products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 164, l-11 (1974)

Products of Rat Liver Mitochondrial Protein Synthesis:

Electrophoretic Analysis of the Number and Size of

These Proteins and Their Solubility in Chloroform : Methanol

JAMES P. BURKE’ AND DIANA S. BEATTIE

Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029

Received December 21, 1973

Rat liver mitochondria were incubated in uitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electro- phoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.

Mitochondria were incubated for 5 min with [3H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the A4, 27,000-40,000 range.

Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform : methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform : methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.

Yeast cells were pulse labeled in uiuo with [3H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform : methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a &fold stimulation of labeling into the band of lowest molecular weight of the chloroform : methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform : methanol.

During the biogenesis of mitochondria, proteins synthesized at two different sites in the cell are integrated into a functional

‘This work is in partial fulfillment of the require- ments for the Doctor of Philosophy degree at the City University of New York. Present address: Sinclair Comparative Medicine Research Farm, University of Missouri, Columbia, MO.

membrane. Although the vast majority (over 90%) of mitochondrial proteins are synthesized in the cytoplasm and trans- ported into the mitochondria in a subsequent step (l), the proteins synthesized within the mitochondria are essential for proper assembly of several enzyme complexes of the inner membrane (2-6).

Copyright Q 1974 by Academic Press. Inc. All rights of reproduction in any form reserved

2 BURKE AND BEATTIE

Thus, cytochrome oxidase (7, 8), oligomy- tin-sensitive ATPase (4), and a membrane fraction containing cytochrome b (3) have been shown to contain proteins synthesized on the chloramphenicol-sensitive mitochondrial ribosomes. In addition to the synthesis of these well-characterized en- zymes, Tzagoloff and Akai (9) have reported that all of the products of yeast mitochondrial protein synthesis are soluble in chloroform-methanol and hence defined as proteolipids. The major proteolipid observed by these workers was a low molecular-weight peptide soluble in neutral chloroform : methanol. Subsequently, we (10) also reported that rat liver mitochondria incorporate amino acids in vitro into proteins soluble in chloroform : methanol. Only 15% of the total radioactivity, however, was extracted into the proteolipid fraction. In addition, analysis by gel electrophoresis revealed that label was present mainly in a band of 40,000 M,. In contrast, Kuiela et al. (11) recently reported that rat liver mitochondria synthesize a single low molecular-weight protein which could be extracted with neutral chloroform : methanol.

Another approach to studying the products of mitochondrial protein synthesis has involved gel electrophoresis of whole or fractionated mitochondrial membranes labeled with radioactive amino acids either in vitro or in vivo in the presence of sufficient cycloheximide to block cytoplasmic protein synthesis. Experiments of this nature have demonstrated that mitochondria from mammalian tissues can synthesize several proteins ranging in molecular weight from 12,000 to 50,000 (12, 13). Furthermore, the proteins labeled by isolated mitochondria in vitro appear to have molecular weights identical to those synthesized by mitochondria in viuo (12-14). In contrast, Michel and Neupert (15) recently reported that the proteins synthesized on the mitochondrial ribosomes of Neurospora are of low molecular weight. These very hydrophobic proteins were apparently modified to proteins of higher molecular weights after or during integration into the mitochondrial membrane.

In the present study, the size distribution of the in vitro products of rat liver mitochondrial protein synthesis has been examined in several polyacrylamide gel systems. Six to eight different polypeptides ranging in molecular weight from 12,000 to 50,000 were noted to contain label after an in vitro incubation; however, a polypeptide of approximately 40,000 was labeled the most extensively even after very short incubation times. The synthesis of proteolipids by rat liver and yeast mitochondria was also investigated.

MATERIALS AND METHODS

Preparation of Mitochondria

Rat liver mitochondria were prepared under sterile conditions in a medium containing 0.25 M sucrose, 0.01 M Tris Cl, pH 7.8, and 0.009 M EDTA (sodium salt) by the method of Beattie (16). This yielded a pellet 3% contaminated with microsomal protein as determined by glucose-6-phosphatase activity.

A diploid strain of Saccharomyces cereuisiae was grown with either 1% glucose or 5% glucose as an energy source in the medium previously described by Kim and Beattie (6). Yeast mitochondria were prepared from cells harvested in late ldg phase in a medium containing 0.25 M mannitol, 0.01 M Tris, pH 7.4, and 0.001 M EDTA by use of a Bronwill Mechani- cal Shaker (17). All steps were performed under strictly sterile conditions; all glassware and solutions were autoclaved prior to use.

