Expression of the mitochondrial genome in HeLa cells

J. Mol. Biol. (1972) 65, 273-289

Expression of the Mitochondrial Genome in HeLa Cells XT. Properties of Mitochondrial Polysomes DEANNA OJALA AND GIUSEPPE ATTARDI

Division of Biology, California Institute of Technology Pasadena, Calif. 91109, U.X.A. (Received 27 August 1971) In the present work, polysomal structures, estimated to contain from two to seven 60 s monomers$, have been identified in HeLa cell mitochondria and some of their properties have been investigated. The study of mitochondrial polysomes has been facilitated by the use of RNase inhibitors, which reduced the oontamination of these polysomes by endoplasmic reticulum-bound polysomes, and by the use of appropriate antibiotics, which allowed the selective labeling of either the nascent chains or the RNA components of the mitoohondrial structures. Mitoohondrial polysomes have revealed an unusual resistance to degradation by RNase; furthermore, only a partial dissociation of ribosomes into subunits and very little release of nascent chains were observed in the presence of even high concentrations of EDTA. The evidence derived from enzymic tests and from the comparison with the effects of RNase and EDTA on endoplasmic reticulum-bound polysomes, has suggested that this unusual behavior of mitochondrial polysomes is due to the nature of their polypeptide products, which makes the nasoent chains particularly “sticky.” These chains would thus, on one hand, interact by secondary bonds with the two ribosomal subunits, stabilizing the ribosomes against dissociation by removal of Mga + ; on the other, they would interact with other chains on the same polysome, thus stabilizing the whole structure against degradation by RNase. The analysis of the distribution of mitochondrial rRNA among different ribosome structures has indicated that, under the present conditions of cell growth and of subcellular fractionation, about 50% of mitochondrial ribosomes are recovered in polysomes, about 20% as monomers and the rest as free subunits.

1. Introduction The existence in mitochondria of 55 to 60 s ribosomes, which are the site of an amino-acid incorporation in vitro and in vivo with the characteristics expected for mitoehondrial protein synthesis, has been reported for rat liver cells (O’Brien & Kalf, 1967; Ashwell & Work, 1970), for Xenopus laevis oocytes (Swanson & Dawid, 1970) and for HeLa cells (Attardi & Ojala, 1971; Brega & Vesco, 1971). The two high t Paper IX in this series is Pica-Mattoccia & Attardi, 1972. $ The values of 60 s, 45 s and 35 s have been used here for the sedimentation coefficients, of the mitochondrial ribosomes and their major and minor subunits, respectively. These values were estimated on the basis of the hydrodynamic behavior of newly synthesized particles, labeled during a Z-hr [5-W]uridine pulse in the presence of 0.1 pg actinomyoin D/ml., relative to that of the cytoplasmic ribosomal subunits. It is likely, however, that the mature mitochondrial ribosomes not bearing messenger RNA have a sedimentation coeffioient somewhat lower than 60 s. A value of about 56 s was in fact estimated after mild RNase treatment (presumably apt to digest most of the attached mRNA) of [3H]uridine-labeled particles and of ribosomes labeled with [sH]leuoine in their nascent protein chains. 273

274

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molecular weight RNA components sedimenting at 16 s and 12 s, coded by mitochondrial DNA, which have been described in several laboratories (Vesco & Penman, 1969; Dawid, 1969; Attardi, 0. et al., 1969; Dubin & Montenecourt, 1970; Attardi & Attardi, 1971; Aloni & Attardi, 1971b), are the structural components of these ribosomes. The occurrence of 40 to 45 s and 30 to 35 s ribonucleoprotein particles containing 16 s and 12 s RNA, respectively, and representing presumably the major and minor subunits of the 60 s ribosomes, has also been detected (Swanson & Dawid, 1970; Attardi & Ojala, 1971; Brega & Vesco, 1971; O’Brien, 1971). In the present work, polysomal structures, estimated to contain from two to seven 60 s monomers, have been identified in HeLa cell mitochondria, and their unusual resistance to RNase digestion and EDTA treatment has been investigated. Evidence has been obtained suggesting that this behavior is due to the stabilizing effect of growing polypeptide chains. At least 50% of mitochondrial ribosomal RNA has been found in polysomal structures.

2. Materials and Methods (a) Method of growth of cells and labeling

conditions

The method of growth of HeLa cells in suspension has been previously described (Amaldi & Attardi, 1968). Likewise, reference is made to previous reports for the conditions used for selective labeling of mitochondrial RNA in the presence of 0.1 pg actinomycin D/ml. (to inhibit cytoplasmic rRNA synthesis (Perry, 1964; Dubin, 1967; Penman, Vesco & Penman, 1968)), and for long-term labeling of total RNA with [2-14C]uridine or [5-3H]uridine, and of DNA with [2-i4C]thymidine (Attardi & Ojala, 1971; Aloni & Attardi, 1971a). Long-term labeling of phosphatidyl choline was carried out by growing cells (‘7 x 104/ml.) for 24 hr in the presence of [1,2-14C]choline chloride (O-07 &%/ml., 2 mCi/m-mole). For pulse-labeling with L-[4,5-3H]leucine, 1.3 to 5.0 x lo8 exponentially growing HeLa cells were collected by centrifugation, washed twice in leucine-free medium with 5% dialyzed serum and resuspended at a concentration of about lo6 cells/ml. in the same medium (warmed to 37’C); after 35 min at 37’C the cells were treated with cycloheximide (200 pgglml.) or emetine (100 pg/ml.) for 5 min, then exposed to n-[4,5-3H]leucine (40 to 58 mCi/pmole, 4 to 16 &i/ml.) for 4 min. In some experiments, as specified below, the cycloheximide or emetine treatment was carried out in the presence of chloramphenicol (200 pg/ml.), or was omitted. (b) Xubcellular

