THE JO~RNAI. OF BIOLOGICAL CHEIMISTRT Vd 247, i%o. lZ,Issue ofJune25, pp. 4063-4073,19X2

Printed zn U.S.A.

Effects of Chloramphenicol and Cycloheximide on the

Biosvnthesis of Mitochondrial Ribosomes in J

Tetrahymena*

(Received for publication, October l-1, 1971)

ALBERT J. T. MILLIS~ AND YOSHITAKA SUYAMA$

From the Department of Biology, University of Penrxsylvania, Philadelphia, Pennsylvania 1910-J

SUMMARY

Effects of two antibiotics, chloramphenicol and cyclo- heximide, on the synthesis of mitochondrlal and cytoplasmic ribosomes in Tefrohymena were studied. The results pre- sented here show that mitochondrial ribosome synthesis was not completely inhibited by chloramphenicol (0.5 IIIM), but was abolished by cycloheximide (1 mM). Acrylamide gel electrophoresis analysis revealed that mitochondrial ribo- somal proteins in a range of molecular weights from 13,000 to 25,000 were found particularly susceptible to chloram- phenicol inhibition. Cytoplasmic ribosomal proteins did not appear to be differentially inhibited by chloramphenicol.

The inhibition of mitochondrial ribosome synthesis in the presence of cycloheximide did not result from the loss of mitochondrial protein synthesis capacity as evidenced by in

vitro (14C]leucine incorporation capacity of mitochondria isolated from whole cells treated with cycloheximide. How- ever, cycloheximide greatly reduces the capacity to incor- porate [zH]uridine into mitochondrial ribosomes as RNase- resistant radioactivity.

These results suggest that some mitochondrial ribosomal proteins are synthesized in mitochondria and others by cytoplasmic ribosomes. The synthesis of those proteins that are produced by the mitochondrial ribosomes appears to be under the control of cytoplasmic protein synthesis. How- ever, mitochondrial ribosome assembly can take place in the absence of cytoplasmic protein synthesis.

In recent years it has become increasingly evident’ that mito- chondria contain ribosomes which differ from cytoplasmic ribo- somes in many physical-chemical properties (1-3).

* This investigation was supported in part by Contract AEC [AT(30-1)3588] from the United States Atomic Energy Commis- sion.

1 Present address, Department of Pediatrics, School of Medi- cine, University of Washington, Seattle, Washington 9810.5.

0 To n-horn all correspondence should be sent. Present address, Centre de Genetique Moleculaire, du C. N. R. S. Gif-sur-Yvette, Essonne, France.

It has been shown that the constituent RNhs of mit,oc~llondri:ll ribosomes are products of the mitochondrinl genorrle (3-O). However, the origin and site of synthesis for the proteins that compose these ribosomes have not beeu satisfactorily resolved.

Current evidence strongly favoring the cytoplasmic origin of mitochondrial ribosomal protein stems from the esperimeuts of Kuntzel (7) and Neupert et al. (8). They demonstrated a high inhibition of mitochondrial ribosomal protein synthesis by cgclo- heximide, a known inhibitor of cytoplasmic protein synthesis in eucaryotes, but no inhibition by chloramphenicol, an inhibit,or of mitochondrial protein synthesis. In support of that evidence, Davey, Yu, and Linnane (9) found that Succhuromyces growu in the presence of chloramphenicol retained mitochondria with the capacity for in vitro protein synthesis. Furthermore, mitochon- dria isolated from Neurospora were incapable of incorporat,ing [14C]leucine into the proteins of their mitochondrial ribosomes (10).

Although less rigorous, conflicting evidence has been obtained in yeast. A cytoplasmic mutation exists which results in resist- ance to erythromycin or other antibacterial ant.ibiot.ics (1 l-13). By analogy with similar mutations in bacteria (14, 15), the resist- ance is supposedly conferred by an alteration of a single mitochon- drial ribosomal protein which is coded by the mitochondrial ge- nome (11, 12).

Recently, Rifkin and Luck (16) reported that :I cytoplasmic “poky” mutation results in the abnormal assrmblg of ribosomnl subunits in mitochondria. Since there appear to be no gross defects in rRNA cistrous, a mitochondrial ribosomal prot.ein mu- tation is suspected.

In view of the above conflicting notions, it is of interest to investigate whether all mitochondrinl ribosomal proteins origi- nate on cytoplasmic ribosomes. Although one must be aware of the limitation in interpreting the effects of inhibition on inberde- pendent protein-synthesizing systems, antibiotics such as chlor- amphenicol and cyclohesimide offer a useful means for stlldying separately the products of each system (17-22), particularly if a genetic approach is not immediately available.

In this paper we studied the effects of those antibiot’ics on the synthesis of mitochondrial and cytoplasmic ribosomes in T&a- hymena. We have analyzed their effects on (a) mitochondrial ribosomal protein synthesis by polyacrylamide gel electrophore- sis, (b) the capacity for protein synthesis by isolated mitochon- dria, and (c) RNA synthesis.

4063

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4064 Biosynthesis of illitochondrial Ribosomes

MATEUI.4LS .4SD METHOIX?