Amino Acid Incorporation Studies

Amino acid incorporation into mitochondrial protein in vitro was determined in a medium containing 50 mM Bicine buffer, pH 7.6, 90 mM KCl, 10 mM MgCl,, 1 mM EDTA, 5 mM potassium phosphate, pH 7.6, 22.5 rg of a complete amino acid mixture minus leucine, as described by Roodyn et al. (18), 5 mM

P-enolpyruvate, 2 mM ATP, 10 pg of pyruvate kinase, 3-4 mg of mitochondrial protein, and radioactive leucine as indicated in the legends to tables and figures. After 30-min incubation at 30°C in a meta- bolic shaker, the incubation was terminated by the addition of 10 mM unlabeled leucine, and the mitochondria were reisolated at 12,OOOg for 10 min. The mitochondrial pellet was then washed two times in sucrose containing unlabeled leucine (10 mM, final concentration) prior to fractionation.

Yeast cells were harvested by centrifugation at 1OOOg and washed once with water. The cells were suspended to a final concentration of 250 mg/ml in 0.05 M potassium phosphate buffer, pH 7.4, containing 0.1% glucose. Cycloheximide (100 &ml) was

MITOCHONDRIAL PROTEIN SYNTHESIS 3

added and the culture incubated for 15 min prior to addition of 50-60 rCi/ml of L-[4,5-3H]leucine. After incubation for 30 min at 3O”C, 10 mM unlabeled leucine was added and the incubation continued for 15 min. The cells were then harvested and mitochondria prepared as described above.

Fractionation Procedures

Reisolated mitochondria were brought to a concentration of 5 mg/ml and extracted with 1.4% acetic acid (final concentration), for 30 min at 0°C as described by Zahler et al. (19). The insoluble residue was removed by centrifugation at 80,OOOg for 1 hr. The pellet was washed once with the original volume of 1.4% acetic acid and recentrifuged at 100,OOOg for 1 hr. The resulting pellet was resuspended to a concentration of 10 mg/ml in isolation medium and the nonionic detergent Lubrol was added at a concentration of 1 mg/lO mg of mitochondrial protein. The suspension was centrifuged at 80,OOOg for 1 hr. The resulting pellet was washed with distilled water and recentrifuged at 80,OOOg for 1 hr.

Sonication

The washed mitochondrial pellet was resuspended in isolation medium to a final concentration of l-2 mg/ml. Sonication was employed at maximum output of an Ultrasonic Model W185D sonifier for 15 sec. The sonicated mitochondria were centrifuged at 164,OOOg for 30 min.

Extraction of Proteolipids

Submitochondrial particles containing 5 mg of protein were suspended to I ml in isolation medium and extracted with 9 ml of chloroform: methanol (2: 1, v/v) for 30 min at room temperature. The extracts were centrifuged at maximum speed in a clinical centrifuge. The lower layer was washed once with 5 ml of water and three times with 10 ml of chloroform: methanol: water (3:48: 47) (20). In some experiments, the initial chloroform : methanol extract was used without the washes.

Alternatively, submitochondrial particles were lyophilized and then extracted with 10 ml of chloroform : methanol (2 : 1, v/v). The chloroform : methanol-soluble fraction was used as such. The extracts and residues from all methods were dried under a stream of nitrogen at 50°C and dissolved in either 1% sodium dodecyl sulfate for counting and protein de- termination or in the medium used for gel electrophoresis. The solutions were heated at 50°C until the proteins were solubilized.

Gel Electrophoresis

The various membranous fractions were resuspended in isolation medium at a concentration of I-2