fractionation

The preparation by differential centrifugation of a crude mitochondrial fraction containing the bulk of HeLa cell mitochondria and rough endoplasmic reticulum, in addition to smooth membrane structures, has been previously described (Attardi, Cravioto & Attardi, 1969). The pellet resulting from the second 8100 g,, oentrifugation of the mitoehondrial fraction was resuspended in lysis medium (usually, 0.5 to 1.0 ml/g cell equivalent): this consisted, unless otherwise specified, of 0.05 M-Tris buffer (pH 6.7 at 25’C), O-1 M-KCl, 0.01 M-Mgcl, (Tris-K-Mg), containing 2% Triton X-100. After 10 min at 2°C the lysate was centrifuged in the SS34 rotor of the Servall centrifuge at 20,000 g,, for 15 min. The 20,000 g,, supernatant was then layered on a 15 to 30% (w/w) sucrose gradient in Tris-K-Mg, and centrifuged in the SW27 Spinco rotor (2.54 cm x 8.83 cm buckets) at 3°C at 27,000 rev./min for 6.5 hr, unless otherwise specified. In the experiments aimed at isolation of mitoohondrial or cytoplasmic polysomes, sodium polyvinyl sulphate was in general added to the homogenization and lysis medium (20 pg/ml.). In most experiments rat liver 229,000 g,, supernatant, prepared as described below (see section (c)), was also added, in the proportion of 25% by volume, to the lysis medium. Components separated by sucrose gradient centrifugation were in some cases dialyzed for 2 to 3 hr verse three changes of 1 1. of an appropriate buffer, as indicated below, and, either directly or after enzymic treatment, re-run in a 15 to 30% (w/w) sucrose gradient (in the same buffer used for dialysis) in the SW27 rotor, under the conditions specified in the legends to the Figures.

MITOCHONDRIAL

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Free polysomes were isolated by centrifuging a portion of the 13,300 g,, supernatant of the cytoplasmic extract of HeLa cells (adjusted to 0.05 M-Tris buffer (pH 6.7), 0.1 MKCl, 0.01 M-MgCI, and 2% T&on X-100, and with the addition of 25% by volume of rat liver supernatant) through a 15 to 30% sucrose gradient in Tris-K-Mg in the SW27 Spinoo rotor (2.54 cm x 8.83 cm buckets) for 2 hr at 25,000 rev./min at 3°C. (c) Prqaration

of rat liver

229,000

g,, supernatant

For the preparation of the 229,000 g,, supernatant, the l.iver from an adult male rat (which had been starved for 20 hr) was homogenized with a Potter-Elvehjem glass homogenizer in about 2.5 vol. of 0.25 M-SUCPOSB, 0.05 M-Tris buffer (pH 6.7), O-025 M-KCl, 0.005 M-MgCl,, until 80 to 90% of the cells were broken. The homogenate was centrifuged at 13,300 g,, for 15 min; the supernatant was recentrifuged under the same conditions, and then in the Spinco 65 tied-angle rotor at 229,000 g,, for 2 hr. Unless immediately used, the supernatant was kept frozen at -20°C. Before use, MgCI, and KC1 were added to 10m2 M and 10-l M, respectively. (d) Extraction

and analysis

of RNA

RNA was released from the submitochondrial structures separated in sucrose gradient with 2% sodium dodecyl sulfate, collected by ethanol precipitation and centrifugation, dissolved in sodium dodecyl sulfate buffer, and run through 15 to 30% (w/w) sucrose gradients in sodium dodecyl sulfate buffer in the SW27 rotor (1.59 cm x 10.16 cm buckets) for 25 to 28 hr at 20°C (Attardi & Ojala, 1971).

3. Results (a) Identi$cation

of mitochondrial

polysomes

(i) 8edimentation pro$le of protein synthesizing structures heavier than 60 s in the mitochondrial lysate The existence in mitoohondrial lysates of structures heavier than 60 s ribosomes, which can be pulse-labeled with [3H]leucine in the presence of specific inhibitors of cytoplasmic protein synthesis, but not in the presence of chloramphenicol, was previously reported (Perlman & Penman, 1970a,b; Attardi & Ojala, 1971; Brega & Vesco, 1971). Figure l(a) and (lo) show labeled structures sedimenting in the region of about 60 to 200 s from cells exposed to a 4-minute [4,EL3H]leucine pulse in the presence of two inhibitors of cytoplasmic protein synthesis, cycloheximide (a) (Ennis & Lubin, 1964) or emetine (b) (Grollman, 1966). The three small peaks in the O.D.260 profile on the heavier side of the 74 s peak (especially clear in Fig. l(a) and (G)) presumably represent endoplasmio reticulum-bound dimers, trimers and tetramers (corresponding to about 113, 147 and 178 s, respectively (Wettstein, Staehelin $ Noll, 1963)), and provide convenient sedimentation markers. One recognizes in the radioactivity profiles in Figure l(a) and (b) a partially resolved component at approximately 80 s and heavier components up to about 200 s, a peak at 60 s and a smaller peak at 45 S, the last one corresponding presumably to the major mitochondrial ribosoma,l subunits. In other patterns (see, for example, Fig. l(f)) the labeled structures were found to form a broad symmetrical band centered around 120 s and extending from about 74 to 200 S. More than 95% of the label is alkali-stable (not shown). Purthermore, most of the radioactivity can be chased by puromycin (Fig. l(c)), indicating that

it is in nascent

polypeptide

chains.