Culture and Gro~fh Jledia

Tefrahynlena pyrijormis, st.rnin ST, was used for all esperi- merits. The stock culture was maintained in culture tubes con- t,aining approsimately 5 nil of 1 CT lxot.eose peptone-0.1 yc yeast e&act, at 28 f 0.5O.

Experimental cells were obtained from a 2-liter Erlenmeyer flask containing 300 ml of 2 ‘:; proteose peptone-0.01 Q& yeast crtract medium. This medium yields 0.6 to 0.8 ml of wet packed cells and the generation time was approximately 23 hours.

Chemically defined growth medium modified after the method of Rosenbaum ef al. (23) was also used. The generation hime was 3 to 4 hours and this medium yields 0.15 t.o 0.3 ml of wet packed cells per flask. This was used for i?> vivo labeliug of ribosomes and ribosomal proteins.

Radioisotope Incorporation Systems

A4w2ino Acid Tncorporafion id0 Proteins-For whole cell iu- corporation, washed cells were resuspeuded in chenlically defined growth medium, without amino acids, at a concentration of 1.5 to 2.0 ml of packed cells per 390 ml of culture medium. The cells were incubated for 5 to 15 min in t.he presence or absence of ;mti- biotic. At that time, 0.05 mCi of W-labeled amino acids (In- terlratiollal Chemical and Nuclear, specific a&i&y = 1 mCi per mg) was added along with 0.5 ml of a 20 mg per ml mixture of five unlabeled amino acids (L-asparagine, L-cystine, L-glu-

tamine, L-methiouille, and L-tryptophan). After brief shaking, the cultures were incubated for i0 min.

For [W]leucine incorporation by isolated mit,ochondria (24, 25), washed mitoclloudrin were suspended in 0.25 M sucrose in IO 1llM ‘l%s~IICl, $1 7.4. XIit,ochondrial suspension (0.1 ml) ~1s added to 0.9 ml of 30 mar Tris-HCl, pH 7.2; 20 m&l KCl; 40 nm IilV.,, pT-I i.2; I 1~1~ Na~EDT;\; 10 m11 ?rIgC&; 1.6 InM

.YI’P; 1 mg per ml of L-amino acid mixture without leucine; and 1 MCi of L-[‘4C]leucine. This mist,ure was incubated at’ 25” with shaking.

[3H] Uridine Incorporafion info RSd---The procedure for RN,1 synthesis was identical wit.11 that used for protein synthesis, es- cept that the medium contained amino acids and did not contain uracil. .\ft,er incubation for 15 min in the presence or absence of cyclohesimide, [LH]uridine (New England Nuclear, specific ac- tivity = 5 Ci per mmole) w-as added and incorporation con- tiuued for 70 more miuutes at 28 * 0.5”.

Isolation of Jritocho&ria

Slitocholldrix were lxepared as preriously described (26) ex- cept that instead of 0.25 RI raffinose we substituted 0.25 M sucrose in 10 mhl Tris~TICl, 111 I 7.4.

Ribosomes

J1i!ocholldrinl---‘l’lle final mitochondrial pellet, (20 to 30 mg of protein) was resuspended ilk 0.5 to 1.5 ml of 2c; Triton X-100 in 13uffer .\ (10 mn~ Tria-IICl, pH 7.4; 10 m1\1 31gClz; 100 n1~1 KCl) and lysed for 15 min at 4”. The lgaate was cent,rifuged at 17,300 x g for 10 min. The supernatant was layered on sucrose gradients.

Cytoplas,)Gc-The cell homogenate in 0.25 JI sucrose-10 rnbf Tris-HCl, pH i.4, was cent,rifuged at 12,000 X g for 10 min. The supernatant was theu mised with an equal Tolume of 47; Ttiton

X-100 in 20 InnI Tris-IICI, pII 7.4; 20 mx XIgCl,; 200 II~I l\(‘l

and treated as the mitochondrial lyaate.

Isolation of R9A

Jlitochondrial RAVA-The mitorhondrial pellet was suspel&d in 30 ml of 10 mM Tris-HCl, pH 7.4; 0.3 M NaCl; and 1“; S1 !$I in a volume equal to 5 times the original packed cell volume, :tntl stored at 0” for 15 min. The lysnte was mixed with an equal volume of phenol-cresol-hydroxyquinoline (27) which 11x1 been saturated wit.11 the same buffer. .\fter shaking for 20 nlin in :ul ice bath, the mixture was centrifuged nt 5000 rpm for j min. The aqueous layer, containing rnit~ocholldrinl Rx.\, W;I~ htored on ice while the interphase was re-est.racted. The t.wo :iqut’orls phases were then pooled and mixed with au equal volume 01 cold 959; ethanol and precipit,nted for 3 hours at -20”.