mg/ml, sonified at maximum output for 15 sec. and centrifuged at 164,000g for 30 min. The pellets were extracted with 5 vol of 5% trichloroacetic acid. The precipitated proteins were washed three times with distilled water and dissolved in a solvent system containing 10% glycerol (w/v), 1% SDS, 1% mercapto- ethanol, 0.01 M sodium phosphate buffer (pH 7.1), and 0.002% bromphenol blue at a protein concentration of 2 mg/ml (4). To insure monomerization and prevent subsequent proteolysis (21). the protein solution was heated at 70°C for 20 min. The proteins were separated on 7- or lo-cm gels according to the method of Weber and Osborn (21). The electrophoresis was carried out at room temperature on gels made 7% with polyacrylamide at a current of 8 mA per gel for approximately 3-4 hr or on 10 or 12% polyacrylamide gels at a current of 3 mA per gel for approximately 16-24 hr. Gels were stained overnight with 2% Coo- massie blue in methanol: acetic acid: water (5: 1: 5) and were destained electrophoretically in the same solvent system. In some cases, the gels were stained after fixation in isopropyl alcohol as described by Fairbanks et al. (22). The stained gels were scanned at 540 nm in a Gilford spectrophotometer, equipped with scanning equipment.

Gels were calibrated according to the method of Weber and Osborn (21). Standards used were insulin (B chain), cytochrome c, chymotrypsinogen, trypsin, pepsin, alcohol dehydrogenase, ovalbumin, and bo- vine serum albumin.

Counting of Gels

Gels were sliced into 1.0.mm or 2.0-mm slices, depending on the experiment, with a Gilson Model B-100 Aliquogel Gel Slicer, and 0.5 ml of hydrogen peroxide was added automatically. The gels were heated overnight at 50°C or until completely dissolved. Ten milliliters of Bray’s solution (23) or 5 ml of toluene counting solution (24) were added, depending on the type of counting vials utilized. The radioactivity was determined in a Packard scintillation counter with an efficiency for “C of 77% and 3H of 25% in Bray’s solution and 908 for “C and 27% for ‘H in the toluene counting solution.

Protein Analysis

Protein concentrations were determined by the method of Lowry et al. (25) or by the Biuret method described by Gornall et al. (26). Proteins were prepared for counting by previously described methods (27).

Materials

Uniformly labeled L- [“Clleucine (250 Ci/mole) and L- [4, 5-SH]leucine (50 Ci/mmole) were obtained from Amersham-Searle. ATP, P-enolpyruvate, pyru- vie kinase, Na dodecyl sulfate, cycloheximide, and

4 BURKE AND BEATTIE

the standards used for gel calibration were obtained from Sigma. Acrylamide, bis-acrylamide, and tetra- methylethylenediamine, were obtained from Eastman. Lubrol was a gift of ICI American, Inc. Triton X-100 was purchased from Rohm and Haas.

RESULTS

Products of Mitochondrial Protein Synthesis

In a previous communication (27), we reported that amino acids are incorporated in vitro by rat liver mitochondria into a heterogeneous membrane fraction obtained by treating mitochondria with 1.4% acetic acid (19). This membrane fraction contained approximately 10% of the total mitochondrial protein and 85% of the total radioactivity incorporated into mitochondrial protein. As seen in Table I, extracting the acid-insoluble membrane fraction with the nonionic detergent Lubrol resulted in a loss of considerably more protein and insignificant radioactivity. The specific radioactivity of the final pellet obtained after extraction with acetic acid and Lubrol was 6-fold greater than the specific activity of the intact mitochondria.

Analysis of this preparation by SDS- polyacrylamide gel electrophoresis revealed 10 or more distinct protein bands indicated by staining observed with Coo- massie blue (Fig. 1). Radioactivity, however, was observed in only three labeled bands of approximate molecular weights equivalent to 40,000, 27,000, and 20,000

daltons. An identical radioactivity and protein profile was observed whether the Lubrol pellet was extracted directly with trichloracetic acid or sonicated prior to extraction and then dissolved in the electrophoresis solvent system. It should be noted that no significant radioactivity was observed in any of the low molecular- weight protein bands observed in the den- sitometric tracing of the same gel.

Addition of cycloheximide, the specific inhibitor of cytoplasmic protein synthesis, had no effect on the amount of radioactivity incorporated either into the intact mitochondria, or into the pellet obtained after extraction with acetic acid and Lu- brol (Table I). In contrast, chloramphenicol, the specific inhibitor of mitochondrial protein synthesis, blocked 85% the incorporation into intact mitochondria. Further- more, there was no increase in specific activity when the chloramphenicol-treated mitochondria were fractionated with acetic acid and Lubrol. As seen in Fig. 2, the same three bands contained radioactivity after gel electrophoresis of the Lubrol pellet obtained from mitochondria incubated in the presence of cycloheximide as compared to control mitochondria. No radioactive label, however, was present in any part of the gel after electrophoresis of the Lubrol pellet obtained from mitochondria incubated with chloramphenicol.