The

incorporation

of [4,5-sH]leucine

into

acid-precipitable material is ost completely sensitive to chloramphenicol, as expected for mitochondrial protein synthesis (Kroon, 1965; Wheeldon & Lehninger, 1966; Linnane, 1967; Lederman & Attardi, 1970), both in the presence of emetine

276

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Cd’ j

0.60

(e) 0.40

Fraction

no

FIG. 1. Pulse-labeling with n-[4,5-sH]leucine, in the presence of cycloheximide or emetine, of mitochondrial protein synthesizing structures, and chase by puromyoin. (a) to (e) Three batches of HeLa cells were exposed for 4 min to n-[4,5sH]leucine in the presence of cycloheximide ((a) and (c)) or of both oycloheximide and chloramphenicol (e), and immediately harvested ((a) and (e)), or treated with puromyoin (150 rg/ml.) for 10 min and then harvested (0). Two other batches of cells were labeled for 4 min with L-[4,5sH]leucine in the presence of emetine (b), or of both emetine and ohlorampheniool (d), and immediately harvested. The mitochondrial fraction was isolated from each batch of cells, lysed with Triton X-100, and run through a sucrose gradient, as detailed in Materials and Methods, section (b). (f) The total mitochondrial lysate from HeLa cells labeled for 4 min with L-[4,5-3H]leucine in through a 15 to 30% the presence of emetine was run, without prior 20,000 g,, oentrifugation, sucrose gradient in Tris-K-Mg (over 5 ml. of 55% sucrose) for 3 hr at 27,000 rev./mm 3H( cts/min). --c--o--, O.D.,G,,,,~; -+--,

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(Fig. l(d)), which inhibits substantially completely cytoplasmic protein synthesis, and in the presence of cycloheximide (Fig. l(e)), which allows a residual protein synthesis on 74 s ribosomes (Perlman & Penman, 19708). The latter observation suggests that relatively little contamination of the mitochondrial protein synthesizing structures by endoplasmic reticulum-bound polysomes, active in the presence of cycloheximide, occurred under the conditions used here. In these experiments, rat liver supernatant had been added to the lysis medium to prevent degradation of endoplasmic reticulum-bound polysomes to smaller aggregates sedimenting in the region of the mitochondrial protein synthesizing structures. That the RNase inhibitor present in the rat liver supernatant (Blobel & Potter, 1966) was effective in the present case, is suggested by the observation that, in experiments in which the supernatant was omitted, the chloramphenicol-resistant amino-acid incorporation in the presence of cycloheximide was considerably higher than in the experiment shown in Figure 1, up to more than 30% of the total. The labeled components sedimenting in the region from 60 to about 200 s in Figure l(a) and (lo) represent the bulk of mitochondria-specific protein synthesizing structures. This is shown by the experiment illustrated in Figure l(f), in which the whole mitochondrial lysate was run, without prior 20,000 g,, centrifugation, through a 15 to 30% sucrose gradient with a dense sucrose cushion at the bottom of the tube. That association with DNA is not involved in determining the sedimentation behavior of the heavy mitochondrial protein synthesizing structures was indicated by an experiment in which the latter were isolated, by centrifugation on sucrose gradient, from the mitochondrial lysate of cells long-term labeled with [2-14C]thymidine, pulse-labeled with [4,5-3H]1 eucine, and then rerun in sucrose gradient with or without pretreatment with 100 pg of electrophoretically purified DNase/ml. No change whatsoever was observed in the sedimentation profile of these structures after DNase treatment, while about 60% of the DNA sedimenting in the same region of the gradient was made acid-soluble, and, of the remainder, about 70% was displaced to the upper region of the gradient. Omission of sodium polyvinyl sulfate from the homogenization and lysis medium had no effect on the presence of the heavy mitochondrial protein synthesizing structures, indicating that these do not result from aggregation of mitochondrial ribosomes induced by this substance.

(ii) Ejfect of RNase on the heavy mitochondrial protein synthesizing structures It seemed likely that the heavy protein synthesizing structures sedimenting between 74 s and about 200 s represent polysomes consisting of 60 s monomers. In order to obtain direct evidence on this point, these structures were treated with a concentration of pancreatic RNase (1 pguglml.)known to break endoplasmic reticulumbound polysomes to monomers (Attardi et al., 1969b). As shown in Figure 2(a) and (b), RNase had little effect on the heavy mitochondrial protein synthesizing structures, with only a relatively small increase in the amount of single mitochondrial ribosomes carrying nascent chains, which now sedimented at about 56 s. In another experiment, under identical conditions, the [4,5-3H]leucine pulse-labeled endoplasmic reticulum-bound polysomes present in the lysate (120 to 350 s) were, in a great majorit*y, broken to monomers and dimers.

278

D.

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ib)

74s I I.(I

i

56 s /

(d)

jN

‘0x 2

60 s

N ‘0 x

“E ) ’.c E \ 1p 0 z 5P

L :, 1

Fraction

74 s !