Cyfoplasmic RNA-Packed cells were suspended it1 ti tinles

their volume of 0.25 M Sucrose in 10 111~ Tris-IICl, pll i.4; 10 I~I lVIgCIZ; 0.3 1\1 NaCl; and 100 pg per ml of n~;~loitl :LII~ Iioi~~o~-

enized through :I ~e:uu l~omogt~nieer. ‘I’lw llolllogcw:l tr \\‘:is

centrifuged at 13,000 rlxn for IO min to reniovc Illitoc’liotlclt,i:l and other cellular debris. l’hr l)o”t,nlit,oc’llotldri:ll sul)t~rll:~t:~l~t, was then decanted :md 1 O(‘; Sl IS :&Id t.0 :i filcl.1 conwntr:itioi1 of 3 ‘i; . This material W:IS tIleI treated ill t.he s:~nle f;lsllion :I:: the mit,ochondri:tl material escept that the iut,erph:~se from the phenol tren.tment was not, eXtWC.td.

lifter precipit.atiou, both RN:\ samples were ceut.rifuged ;It. 15,000 rpm for 15 min t’o pellet the RN;\. The pellet was tlis- solved in a small volume of 10 ~JI Tris-I-ICI, pII 7.4, and 0.3 SI NaCl. That solut’ion was mixed I\-it11 an equal volume of rthel three times and consecutively aspirated t,o remove all tr;lce:: of phenol. Finally, air xas bubbled through the solution to remn~e remaining ether.

Preparation and Analysis of Ribosomes awl Ribosonwl RSA

Discontinuous Sucrose Gradients (for Gel Blectrophoresis)

Sucrose (1.5 ml, 2.05 11) in 10 nx~ Tris~ITCl, l)N i.4; 10 II~JI MgClz; 100 rn%f KC1 n-as o-vedayed with 0.1 ml of l.iG nr sucrose in the same buffer. blitochondrial or eytoplaamic lysates were carefully layered on top. Centrifugation was a.t 38,000 rpm for 17 to 20 hours in the So. 40 fixed angle rotor in t,lle Spiuco model L preparative ultracent’rifuge at 4”. This procedure results ill :I ribosome pellet, whereas nonribosonxd nxrtrrial renr:litls ill the soluble phase (3, 28).

Linear Sucrose Gradients

Gradient,s were prepared :lccortling to t,he met,hotl of l<rittell aud Robert,s (29), but varied in c*omposition deprlldillp OII tile material to be analyzed.

Ribosomes-These gradients contained 10 to 30’ ( ~ucro~; 10 nlM Tris-HCl, pH 7.4; 10 n1n1 MgCI?; 100 mnr KC1 buffer. The volume of the gradient :u~d time of centrifug:\t.ion were variable and thus are described with each experiment. MteI formation the gradients were cooled for at least 60 min it1 :I w

frigerator. RIVA-The 36 ml of 10 to 30(:; R?;ase-free sucrose gradielltc;

were made in 10 rnlr Tris-IICl, pH 7.4; 0.3 n1 KaCl; O.;i(‘; SW;. Centrifugation was for 14 hours in the SW 27 rotor at 26,000 rpm at a temperat’ure of 25”.

1 The abbreviation used is : SDS , sodiu1n do&c)-1 sulfatc~.

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A. J. ?‘. Millis and Y. Suyama. ‘$065

Fractions were rollected from each gradient by placiq the nitrocellulose t,ube ill a holder and puncturing the bottom of the tube with a needle. The lnaterial was then pumped through a 0.5cm flow c?ell and collected in 1.0.ml fractions. .Ibsorption was monitored at 260 nm with :I Gilford automa,tic recording ~pectro])hototneter. .2bsorption at 260 nm was later measured iu each fraction. For mdiouctivity determinations, each fract’ion \V:W treated \vith 5”~’ c cold trichloroacetic acid, and precipitates were collected by IsacT membrane filters, which were then dried :utd counted in a Packard scintillation spectrometer.

Polyacrylamide Gel Electroph~oresis

Sa,,lple Preparation-The ribosomal pellet from the discoa- t,illuous sucrose gradient was carefull:- rinsed in Buffer A to re- move all traces of sucrose. The pellet was then suspended in 10 nlhl Tris-HCl in 25, SIX3 and placed in a boiling wa.ter bath for 2 min. The heated sample was then mixed with P-mercapto- ethanol to 37; and glycerol to IOr; This material was applied to the top of the gel ~~lunlt~ ill :I volume of 20 to 60 ~1 atld over- layed with bromphenol blue.

The gels were pr.el)ared in glass columns (10 x 0.6 cm) (irmrl diameter) that had been arid-washed ;uld treated wit’11 1 ?; col- umll coat, solution. They collsist,ed of a small (one-fifth to one- sisth total volume) 3$; acrylamide npl)er Kel and a lo?; acryl- amide lower gel. Ilasically, they were constructed as described by Jt’eber and Osborn (30).

Elect.roplloresis was ruti for 120 to 150 miu, at room tempera- ture, at 8 111.1 per tube (24 mh t.otal) in a tray buffer composed of 6.0 g of Tris, 2X.8 g of glyrine, and 0.1’:; SIX made t,o 1 Mer. -1fter elect,royhorrsis, the gels were &ained in 0.5“; Coomassie brillinilt blue, made in 50c; trichloroacetic acid, for 60 min. A\t that tinle t,hey were rinsed in ‘ic; h 4tcial acetic acid and destained overnight in a large volume of 7~; acetic acid.

Coml)lete gels were scauned at 600 nm at a rate of 1 cm per mill, in the Gilford recording sl)ectropllot,ometer with gel scan attachment.

