar experiment by using a strain related to that employed by Terpstra and Butow [25] (strain 5DSS) and grown under conditions described in their report (batch cultures in the presence of 2% galactose as an energy source, and 2 m g / m l erythromycin). Under these conditions, we could not find any noticeable amounts of the 30-S particle. This particle can be observed when strain 5DSS was grown in glucoselimited chemostat cultures in the presence of the antibiotic. The details of these experiments will be published elsewhere. Our results indicate that the 30-S particle is a defective small subunit which is formed in the absence of mitochondrial protein synthesis. When the 30-S particle isolated from petite strain Y8 was analyzed by two-dimensional gel electrophoresis, all the protein components of the small subunit were present with the exception of 3-5 proteins (including varl). It is possible that these missing proteins are the last to enter the assembly pathway of the small subunit, and these final steps of assembly cannot proceed in the absence of varl protein. Alternatively, some of these proteins might have been lost during extraction and the isolation of the 30-S particle. The absence of the varl protein may result in some proteins being only loosely associated with the small subunit, or in the instability of this subunit. Our results are in agreement with the observation in Neurospora crassa [26] that a product of the mitochondrial translation system designated S-4a is involved in the assembly of the small subunit of

163 the mitochondrial ribosomes. Thus, when Neurospora cells were treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of the small subunit (30 S in Neurospora) was found to be rapidly inhibited. As a result, there was an accumulation of a ribosomal particle (CAP-30S) which sediments slightly behind the mature small subunit. Electrophoretic analysis suggests that the CAP-30S particle was deficient in several proteins, including S-4a. It was further suggested that the CAP-30S particle is enriched in a precursor rRNA species which is slightly longer than mature 19-S rRNA (small subunit rRNA) [27]. Recently, Osinga et al. [28] reported that in a wild-type strain of S. cerevisiae mature 15-S rRNA is derived from a 15.5-S RNA transcript by the removal of approximately 80 nucleotides at the 5' end. In vlvo processing of the 15.5-S RNA transcript appears to be restricted in a petite strain which retains the 15-S rRNA gene [29,28]. It has not been established in the present study whether the RNA species observed in petites Y6 and Y8 is the 15.5-S RNA precursor or the mature 15-S rRNA. When RNA extracted from mitochondria of petites Y6 and Y8 was analysed on agarose-urea gels, the small subunit species of the mitochondrial rRNA appeared to have a mobility which in our gel system was indistinguishable from that of the mature 15-S rRNA.

Assembly of the large ribosomal subunit in the absence of mitochondrial protein synthesis The results of the present study clearly show that there is no involvement of the mitochondrial protein synthesis in the assembly of the mitochondrial large ribosomal subunit (54 S). The large subunit from petite strain Y8 is indistinguishable from that of the wild-type strain on sucrose density gradients and they have identical protein patterns on two-dimensional gel electrophoresis. Furthermore, the large subunit from petite strain Y8 is capable of translating poly(U) when reconstituted with the small subunit from a wild-type strain. This result confirmed and extended a previous observatioan [24] that petite strains retaining 21-S r R N A gene contain mitochondrial large ribosomal subunit. The presence of the large ribosomal subunit has also been

reported in mitochondria of wild-type ceils which have been grown in the presence of erythromycin [25]. The present study shows that the amount of the large ribosomal subunit in petites retaining the 21-S rRNA is significantly higher than that in the wild-type strain. This observation might simply be a reflection of the amplification of the ribosomal R N A gene in the petite mutants studied. However, the result may also indicate that the small subunit is involved in the regulation of the amount of the large subunit being synthesized, and that the regulatory mechanism might have been disrupted by the absence of the small subunit. Acknowledgements

We wish to thank Professor Anthony W. Linnane for his support and constructive discussions. The excellent technical assistance of Anne Thomas and Linton Watkins in operating the continuous cultures, and of Anne C. Muntz in nucleotide analysis is gratefully acknowledged. This work was supported by grant 82/15245 from the Australian Research Grants Scheme. References 1 Butow, R.A., Vincent, R.D., Perlman, P.L., Terpstra, P. and

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