,

no

FIG. 2. Effect of RNase, pronase and phospholipase C on heavy mitoehondrid protein synthesizing structures pulse labeled with L-[4,5-“Hlleucine. (a) to (d) The mitochondrial lysate from a mixture of HeLa cells labeled for 4 min with I,-[4,5-sH] leucine in the presence of emetine and cells long-term labeled with [2-W$ridine was run through a sucrose gradient, as in Fig. l(a). The fractions of the polysome region from ~74 to ~180 s (see insert) were pooled, dialyzed against Tris-K-Mg and divided into 4 equal portions; (a) control, incubated with no addition of enzymes; (b) incubated with preheated (SO”C, 15 mm) pancreatic RNase, 1 pgglml., 2”C, 15 mm; (c) incubated with pronase (Calbiochem), 40 pg/ml., 2X’, 45 min; (d) incubated with pronase, 40 pg/ml., 2”C, 30 min, and then with RNase, 1 pg/ml., 2”C, 15 min. The four samples were then run through sucrose gradients, as in Fig. l(a). (e) and (f) Heavy mitochondrial protein synthesizing structures, isolated as described above in a simiIar experiment, after dialysis against Tris-K-Mg, were re-run through sucrose gradients either

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CELLS

(iii) Sign@cance of the RNase resistance of the heavy mitochonclrial protein synthesizing structures

The unusual behavior of these structures after mild RNase treatment suggested the possibility that the presumptive mitochondrial polysomes might be stabilized against RNase attack by residual membrane material or by nascent protein chains. The analysis of the distribution in a sucrose gradient of phosphatidyl choline containing phospholipids, after centrifugation of a lysed mitochondrial fraction from cells long-term labeled with [1,2-14C]choline, showed no correspondence with the profile of the heavy protein synthesizing structures, arguing against the association of these with membrane material. Likewise, the lack of effect on the sedimentation behavior and on the RNase sensitivity of these structures of varying the amount of lysis medium from 0.5 to 4.0 ml. per gram cell equivalent of mitochondrial fraction, or of using, instead of Triton X-100, another non-ionic detergent, Brij 58 (Atlas Chem. Ind.) (0*5%), in combination with sodium deoxycholate (OGo/o), or of treating the mitochondrial structures with phospholipase C, tended to exclude that the relative RNase resistance of these structures was due to stabilization by residual membrane material. By contrast, experiments of proteolytic digestion indicated that some protein component was involved in this stabilization. In fact, treatment of the heavy mitochondrial protein synthesizing structures with pronase, using conditions under which the sedimentation behavior of typical polysomes is not affected, but the exposed portion of the nascent chains on the ribosomes is digested (Malkin & Rich, 1967), made the structures to a great extent sensitive to RNase (Fig. 2(d) and (e)). After pronase treatment alone, only a relatively small increase in the amount of monomers carrying nascent chains was observed (Fig. 2(c)). In the experiments shown in Figure 2(d) and (e), the breakdown of the heavy mitochondrial protein synthesizing structures was not complete. That this is not due to insufficient RNase action, but rather to incomplete hydrolysis of the stabilizing protein(s) by the proteolytic enzyme, is suggested by the observation that a similar proportion of resistant structures was found if exposure to RNase followed (Fig. 2(d)) or started simultaneously with the pronase treatment (Fig. 2(e) ) . A partial degradation of the 74 s cytoplasmic ribosomes to 60 and 45 s subunits was observed after pronase treatment (Fig. 2(c), (d) and (e)), in confirmation of previous observations (Malkin & Rich, 1967). The results discussed above strongly suggested that the heavy mitoohondrial protein synthesizing structures are polysomes consisting of 60 s monomers, which are stabilized by some protein component, possibly nascent protein chains. (iv) Effect of EDTA

on the heavy mitochondrial

protein synthesizing

structures

Figure 3(a) shows the effect of treatment with 3 x 10e3 M-EDTA on [4,5-3H]leucine pulse-labeled mitochondrial polysomes previously separated on a sucrose gradient. It appears that the majority of the 3H label sediments in the region heavier than 50 S, with a well-defined but relatively small peak sedimenting at about 43 S, and a small amount of label sedimenting in the region 4 to 6 s. The latter is presumably without treatment, or after incubation with pronase, 40 pg/ml., and pancreatic RN&se, 1 pg/ml., 2W, 45 min (e), or with phospholipase C (Sigma), 40 pg/ml., and RNase, 1 pg/ml., 2’33, 45 min (f). -e-O---, 3H (cts/min); -O-O--, 14C (cts/min); (..a. * .*.), superimposed profile of 3H(cts/min) of control sample in (e) and (f).

280

D.

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(a)

I 01

0.51

N bx i E -. .E : ‘; ‘f n

0.7

0.5

02

Voiume

(ml.)

FIG. 3. Effect of EDTA treatment on heavy mitochondrial protein synthesizing structures pulse-labeled with L-[4,5-3H]leuoine. Heavy mitoohondrial protein synthesizing structures, isolated as in Fig. 2 from a mixture of HeLa cells labeled for 4 min with L-[4,5-3H]leucine in the presence of emetine and cells long-term labeled with [2-W7@ridine, were dialyzed against 0.05 M.Tris buffer (pH 6.7), 0.025 M-KCl, containing either 0.003 M-EDTA (a) or 0.05 M-EDTA (b). 0 ne 5-ml. portion of each dialysate was run through a 15 to 30% sucrose gradient in the same buffer used for dialysis, at 27,000 rev./min for 8 hr at 3°C. -•--•--, ?H(ots/min); -- 0 -- 0 - -, 14C(cts/min).