The distribution of radioactivity wan determined as follows. The gel ~1s hand-&red into about, 80 equal t-mm thick pieces with a razor blade. The slides were placed into scintillation I-ials with 0.5 ml of 30’:; H,O, and tightly capped. A\ft.er heating in an 80” oven for 60 min the slices were completely solubilized :IIKI blem+ed. ‘I’he cttps were removed from each vinl and t.he hydrogen lleroside solutiotl was mised with 5 ml of :I 5 : I .5 mis- tilre of scintillation fluid (IX Spectrofiuor, .1mershum) to Triton X-l 00. The resulting solutiou W:IS agitated until clear. The C:IJIS were replaced and the samples rounted in a Packard 3000 ‘I‘ri-Garb scintillatio~l spectrometer,

‘I’o obtain molec~ular \veigllt, estimates of samples, a standard calibration cur\‘e nxs collstructetl with the use of bovine serum :dl~utnin, oralbur~~iu, tryp&l, and cytochronie c.

I’rot.eill collctetlt.rat,ioll 1~s assayed by t.lte Folin method (31) 1vit.h bovine serum albuntin as a st.andard. For gel elect,rophore- Sk;, :I ~tantlard wrve was const,rnct,ed relating bovine serum al- bunritl cotlcentratio~l t.o :~bwrb:mre at 600 run. =Imounts of bovine serum albumill raiigilig from 5 to 70 j.48 were simultnne- ousl~ run on gels of the same cons;truction as those used for ribo- somu proteius. --ifter scaluliug, the area under the peak was traced on glassine \veighing leaper, cut out, and paper weight plotted wrsus proteili. Also, the absorbance at the highest point of the peak was plotted t’ers~(s protein. The I\-eight of the ,):\,,er is linear with respert to proteil[ concentration.

Minutes

FIN. 1. Effect.sof ~hlonrmpherricol (C,4P) and eyclohesimide (CH) on prot,ein synthesis by isolated mitochondrin. Isolnted nlit o- chondria were incubated at 4 mg per ml of in. vif~ :~nlil~o :lcsid incorporation medium containing [‘~‘(:lleuciuc (see “M:ltcri:ds :111d Methods”). Alicl(lots (0.10 nil) were sarnplcd at vrlriorls tinlcs and hot 10%: trichloro:lcetic :rcid-~recil)it;lt)le (bollnts \vcrr tlctcr- mined by t,ho method of XIalls and Novrlli (32). The COIIVCII- trations of chloramphenicol :\lld c*ycloheximid(: wcrc O.,Y :tlld 1 .O nmr, respectively.

Fractions 20 to 32 were collected after sucrose gradient centrifrl g&ion (see Fig. 2), pooled, a.nd assayed for protein and acitl-ill soluble radioactivity. L)atn for chloramphenicol-treated mile- chondrial and cytoplasmic soluble proteins were obtained from the same culture with a parallel culture providing the tintreated data. A similar procedure was used to obtain data for cycle- heximide-treated soluble proteins.

Source of soluble protein

Mitochondrin. cytop1ssm. Mitochondria. Cytopl:tstn.,

I.- /

.- Antibiotic treatment

i Chloram- Untreated j phenicol

“Y,$,,-

, (0.5 mu) / (0.1 lmf)

-

Percentage of inhibition

Chloramphenicol-The effects of chlora~lll~l~e~~icol oII i,/ &To mit.ochondrial prot.ein synthesis are lye11 documented (I) md

appear t.o be sirnilar to x&at, is observed in ?%trahywer.:n (Fig. I). However, it. is difficult. to assess t,he effects of this antibiotic, in viva, since only a.bout, 2“; of cellu1a.r prot.ein synthesis is mit.o- chondrial (20). We have fractionated IT-hole cells, labeled \vith ‘Gamine acids in the presence of 0.5 mhf chlor~lltlpllellit~ol. Soluble proteins collected from t,he mitochondrial fraction appeal

slightly more inhibited than those from the corresponding cayto- plasmic fraction (Table I). However, because mitocltondrinl protein synthesis accounts for such a small minority of total cellli- lar prot,ein sytAlesis, it is likely that the iuhibiticm of c-ytol>huic

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Biosynthesis of Mitochondrial Ribosomes Vol. 247, Ku. 1”

proteins by chloramphenicol resulted from a secondary effect of this antibiotic (33, 34).

In the following experiments we selected a concentration of chloramphenicol (0.5 mM) that inhibits over 95cG of [i%]leucine incorporation by isolated mitochondria (Fig. 1). That concen- tration inhibits only 307, of whole cell uptake of amino acids.

Sucrose gradient centrifugation analysis of cytoplasmic and mitochondrial extracts revealed that ribosomal regions from both sources receive significant radioactivity (Fig. 2). In these ex- periments the culture was treated with antibiotics for 15 min

I 0.m

I -1

cl

A

1000

800

400

200

10 20 Fraction Number

FIG. 2. Sucrose gradient profiles of mitochondrinl and cyto- plasmic extracts after 70.min incorporation of ‘%-amino acids with or without 0.5 mM chloramphenicol. Two-milliliter packed cells grown in chemically defined medium were inoculated into fresh medium without amino acids. After 5 min in the presence of chloramphenicol, 0.05 mCi of 14C-amino acids was added and incubation continued for 70 min. After the 38-ml gradients had been centrifuged at 26,000 rpm for 4% hours in the SW 27 rotor, fractions were collected from the bottom of the sucrose gradients and assayed for At60 and for 10% trichloroacetic acid-precipitable radioactivity. The bottom of the tube is represented in Fraction 1. Mitochondrial ribosomes without (A) or with (B) chloram- phenicol. Cytoplasmic ribosomes without (C) or with (D) chlor- amphenicol.