Several recent studies have indicated that mitochondria isolated either from

TABLE I

DISTRIBUTION OF RADIOACTIVITY AFTER FRACTIONATION WITH ACETIC ACID AND LUBROL~

Fraction

Specific activity

(cpmhd

Control + + Chloramphenicol Cycloheximide

Protein Total cpm (cpm/mg) (cpmhg) (md

Mitochondria 4,100 50 205,000 492 3,800 Acetic acid-soluble 74 28 2,070 - -

Acetic acid-insoluble 14,200 12 170,400 480 11,100 Lubrol-insoluble 25,100 6.5 163,200 530 24,900

a Mitochondria from one liver were incubated for 30 min in 25 ml of incubation medium containing 50 mM Bicine buffer, pH 7.6, 90 mM KCI, 10 mM MgCl,, 1 mM EDTA;5 mM phosphate, pH 7.6, 22.5 pg of an amino acid mixture minus leucine (la), 5 mM p-enolpyruvate, 2 mM ATP, 10 pg of pyruvate kinase, and 0.25 pCi/ml of [Wlleucine. After the incubation, mitochondria were reisolated at 12,OOOg, washed once in 0.25 M sucrose containing 10 mM unlabeled r.-leucine, and fractionated. In parallel experiments either 1.6 mM cycloheximide or 0.47 mru chloramphenicol was also present.

30 the mitochondrial membrane fraction pre-

27 pared with Lubrol to permit resolution of

GEL SCAN peaks containing very low counts. To test 2.4 i these possibilities, rat liver mitochondria

E2 ’ were incubated with sufficient [3H]leucine 6 1.8 of high specific radioactivity, to ensure the ::I5 presence of a membrane fraction contain-

2 1.2 ing very high counts. Submitochondrial

.9 particles were then prepared by sonication and were subsequently analyzed on gels

6 containing a final concentration of 12% 3 acrylamide for 24 hr. The gel profiles 0 revealed radioactivity in the three bands

180 corresponding to molecular weights of AA INCORPORATION 40,000, 27,000, and 20,000, as previously

LUBROL EXTRACTION observed in electrophoretic pattern of the Lubrol pellet on the shorter gel (Fig. 3). However, the predominant band of 40,000 molecular weight revealed two distinct shoulders of higher molecular weight while two radioactive peaks were observed in the M, 27,000 range. In this gel, significant radioactivity was also observed in several bands of radioactivity in the M, range

0 IO 20 30 40 50 60 70 below 20,000. FRACTION NUMBER

FIG. 1. Electrophoretic profile of radioactivity and In a recent report, Michel and Neupert

optical density scan of the residue obtained after (15) suggested that Neurospora mitochon-

extraction of mitochondria with acetic acid and Lu- dria may synthesize low molecular-weight

brol. Mitochondria were labeled and fractionated as polypeptides which are modified to larger described in the legend to Table I. The final residue proteins after integration into the mem- was prepared for electrophoresis as described in brane. We have attempted to test this Materials and Methods. Approximately 100 fig of hypothesis by incubating isolated mito- protein with a specific activity of 25,100 cpm/mg was chondria with radioactive leucine for very applied to each gel. The upper trace is the optical scan short times (5 or 10 min) followed by a of the stained gel and the lower trace shows the number of counts in each gel slice. I , I I I I

mammalian cells (12, 13), Neurospora (15, 28) or yeast (2,9, 17, 29, 30) may synthesize proteins with molecular weights in the lO,OOO-15,000 range. The absence of label in this region in our gels (Fig. 1) may result from several factors. Perhaps, these low molecular-weight proteins are not resolved on the 7% polyacrylamide gels in the short time allowed for electrophoresis. Secondly, the original studies of mammalian mito- I 5 IO 15 20 25 30 35

chondrial protein synthesis indicated that GEL SLICE NUMBER

their low molecular weight proteins may FIG. 2. Electrophoretic profile of Lubrol pellet

contain much less radioactive label than obtained from control mitochondria and mitochon-

proteins of higher molecular weight. dria incubated with chloramphenicol or cyclohexi-

Possibly, in our previous experiments mide. Incubations were performed as described in the legend to Table I. Approximately 100 c(g of protein


insufficient radioactivity was present in was applied to each gel.