MITOCHONDRIAL

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associated with nascent chains linked to 4 s RNA (Gilbert, 1963). By comparison with the pattern of Figure l(c), it appears that removal of Mg2+ by the chelating agent has released little of the nascent chains. The 43 s labeled peak presumably represents the major subunits of mitochondrial ribosomes with still attached nascent chains. Increasing the concentration of EDTA to 10V2 DTand 5 x 10m2 M (Fig. 3(b)) results in a progressive displacement of radioactivity from the heavier regions to the 55 s to 65 s region and in an increase in the size of the labeled 43 s peak. However, also with the highest EDTA concentration used here, there is a relatively small release of nascent chains, and the majority of label sediments faster than 50 S. In striking contrast, the great majority of cytoplasmic ribosomes present in the lysate are dissociated into 50 s and 30 s subunits (Fig. 3) ; in another experiment the bulk of nascent chains labeled in a 4-minute [4,5-3H]leuoine pulse on these cytoplasmic polysomes was, under identical conditions (10m2 M-EDTA), released free, and sedimented close to the meniscus. As previously reported for reticulocyte polysomes (Malkin & Rich, 1967), treatment of the mitochondrial polysomes with pronase (40 pg/ml.) in the presence of 3 x 1O-3 M-EDTA resulted in a more extensive digestion of nascent chains than observed in the presence of Mg2 + , presumably due to disruption of the ribosomes: in fact, there was a 40% decrease in the radioactivity associated with the heavy protein synthesizing structures. However, also in this case, the majority of the remaining label sedimented faster than 50 S, with only a small peak at 43 s. The experiments discussed above indicated that EDTA treatment of mitochondxial polysomes produced only a partial release of the major subunits of mitochondrial ribosomes, as judged from the sedimentation behavior of the nascent chains. In these experiments the fate of the minor mitochondrial ribosomal subunits could not be followed on the basis of their long-term [2-14C]uridine label, because their possible release by EDTA would have been obscured by the large excess of small cytoplasmic ribosomal subunits. An analysis was therefore carried out on the distribution of long-term [2-14C]uridine-labelecl 12 s RNA among components separated by sucrose gradient centrifugation of mitochonclrial polysomes treated with 3 x low3 M-EDTA. This analysis showed that only about 18% of the 12 s RNA was in components sedimenting slower than 40 S. These results indicated that also the small mitochondrial ribosomal subunits had been released only in minor part by the EDTA treatment. (b) Labeling of mitochonclrial polysome RNA during a 2-7tour [5-3H+cridine pulse In order to quantitate the distribution of the newly synthesized mitochondrial rRNA among different Triton X-100 lysis products of mitochonclria, the mitochonaria1 lysate from cells labeled for 2 hours with [5-3H]uridine in the presence of 0.1 pgcglml.actinomycin D was run for 3 hours through a P5 to 30% sucrose gradient with a dense sucrose cushion at the bottom of the gradient (Fig. 4(a)). The [3H]uridine profile showed some slow-sedimenting components near the meniscus, a peak corresponding to non-resolved monomers, 45 and 35 s subunits, and a broad peak in good correspondence with the peak (centered at about 120 s) of [4,5-3H]leucine pulse incorporation (as determined in a parallel sucrose gradient run) ; a considerable amount of radioactivity (about 20%) had sedimented near the bottom of thegradient, and had been prevented from pelleting by the dense sucrose cushion. In another experiment, the labeling with [5-3H]uridine of the structures up to

282

D.

OJALA

AND

G. ATTARDI

Cd)

(e)

i---)c-

p

J

4 60

120 s

I 4.0

2.0

I

N

IO

20

30 Fraction

(d)

40

50

rr) ‘0

no.

18~125 116s

(e)

FIG. 4. (a) Labeling with [S3H]uridine, during a 2-hr pulso in the presence of 0~1 pg aotinomycin D/ml., of components sedimenting in the polysome region and of heavier structures from the mitochondrial lysate of HeLa cells. The mitochondrial lysates from (expt. A) a mixture of cells labeled for 2 hr with [S-sH]uridine in the presence of 0.1 pg actinomycin D/ml. and cells long-term labeled with [1,2-l%]choline chloride, and (expt. B) a mixture of cells labeled for 4 min with L-[4,5-3H]leucine in the presence of emetine and cells long-term labeled with [1,2-14C]choline chloride, were run through sucrose The superimposed sedimentation gradients, as in Fig. l(f) (after a 20,000 g,, centrifugation). profiles are shown. Expt. A: --O--O--, [3H]uridine (ots/min); -A--&-, 14C (ots/min); ** l .* l . . , o.D.~~,,~~. superExpt. B: -a-e--, [3H]leuoine (ots/min) (the 14C profile, not shown, was virtually imposable on that of expt. A).