%

C

0.

C 80.5

c &I

lb ’ 2b ’

Fraction Number

FIG. 2C.

lo 20

Fraction Number

FIG. 20.

800

600

i

Fraction Number

FIG. 2B.

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Issue of June 25, 1972 A. J. T. Millis and Y. Suyanza 4067

before addition of W-amino acids and subsequently incubated for 70 min. In a typical experiment, it was found that the radio- activity of cytoplasmic ribosomes was reduced by 29yc,, when compared with the control, whereas that of the mitochondrial ribosomes was reduced by 64$&. In general, the synthesis of mitochondrial ribosomes appears to be more susceptible than cy- toplasmic ribosomes to inhibition by chloramphenicol (Table II).

It was repeatedly observed that mitochondrial ribosomes ex- hibited a specific radioactivity several-fold greater than cyto- plasmic ribosomes. Our evidence suggests that the radioactivity of both samples represented counts incorporated into the proteins that compose the ribosome and is not the result of incorporation into RNA or present only in nascent polypeptides.

Ribosome preparations obtained from whole cells previously subjected to alkaline hydrolysis or treated with puromycin did not show significant reduct,ion of their specific radioactivities (Table III). Further, the results of a long pulse iu which cells were labeled for 25 hours demonstrated that the specific radio- activities of the two t,ypes of ribosotnes tend to approach the same value.

T.\DLE II

Specilk ra&oactivit~J (cp~/~-t 200) of mitochondrial and cyloplusn2ic

ribosomes after chloramphenicd or cycloheximitle trenlnaent

I- blitochondrial 4214 1533 64

0.1 rnM 3243 120 >96

1.0 rnM 1030 58 >94 Cyt oplasmic 435 309 29

0.1 mnl 341 85 75

1.0 rnM 386 24 94

Specific activity (cpm/A 260) of mitochondrial and cytoplasmic ribosomes after puromycin chase, alkaline hydrolysis, .-d _’

and long term labeling ~-xFT

Ribosomes were prepared as described in the legend to Fiz The data are the result of five inde

Treatment

- I Mitochondrial (Ml

:p

I

Control Treated

Puromycin” Puromycin and ex-

cess unlabeled amino acids

Alkaline hydrolysisb Long term labeling

12 hours” 25 hour@

1,874 3,420

3,540

15,400

4,580

l,G50 6,620

2,730

I

2ndent experiments.

Cytoplasmic (C)

Control T

340

Treated

400 450

458 423

1,400

2,848 I

Ratio (M/C)

Zontrol Treated -__

4.1 10.0 14.7

7.7 6.4

3.9 1.6 /

* Puromycin treatment. (0.18 miq for 15 min) after W-amino acid incorporation. Puromycin, at this concentration, com- pletely inhibit,s further amino acid incorporation into whole cell protein as well as mit,ochondrinl protein.

b Alkaline hydrolysis (0.3 N KOH, 16 hours at 37”) and hot trichloroacetic acid-precipitable radioactivity.

c *G-Amino acids (20 pCi) present at the start of the labeling period.

d ‘Y-Amino acids (8 &i) present at the start of the labeling

period.

It appears that the differential specific radioactivity was not due to an artifact of ribosome isolation or specific radioactivity determination, rather that the amino acid or protein precursors for the ribosomes are drawn from distinct pools or that mito- chondrial ribosomes turn over more rapidly than cytoplasmic ribosomes. In these experiments contamination of mitochon- drial ribosomes by cytoplasmic ribosomes has been shown to be less then 27, (3). As seen in a later section, our analysis of RNAs isolated from mitochondrial ribosomes shows very little contamination with cytoplasmic RNA. Furthermore, the gel

IA

I- ZOl- Q

10 20

Fraction Number

1 Q 200

loo

Fraction Number

FIG. 3. Sucrose gradient profiles of mitochondrial and cyto- plasmic ext,racts after 70..min incorporation of ‘“C-amino acids with 1.0 IIIM cycloheximide. Packed cells (1.80 ml) grown in pro- teose-peptone medium were suspended in chemically defined me- dium without amino acids. Aft.er 15 min in the presence of 1.0 mu cycloheximide, 0.0.5 mCi of 14C-amino acids was added and in- cubat,ion continued for 70 min. After the 38.ml gradients had been centrifuged at : S,OCO rpm for 4$ hours, fr;l,ciions were col- lected and assayed 3s i:i 1 i:.;. 2. A, mit,ochondri:il; Li’, c,, toplnsmic.

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4065 Biosynthesis of Mitochondrial Ribosomes Vol. 247, x0. 12

patterns of these ribosomal proteins from the two types of ribo- somes are quite distinct (see Fig. 6).