6 BURKE AND BEA’M’IE

sooj.,,.,-,.,,.,.,l

0 IO 20 30 40 50 60 70 SO 90

FIG. 3. Electrophoretic profile of submitochondrial particles obtained from mitochondria incubated with [‘Hlleucine as described in Materials and Methods. The concentration of acrylamide was 12% and the time of electrophoresis was 24 hr. Approximately 150 pg of protein with a specific activity of 109,000 cpm/mg was applied to each gel.

pulse of unlabeled leucine for various periods of time.

As seen in Fig. 4, the radioactivity incorporated into protein increased significantly for approximately 5 min after addition of the unlabeled leucine chase to the incubation medium. The lag observed before the synthesis of radioactive protein ceased probably is a reflection of the time required for complete uptake of the amino acid into the matrix of the mitochondria (31). It should also be noted that in these experiments there was no loss of radioactivity in acid-precipitable protein during the 20 or 25 minutes studied after addition of chase.

The gel patterns of mitochondrial membrane proteins labeled for 5 and 20 min are compared to that of mitochondria labeled for 5 min followed by a 15min chase (Fig. 5). After a 5-min incubation, significant radioactivity was observed in the bands corresponding to molecular weights of 43,000 and 37,000. After addition of the chase, more counts were present in both these bands as well as in a well-defined band of h4, 26,000. The continued increase of acid-precipitable counts after addition of unlabeled amino acid probably results from continued synthesis of these proteins. The radioactive leucine incorporated into protein of lower molecular weight was not clearly resolved into peaks at the short times of incubation with or without the

chase despite significant total radioactivity. Moreover, no decrease in radioactivity in these peaks was observed after addition of the chase. After a ZO-min incubation, several radioactive bands of low molecular weight are clearly distinguished in mitochondrial membranes in addition to the three bands of higher molecular weight.

Proteolipids

Three different methods were used to prepare the so-called proteolipid fraction, those proteins soluble in chloroform : methanol. As seen in Table II, nearly 17% of the total radioactivity and 8.2% of the total protein were extracted from the submitochondrial particles by chloroform : methanol and were present in the chloroform layer. Washing the chloroform layer with aqueous solvents had little effect on the amount of protein present in the chloro-

I5

/

IO

‘: 0 -

; - ,”

5

:/

/’ p---~-----“--r

[%I leuclne- /’

.O-’ _,_. a-------o---.-.- ._,_, -o

,A

0 5 IO I5 20 25 30

FIG. 4. Time course of incorporation of [8H]leu- tine into rat liver mitochondrial protein. Mitochon- dria were incubated at 30°C in the medium described in the legend to Table I. At the times indicated by arrows, unlabeled leucine was added. (O--O) control, (A---A) chase added after 10 min, and (Om .- .O) chase added after 5 min.


form layer or in the amount of radioactivity present. In addition, when the submito-

375

600 - ! : :: ;: Ij 500 - :e

I: I !:

Gel slice (mm)

FIG. 5. Electrophoretic profile of mitochondrial membranes obtained from mitochondria incubated with [3H]leucine for various time periods. (O--O) indicates an incubation time of 5 min; (0-O) indicates an incubation time of 5 min, followed by an addition of a chase of unlabeled leucine and 15 min additional incubation; and (A- - -A) indicates an incubation time of 20 min. Numbers above the peaks indicate the molecular weights x 103.

chondrial particles were lyophilized prior to extraction, 60% of the original protein and 15% of the original radioactivity were extracted into the chloroform layer.

Gel electrophoresis of all the chloroform extracts was performed for 16 hr on gels containing 10% acrylamide. The gel pattern of the extract which was not washed (Fig. 6) appeared very similar to that of the extract subject to extensive washing (not shown). Four or five clearly distinguished bands of molecular weights ranging from 10,000 to 40,000 are apparent; however, the greatest label was again present in the band of approximately M, 40,000. In contrast, gel electrophoresis of the chloroform : methanol extract of the lyophilized mitochondrial membranes revealed only three broad peaks in the low molecular- weight region of 14,000-10,000 (Fig. 7). Insignificant radioactivity was observed in regions of the gel corresponding to molecular weights greater than 14,000.