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180 s fractionated in a sucrose gradient was found to be sensitive (80 to 95%) to I pg ethiclium bromide/ml., an intercalating phenanthridine dye, which has a selective inhibitory effect on mitochondrial nucleic acid synthesis (Zylber, Vesco & Penman, 1969; Goldring, Grossman, Krupnick, Cryer & Marmur, 1970). Analysis of the RNA extracted from various regions of the gradient in Figure 4 showed that, excluding the relatively small amount of not well resolved [5-3H]uridine-labeled components sedimenting between 12 and 18 s in the cushion material (Fig. 4(b)), from 30 to 40% of the newly synthesized 12 s and 16 s RNA was in the polysome region (Fig. 4(c) and (cl)), and the remainder in the monomer-subunit peak (Fig. 4(e)). The substantial absence of newly synthesized 12 s and 16 s RNA in the sucrose cushion material and the rough proportionality between amount of [4,5-3H]leucine pulse-labeled structures and amount of [5-3H]uridine-labelecl 12 s and 16 s RNA in the two cuts ((c) and (cl)) of the polysome region (containing, respectively, about 15 and 60% of total [3H]leutine label (excluding the slow sedimenting material near the meniscus), and about 10 and 20 to 30% of total 3H-labeled 12 s + 3H-labelecl 16 s RNA) suggest strongly that the newly synthesized mitochondrial rRNA species in the polysome region are indeed in functioning polysomes. (c) Steady-state distribution of mitochondrial ribosomal RNA among different ribosome structures Figure 5 shows the sedimentation profiles of RNA extracted from the structures seclimenting in the 60 s, 45 s and 35 s regions in a sedimentation run of the mitochondrial lysate from HeLa cells long-term labeled with [5-3H]uricline. It appears that the 60 s peak contains, in addition to 28 s rRNA, both 16 s and 12 s RNA, in the proportion of about 1.5: 1 expected for equimolar amounts of the two species (Robberson, Aloni, Attardi & Davidson, 1971), while the 45 s peak contains, besides 18 s rRNA, a major 16 s RNA component and a small amount of material seclimenting in the 12 s region, presumably deriving from breakdown of 16 s RNA (Attarcli & Attardi, 1971). The analysis of the distribution of 3H radioactivity between mitochondrial and cytoplasmic rRNA species reveals that 80 to 90% of the material in the 60 s peak, and 40 to 50% of the material in the 45 s peak is represented by cytoplasmic 60 s and 45 s ribosomal subunits, respectively. This is in agreement with the observation (unpublished) that very little o.D.~~~, relative to radioactivity, is associated with labeled 60 s and 45 s particles isolated ‘by sonication, presumably due to a smaller release of cytoplasmic subunits by the latt’er procedure. The material in the 35 s peak contains predominantly 12 s RNA and, therefore, presumably consists mainly of 35 s subunits. Table 1 shows the distribution of the 16 s and 12 s RNA components among mitochonclrial polysomes, monomers and ribosomal subunits, estimated by reconstructing the profiles of these components in sedimentation patterns of RNA pertaining to the structures isolated from the mitochondriaP lysate of cells long-term labeled with [5-3H]uricline or [2-14C?]uridine. It appears that roughly 50% of mitochondrial rRNA is in polysomes, 20% in monomers ad 30% in subunits. Very (b), (c), (d) and (e) The fractions indicated by labeled components shown in (a) were pooled, by sodium dodecyl sulfate and, after addition awsucrose gradient, as detailed in Materials and -•-a---, 3H (cts/min); --O--O--, 14C

arrows in the sedimentation pattern of [3H]uridineand the RNA in each pooled sample was released of 14C-labeled 18 s RNA marker. run through Methods, section (d). (ots/min).

284

D. OJALA

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G. ATTARDI

I( I5 I 30.0 IO.0

9I.0

0.3

6‘.O

0.2

3 .o

0, I

(b)

18 s I

: d 6

0.21

-8

O,I(

8,

0.3( 0,2(

- 4. @I( 10

20

30

40

Fraction

no

50

60

FIG. 5. Sedimentation pattern of the RNA extracted from ribosomes and ribosomal subunits released by Triton X-100 from the mitochondrial fraction of HeLa cells long-term labeled with [5-%]uridine. The Triton X-100 lysate of the mitochondrial fraction from cells long-term labeled with [PL3H]uridine was run in a sucrose gradient at 27,000 rev./min for 13.5 hr. The portions of the gradient indicated by arrows (insert) were pooled, and the RNA in each pooled sample was released by sodium dodecyl sulfate and run through a sucrose gradient in the presence of unIabeled I8 s RNA as a marker. (a) 60 s region; (b) 45 s; (c) 30 s. --o--o--, O.D.Zaonm; -+-a--, “H(ots/min).

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TABLE 1

Distribution

of mitochondrial ribosomal RNA among different structures

Region of the gradient

16 s + 12 s RNA (O/o cts/min)

70-180 s (polysomes) 50-70 s (monomers) 20-50 s (ribosomal subunits) These data were with [5-3H]uridine of RNA pertaining The radioactivity by reconstructing

52 19 29

12 s RNA (% cts/min) 47-50 17-20 33

compiled from the results of three experiments utilizing cells long-term labeled or [2-%$ridine, in which an analysis was made of the sedimentation profiles to the structures released by Triton X-100 from the mitochondrial fraction. associated with the mitochondrial 16 s and 12 s RNA species was estimated their profiles, as exemplified in Fig. 5.

similar results were obtained by estimating the distribution of the 12 s RNA component alone, which is better resolved from other components than the 16 s RNA. (d) EJect of ethidium bromide on amino-acid incorporation into naxent chains by mitochondrial polysomes Figure 6(a) shows that a 20-minute pre-treatment of the cells with 1 pg ethidium bromide/ml. suppresses almost completely the capacity of mitochondrial polysomes to incorporate amino acids. No such effect of ethidium bromide, or a very slight one, was observed in endoplasmic reticulum-bound polysomes (Fig. 6(b)) or free polysomes (Fig. 6(c)).