Cyclohezimi~e-Similar experiments were conducted with cy- cloheximide. Concentrations of cycloheximide up to 1.0 mM showed no effect on [Wlleucine incorporation into protein by isolated mitochondria (Fig. 1).

The effects of cycloheximide on amino acid incorporation into ribosomes were examined by sucrose gradient analysis of cyto- plasmic and mitochondrial extracts (Table II and Fig. 3). Amino acid incorporation into both types of ribosomes was found to be drastically inhibited by 0.1 mM and 1.0 mM cycloheximide. At both concentrations radioactive peaks were not detected in mitochondrial ribosomes. However, it was not possible to eliminate all of the radioactivity from the cytoplasmic ribosomes.

Examination of soluble protein fractions for acid-insoluble radioactivity shows that both fractions mere greatly inhibited (Table I). The mitochondrial soluble proteins were less inhibited under these conditions, but the difference was small.

1Qjixt of Cycloheximide on Capacity for Amino Acid Incorporation by Isolated Mitochondria

The implicat.iorw of the drastic inhibition by cycloheximide, in viva, are that the contribution of mitochondrial protein syn- thesis to tot.al cellular protein synthesis is small (17, 19, 20) and that the ability of the mitochondrion to synthesize proteins

r 0.001 mM

SK) 20 30 40

Minutes FIG. 4. Effect.s of cycloheximide pretreatment of whole cells

on protein synthesis by isolated mit,ochondria (A) and whole cells (B). Aft.er 30-min incubation of whole cells with various con- centrations of cycloheximide, cells were harvested and washed twice with 0.25 M sucrose, Tris-HCI, pH 7.4 buffer. [lG]Leucine incorporation into acid-insoluble material by whole cells was de- termined in chemically defined medium and by isolated mitochon- dria in in vitro amino acid incorporation medium.

greatly depends upon cytoplasmic protein synthesis (21, 22). Under conditions in which cytoplasmic protein synthesis becomes limiting, the mitochondrial system becomes defective. There- fore, studies of protein synthesis by isolated mitochondria provide a means for investigating these defects.

Mitochondria u-ere isolated from whole cells which had been treated with cycloheximide in viva (previously treated) and es- amined for the capacity to incorporate [14C]leucine into protein. As shown in Fig. 4, incorporation capacity was affected by in- creasing concentrations of cycloheximide up to 1.0 m&r. Hom- ever, inhibition increases only slightly in the range above 0.005 mM, and 50% of the activity remains. A similar prior treatment of whole cells, however, resulted in increased inhibition with increasing cycloheximide concentration (Fig. 4). Fig. 5 shows

30.

P e 25

5 20

15.

10.

5

.g 1000

F a

i? 2 e

500.

3

4 . l.OmM

lo 20 30 40 50 Minutes

FIG. 4R.

/‘+-- CONTROL

Minutes FIN. 5. Effects of preliminary incubation time with cgclohexi-

mide on [W]leucine incorporation into protein by isolated mito- chondria. After addition of 5 mM cycloheximide, cells were harvested at various times from 5 to 60 min. [‘4C]Leueine incor- poration by isolated mitochondria was measured, as in Fig. 4-4, by sampling 0.10 ml of each reaction every 10 min.

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that mitochondrial inhibition does not increase after 30 min of prior treatment.

These results suggest that the inability of mitochondrial ribo- somes to incorporate 14C-amino acids, in the presence of cyclo- hesimide, was not due to the complete loss of mitochondrial synthesizing capacity, but rather due to the reduction of some other cytoplasmically synthesized factors specifically responsible for ribosome production.

E$ect of Chloramphenicol and Cycloheximide on Synthesis of Individual Ribosomal Protein

The observation that chloramphenicol does not prevent mito- chondrial ribosome synthesis can be interpreted to mean that mitochondrial ribosomal proteins are synthesized by the cyto- plasmic system (7, 8). Whether all ribosomal proteins are syn-

I

20 40 60 80’

20 40 60 BOTTOM GEL LENGTH (MM1

-

TOP

FIG. 6. SDS-polyacrylamide gel electrophoresis patterns of mitochondrial and cytoplasmic ribosomal proteins. Gels were scanned at 600 nm in a Gilford automatic gel scanner for AWI profile. They were subsequently sliced from the top into l-mm pieces and 14C radioactivity was measured as described under ‘<Materials and Methods.” Mitochondrial ribosomal proteins without (A) or with (B) chloramphenicol. Cytoplasmic ribo- somal proteins without (C) or with (II) chloramphenicol.

thesized under the chloramphenicol inhibition has never been tested. We tested this point by SDS-polyacrylamide gel electro- phoresis. Fig. 6 shows the results of electrophoresis of bulk ribosomal proteins obtained from cells treated with or without chloramphenicol in viva.