4. Discussion The evidence presented in this paper indicates that HeLa cell mitochondria contain, in addition to 60 s ribosomes, and 45 s and 35 s ribosomal subunits, large protein synthesizing structures which, in the most intact preparations, sediment in a sucrose gradient in the form of a broad symmetrical band centered at about 120 s and extending from about 74 to 200 S. The mitochondrial fraction from HeLa cells is known to be highly contaminated by endoplasmic reticulum-bound ribosomes (Attardi, Cravioto & Attardi, 1969). The size of endoplasmic reticulum-bound polysomes in these cells covers the range of about 120 to 350 s. The heavy mitochondrial protein synthesizing structures thus overlap in sedimentation properties with the slower-sedimenting endoplasmic reticulum-bound polysomes, from dimers to tetramers. In the present work, the use of RNase inhibitors (sodium polyvinyl sulfate and rat liver supernatant factor (Blobel & Potter, 1966) ) prevented degradation of endoplasmic reticulum-bound polysomes to smaller aggregates, and thus reduced the contamination of the heavy mitochondrial protein synthesizing structures by cytoplasmic polysomes. The relatively low level of ethidium bromide-resistant [5-3H]uridine incorporation (less than 20%) found in this work in components sedimenting in the region of the heavy mitochondrial protein synthesizing structures, points to the presence in this region of only a small amount of labeled mRNA associated with contaminating cytoplasmic polysomes. Evidence that the heavy mitochondrial protein synthesizing structures are indeed polysomes consisting of 60 s monomers has come from two types of observations. 19

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FIG. 6. Effect of ethidium bromide on amino-acid incorporation by mitochondrial, endoplasmic reticulum-bound and free cytoplasmic polysomes. (a) The mitochondria1 lysates from mixtures of HeLa cells labeled for 4 min with L-[4,5-3H]leuoine in the presence of emetine, with or without 20 min pre-treatment with L pg ethidium bromide/ml., and cells long-term labeled with [2- l*C]uridine were run through sucrose gradients as in Fig. l(a). (b) and (c) Two batches of HeLs cells long-term labeled with [2-Wluridine were centrifuged, resuspended in leuoine-free medium, then labeled for 4 min with L-[4,5-3H]leucine, with or without pre-treatment for 20 min with 1 pg ethidium bromide/ml. The mitochondrial fraction and the free polysome fraction were isolated from each of the two batches, as described in Materiala and Methods, section (b). The two mitochondrial frsotions, lysed as usually, and the two free polysome

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In the first place, from 30 to 40% of the 12 s and 16 s rRNA labeled during a 2-hour [S3H]uridine pulse, and about 50% of the pre-existing 12 s and 16 s rRNA were found to be associated with structures sedimenting in the mitochondrial polysome region. In the second place, it has been possible to break the bulk of these heavy protein synthesizing structures into monomers by appropriate enzyme treatment. In this connection, mild digestion with pancreatic RNase, which is sufficient to break apart most polysomes, proved, surprisingly, to be very slightly effective, producing only a small increase in monomers. Under the same conditions, endoplasmic reticulum-bound polysomes contaminating the mitochondrial fraction were completely susceptible to RNase, in confirmation of earlier findings (Attardi, Cravioto & Attardi, 1969). Treatment with pronase, using conditions under which the exposed portion of the nascent chains should be digested, while the integrity of ribosomes should be preserved (Ma&in & Rich, 1967), was found to be sufticient to make the heavy protein synthesizing structures sensitive to RNase. This finding strongly suggested that nascent chains, or some other extra-ribosomal protein component, stabilized the polysomes against RNase attack. The behavior of mitochondrial polysomes towards EDTA treatment also proved to be unusual. In the 6rst place, only a partial release of large and small mitochondrial ribosomal subunits was observed with EDTA concentrations sufficient to break into subunits the endoplasmic reticulum-bound ribosomes contaminating the mitochondrial fraction. In the second place, very little release of nascent chains from mitochondrial polysomes was found in the presence of EDT.A, again in contrast to the behavior observed for the endoplasmic reticulum-bound polysomes. It seems likely that the peculiar properties of the mitochondrial polysomes, i.e. their resistance to RNase and their behavior towards EDTA treatment, have the same structural basis. An interesting possibility is that this is related to the nature of the polypeptide products of these polysomes, which makes the nascent chains particularly “sticky”. It is conceivable that these chains would interact by secondary bonds with the two ribosomal subunits, stabilizing the ribosomes against dissociation by removal of Mg2+, and stabilizing at the same time their own association with the large subunits. Furthermore, interactions between different nascent chains on the same polysome would make this structure relatively resistant to RNase: a similar situation has been previously reported for collagen-producing polysomes (Goldberg & Green, 1967). On the other hand, digestion of the exposed portion of the nascent chains by pronase would render the polysomes sensitive to RNase. The observation that only a moderate decrease in the [4,5-3H]leucine radioactivity associated with nascent chains was observed after pronase treatment of the mitochondrial polysomes is not in disagreement with this interpretation. In fact, since the [4,S3H]leucine pulse (4 min) was short, relative to the time required to label completely nascent peptides in HeLa cell mitochondria (estimated to be about 10 minutes (Perlman & Penman, 1970a)), and to the time presumably necessary to equilibrate the intramitochondrial leucine pool with the exogenous [4,5-3H]leucine, it is reasonable to assume that the majority of the label incorporated in a 4-minute pulse was in the fractions were then run through sucrose gradients, as detailed in Materials and Methods, section (b). Control samples: --O-O-, 3H (cts/min); (*a*.....), l*C (cts/min). Ethidium bromide-treated samples: --O--O--, 3H (ots/min) (normalized for the difference from the controls in the amount of 14C cts/min associated with the 74 s peak (a) or the polysome region (b) and (c)).

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protected portion of the nascent chains (Ma&in & Rich, 1967). Likewise, the fact that no accumulation of dissociated major ribosomal subunits was detected after pronase digestion in the presence of EDTA is not inconsistent with the proposed stabilizing role of the nascent chains : in fact, it is known that under these conditions the nascent chains are completely digested (Malkin & Rich, 1967), and any released major subunits originally labeled in the growing chains would thus be unrecognizable. The above proposed stabilizing role of nascent chains on mitochondrial polysomes is in keeping with the available knowledge concerning the nature of the protein product of these polysomes. In fact, a considerable amount of evidence (Haldar, Freeman & Work, 1966; Beattie, Bosford, &‘zKoritz, 1967; Neupert, Brdiczka & Biicher, 1967; Yang & Griddle, 1970) has indicated that this product belongs to the class of the so-called “structural proteins,” i.e. proteins characterized by their insolubility in water at neutral pH, and by their ability to complex with each other, with other proteins and with phospholipids by hydrophobic bonds (Griddle, Bock, Green & Tisdale, 1962; Richardson, Hultin & Green, 1963). Interactions between nascent polypeptide chains of this type of protein on the same polysome could occur as a result of the abnormal environment in which the polysomes are found after extraction, or could have a physiological significance related to the initial stage of formation of functional aggregates. Similar intrapolysomal interactions between the nascent chains have been proposed as a mechanism of monomer assembly in large molecules (Kiho & Rich, 1964; Goldberg & Green, 1967). An estimate of the size of mitochondrial polysomes has been made by applying the relationship between relative particle weights and experimentally determined S values, which has been shown to hold for 74 s ribosomes and their oligomers (Wettstein et al., 1963), and by using a value of 56 s for the sedimentation coe&cient of the monomers. Thus, the center of the polysome distribution has been estimated to correspond to a mixture of trimers and tetramers, and the heaviest polysomes to heptamers. The mitochondrial protein synthesizing structures sedimenting at 95 s, sensitive to ethidium bromide, which were described in HeLa cells by Perlman & Penman (197Oa), very likely correspond to a mixture of dimers and trimers. The smaller size of the polysomes observed by these authors could result from partial degradation of the original polysome distribution or from a less close packing of ribosomes on intact mRNA strands. Failure of EDTA to release nascent chains from the 95 s structures was reported also by Perlman & Penman (1970a). The specificity of inhibition of protein synthesis on mitochondrial polysomes by ethidium bromide would tend to exclude that intercalation of this drug into transfer RNA (Bittman, 1969) is responsible for the effect. The half-life of in vivo mitochondrial protein synthesis in the presence of ethidium bromide estimated here (less than 20 min) is much shorter than that esitmated for an in vitro mitochondrial protein synthesizing system after in vivo treatment with ethidium bromide (1 to 1.5 hr (Lederman & Attardi, 1970)), or that estimated in vivo after treatment of the cells with a dose of actinomycin D or cordicepin sufficient to inhibit mitochondrial RNA synthesis (about 2 hr (Perlman & Penman, 1970b)). This strongly suggests that the major part, if not all of the effect of ethidium bromide is not directly related to the inhibition by this drug of mitochondrial RNA synthesis (Zylber et al., 1969), and thus to mRNA decay, but to some specific interference with mitochondrial protein synthesis, which is reversed when the drug is removed.

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These investigations were supported by grant GM-11726 from the National Institutes of Health. Actinomycin D was a generous gift of Dr McCormick from Merck, Sharpe & Dohme. The invaluable help of Mm La Verne Wenzel and Mrs Benneta Keeley is gratefully acknowledged. REFERENCES Aloni, Y. & Attardi, G:. (1971a). J. Mol. Biol. 55, 251. Aloni, Y. & Attardi, G. (1971b). J. Mol. Biol. 55, 271. Amaldi, F. & Attardi, G. (1968). J. Mol. Biol. 33, 737. Ashwell, M. A. & Work, T. S. (1970). Biochem. Biophys. Res. Comm. 39, 204. Attardi, B. & Attardi, G. (1971). J. Mol. Biol. 55, 231. Attardi, B., Cravioto, B. & Attardi, G. (1969). J. Mol. Biol. 44, 47. Attardi, G., Aloni, Y., Attardi, B., Lederman, M., Ojala, D., Pica-M&to&a, L. & Storrie, B. (1969). In International Synvposium on Autonomy and Biogenesis of Mitochondria and ChZoropZasts, p. 293. Canberra, Amsterdam: North-Holland Publ. Co. Attardi, G. & Ojala, D. (1971). Nature New Biol. 229, 133. Beattie, D. S., Bosford, R. E. & Koritz, S. B. (1967). Biochemistry, 6, 3099. Bittman, R. (1969). J. Mol. Biol. 46, 251. Blobel, G. & Potter, V. R. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1283. Brega, A. & Vesco, C. (1971). Nature. New Biol. 229, 136. 1, 827. Ciddle, R. S., Bock, R. M., Green, D. E. & T&dale, H. D. (1962). Biochemistry, Dawid, G. B. (1969). Syrqx Sot. Expt. Biol. 24, 227. Dubin, D. T. (1967). Biochem. Biophys. Res. Comm. 29, 655. Dubin, D. T. & Montenecourt, B. S. (1970). J. Mol. BioZ. 48, 279. Em&, H. L. & Lubin, M. (1964). Science, 146, 1474. Gilbert, W. (1963). J. Mol. Biol. 6, 389. Goldberg, B. & Green, H. (1967). J. Mol. Biol. 26, 1. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R. & Marmur, J. (1970). J. Mol. Biol.

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