Mitochondrial and cytoplasmic ribosomal proteins were re- solved into at least 32 protein ba.nds ranging in molecular weight from 100,000 to 13,000; but they show different patterns of molecular weight distribution. In separate experiments, mito- chondrial ribosomal proteins were analyzed after washing the ribosomes with 1 M NH&l, and the patterns were quite similar. Comparison of the radioactivity profile of gel slices from chlor- amphenicol-treated mitochondrial ribosomes reveals that syn- thesis of mitochondrial ribosomal protein in the range from 13,000 to 25,000 molecular weight appears completely suppressed in relation to those of high molecular weight (Fig. 6). With cyto- plasmic ribosomes, no such effect was apparent. Similar esperi- ments with cyclohesimide-treated samples of both mitochondrial and cytoplasmic ribosomes did not show any significant radio- activity (no data is shown here). These data demonstrate that some mitochondrial ribosomal proteins are susceptible to chlor- amphenicol inhibition, but the extent of inhibition of other indi- vidual proteins cannot be accurately evaluated at present be- cause of considerable overlap between protein bands.

Efect of Cyclohexirnide on Mitochondrial RN=l Synthesis and Ribosome Assembly

Since RNA-DNA hybridization studies demonstrated t,hat mitochondrial rRNAs (21 S and 14 S) are transcriptive products

TOP 20 40 60 go ‘BOTTOM

GEL LENGTH (MM1

FIG. 6B.

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Biosynthesis of iifitochondrial Ribosonzes Vol. 247, Ko. 12

rr

00

h

L 1 TOP 20 l l 60 BOTTOM

GEL LENGTH IYYI

FIG. GD. 0’ 1 TOP 20 .o 60 BOTTOk

Cycloheximide inhibits both mitochondrial and cytoplasmic rRNA species. However, mitochondrial rRNA was more es- tensively inhibited than cytoplasmic rRNA (Table IV). In the present experiments, cytoplasmic rRNA was inhibited lo?;,, whereas mitochondrial21 S was inhibited 78y0 and 14 S inhibit,ed 60yG. It is not certain what proportion of these labeled mnt,e- rials represents mRNA.

The question of whether or not mitochondrial ribosomes are assembled in the presence of 0.1 mM cycloheximide was resolved by a study of [3H]uridine incorporation into 80 S ribosomes (Fig. 8). Whether or not cycloheximide was present, both cytoplasmic and mitochondrial ribosomes incorporate radioactivity which resists treatment with 1 pg per ml of RNase before centrifugation.

An additional peak of radioactivity appears with the cyto- plasmic ribosomes. This peak is suspected to be of 70 S, a sta.te of Tetrahymena ribosomes that is known to exist both during synchronous conditions and at 10e4 M Mg2+ (3, 4, 35). The sig- nificance of this peak requires further study.

These data establish that a pool of ribosomal proteins exists within the cell and these proteins may be used, in the absence of

GEL LENGTH IHM,

FIG. 6C.

of the mitochondrial genome (3, 4), it is particularly important to see how cycloheximide affects the synthesis of these molecules in cells.

Initially, bulk RNA was isolated from mitochondrial and cytoplasmic fractions after incorporation of [sH]uridine into whole cells and subsequently analyzed by sucrose gradient cen- trifugation. The results show that mitochondrial 21 S and 14 S and cytoplasmic 26 S and 17 S received significant radioactivity (Fig. 7). Material sedimenting at about 4 S was also labeled. Absorbance profiles of mitochondrial and cytoplasmic rRNAs approximate the 2: 1 mass ratio expected for the large and small molecular weight rRNA. The specific radioactivity of mito- chondrial 14 S was much higher than that of 21 S RNA, whereas the radioactivity of the cytoplasmic material closely follows the absorbance profile. Also, it was noted that the specific radioac- tivity of the two mitochondrial rRNA species was 5 to 10 times greater than the corresponding cytoplasmic rRNA species.

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Issue of June 25, 1972 A. J. T. Millis and Y. Xuyama 4071

further synthesis, to form new ribosomes. Further, since no incorporation of amino acids was detected in cycloheximide- treated mitochondrial ribosomes, proteins regulated by cyto- plasmic protein synthesis may control the synthesis of mitochon- drial ribosomal protein.

DISCUSSION

The results presented in this paper establish that. mitochondrial ribosomes can be synthesized during conditions of chloramphen-

TABLE Iv

Specific radioactivity of mitochondrial and cytoplasmic RXA I I I

Control Cycloheximide

Mitochondrial RNA 21 s 14 s

Cytoplasmic RNA 26 S 17 s

1708 376 3378 1323

271 246 243 251

!260

Percentage of inhibition

78 60

10 0

P I I

0

Fraction Number

FIG. 7. Synthesis of mitoehondrial and cytoplasmic RNA in the presence (B and D) or absence (A and C) of 0.10 mM cyclo- heximide. Cells were suspended in chemically defined medium without uracil and conditioned to 0.10 mM cycloheximide for 15 min before the addition of 0.2 mCi of [3H]uridine. Incubation was continued for 70 min at 28” f 0.5. Bulk RNAs were isolated from parallel cultures and analyzed by sucrose gradient centrifu- gation. Mitochondrial RNA without (A) and with (I?) cyclo- heximide; Cytoplasmic RNA without (C) and with (II) cyclohexi- mide.

c 8

0.5

1 10 2b ’ 3b

I 2.0

8 1.5

c

1.0

0.5

Fraction Number

265

? A

500

E 8

IO00

500

io . 2cl . 3b ’

Fraction Number

FIGS. 7B AND 7C.

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4072 Biosynthesis of Mitochondrial Ribosomes Vol. 247, No. 12

icol inhibition, but not during cycloheximide inhibition. Al- though these results are in good agreement with those obtained in Neurospora (7, 8), we present further evidence that some mitochondrial ribosomal proteins are susceptible to chloram-

I 2.0

3 1.5 e?

1.c

0.5

500

-400

k

-300

-200

7

0

I

:,

-600

Fraction Number

FIG. 70.

A

80s i

Fraction number

FIG. 8. Incorporation of [3H]uridine into mitochondrial and cytoplasmic ribosomes. Wet packed cells (1.7 ml) were suspended in chemically defined medium and incubated with cycloheximide for 15 min before the addition of 0.2 mCi of [3H]uridine. Gradi- ents (A and B) containing 37 ml of buffered sucrose were centri- fuged in a SW 27 rotor at 26,000 rpm for 41 hours. Gradients (C and D) contained 28 ml of sucrose and were centrifuged in a SW 25.1 rotor at 23,000 rpm for 7 hours. A, mitochondria with cyclo- heximide; B, mitochondria with cycloheximide and RNase; C, cytoplasm with cycloheximide; D, cytoplasm with cycloheximide and RNase.

0.4

-\

I 0.3

L

% -2

0.2

0. I

2 0

I 5

-.

!_

9 I.0 p”

0.5

20

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1

2 1.0

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Fracl~on number -”

IO 20 Frocl~on number

FIGS. 8R-8D.

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Issue of June 25, 1972 A. J. I’. Millis and Y. Suyama

phenicol inhibition. The present studies demonstrate that during incubation with 0.5 ma% chloramphenicol mitochondrial ribosomal protein with molecular weights between 13,000 and 25,000 do not incorporate radioactive amino acids into acid- insoluble product. In contrast, chloramphenicol does not appear to differentially inhibit individual cytoplasmic ribosomal pro- teins.

The question of whether these mitochondrial ribosomal pro- teins that are susceptible to chloramphenicol are synthesized exclusively by the mitochondrial protein synthesizing system cannot be satisfactorily answered, since such differential inhibi- tion may result from variation in the pool sizes of individual mitochondrial ribosomal protein. If chloramphenicol treatment makes the pulse time significantly short with respect to the dou- bling time, only certain proteins might be labeled regardless of their site of synthesis (36).

Subject to the shortcomings of our experimental data, if we postulate that the proteins susceptible to chloramphenicol inhibi- tion are synthesized by the mitochondrial system, why do those proteins not incorporate amino acids in the presence of cyclo- heximide? We have examined RNA synthesis in this connection to see what effect cyclohesimide would have on macromolecules known to be transcriptive products of the mitochondrial genome. Assuming that the mitochondrial transcription process itself is not. affected by cycloheximide, any effect that can be generated by cycloheximide on RNA synthesis would result from a second- ary process due to interdependencies between the two systems for protein synthesis. Indeed, we found that cycloheximide inhibits mitochondrial RNA synthesis to a greater extent than it inhibit,s cytoplasmic (uuclear) RNA synthesis.

The simplest interpretation of these results would be that some mitochondrial ribosomal proteins are synthesized by mito- chondrial and others by cytoplasmic ribosomes. The lack of incorporation of X-amino acids into mitochondrial ribosomes, under cyclohesimide inhibition, is interpreted as evidence that the synthesis of those proteins is under stringent cont.rol by the cytoplasmically synthesized prot.eins. A violation of this theory stems from the observat,ion made in our laboratory that mito- chondria isolated from Tetruhymena incorporate [%]leucine into ribosomes (25, 37). Neither chase with puromycin and cold leucine nor washing ribosomes with CsCl or 1 M NH&l after incorporation elimirlates radioactivity. However, the amount of synthesis that occurs in vitro may be too low to be physiolog- ically significant or detectable by the in viva system present.ed in this paper. The complete in viva inhibition of amino acid in- corporation by cyclohesimide into mitochondrial ribosomes would be due to a secondary inhibition resulting from the short- age of some cytoplasmically synthesized proteins that regulate mitochondrial ribosomal protein synthesis.

* There is experimental precedence for the interpretation t.hat some mitochondrial protein synthesis requires a cytoplasmically regulated protein product (21, 22). Whether this applies to ribosomal protein synthesis in other organisms needs further iurest,igut,ion. However, the recent experimental results of Rifkin aud Luck (16) are particularly interesting in this connec- tiou. It, has been shown that a cytoplasmic “poky” mutation in Newospora exhibits defects in the assembly of its mitochon- drial ribosomes. Woodward and Munkres (38) previously showed that the same mutation results in an alteration of the amino acid composition of mitochondrial structural protein. Although t,he nature and the significance of such a protein com-

ponent are presently under dispute (1)) the aforementioned data,

together with those presented here, allude to the interesting possibility that mitochondrial ribosomal protein synthesis re- quires the concomitant synthesis of a membranous component whose synthesis is, in part, dependent on cytoplasmic ribosomes.

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Albert J. T. Millis and Yoshitaka SuyamaTetrahymenaMitochondrial Ribosomes in

Effects of Chloramphenicol and Cycloheximide on the Biosynthesis of

1972, 247:4063-4073.J. Biol. Chem. 

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