In a previous study, Ibrahim et al. (17) reported that the rate of yeast mitochondrial protein synthesis measured both in vitro and in vivo was stimulated when yeast cells were allowed to accumulate products of cytoplasmic protein synthesis by growth of the cells for various times in chloramphenicol. It was hoped that this increased rate of labeling of mitochondrial membranes in vivo might be a reflection of an increased rate of proteolipid synthesis.

TABLE II

EXTRACTION OF LABELED MITOCHONDRIAL PROTEINS BY CHLOROFORM:METHANOL~

Nonwashed SMPb C : MC Extract

Washed SMPb C : MC Extract

Lyophilized SMPb C : MC Extract

Specific activity Protein (cpm/mg) (md

48,700 11.0 100,000 0.9

46,300 10.0 86,500 0.84

40,000 9.0 98,000 0.54

Total cpm

536,000 90,000

463,000 72,700

360,000 52,920

9% Total

16.8

15.7

14.7

D Mitochondria were incubated for 30 min in the medium described in the legend to Table I with 20 FCi/ml of [3H]leucine, reisolated, and submitochondrial particles prepared by sonication. The extractions with chloroform : methanol were performed as described in Materials and Methods.

b SMP, submitochondrial particles. ’ C : M, chloroform : methanol.

8 BURKE AND BEATTIE

65C ‘r 6OC

‘t 550

‘C 500

450

400 t

T 350

% 300

250 i t

200

I50

100,

OLI ’ ’ I I I ’ ‘1 0 IO 20 30 40 50 60 70

GELSLICE

FIG. 6. Electrophoretic profile of chloroform: methanol extract obtained from submitochondrial particles. The extraction was performed as described in the Materials and Methods without any washes of the chloroform layer. Approximately 100 pg of protein with a specific activity of 100,000 cpm/mg was applied to the gel.

To test this possibility, yeast cells were grown for 13 hr in 5% glucose at which time the culture was divided into two equal parts. To one was added chloramphenicol (4 mg/ml), while the other was used as the control. After another 3 hr of growth, both cultures were harvested, washed, and incubated in uiuo as described in Materials and Methods. Submitochondrial particles were prepared from both preparations and ex-

tracted with chloroform : methanol. An increased specific radioactivity was observed in mitochondrial membranes obtained from cells grown for 3 hr in chlorampheni- co1 compared to that of the control (Table III). Approximately 13% of the total radioactivity and less than 10% of the protein in the mitochondrial membranes was extracted with chloroform : methanol from the submitochondrial particles prepared from control cells or those preincubated in chloramphenicol. The gel profile of the chloroform extracts from the latter cells revealed an almost B-fold stimulation of

500” , , , , ,

7

oL, 1 I / 1 I I '...a A 0 IO 20 30 40 50 60 70

GEL SLICE (mm1

FIG. 7. Electrophoretic profile of chloroform: methanol extract obtained from lyophilized submitochondrial particles as described in Materials and Methods. Approximately 100 rg of protein with a specific activity of 98,000 cpm/mg was applied to the gel.

TABLE III

EXTRACTION OF YEAST SUBMITOCHONDRIAL PARTICLES GROWN IN CHLORAMPHENICOL FOR 3 HR AND TRANSFERRED TO FRESH MEDIUMS

Specific activity (cpmhg)

Protein (mg)

Total radioactivity (cpm)

% of Total

Control SMPb C : MC extract

Chloramphenicol treated SMP C : M extract

64,300 12 731,000 125,000 0.8 100,000 13.6

100,000 8 806,000 198,006 0.5 99,000 12.2

a Partially derepressed yeast cultures were grown for 3 hr in the presence or absence of chloramphenicol. The cells were washed, harvested, and incubated in uiuo as described in Materials and Methods. Submitochondrial particles were prepared by sonication and extracted with chloroform:methanol followed by the washes described in Materials and Methods.

* SMP, submitochondrial particles. ’ C : M, chloroform : methanol.


the radioactivity in the band of low molecular-weight compared to the control cells (Fig. 8). Furthermore, significantly less radioactivity was present in the two bands of highest molecular weight. This observation suggests that the difference in incorporation rates does not result from a change in the leucine pool size.

DISCUSSION

Gel electrophoresis of submitochondrial particles obtained from rat liver mitochondria labeled in vitro with radioactive leu- tine has revealed a fairly simple profile of labeled bands. Approximately 6-8 polypeptides ranging in molecular weight from 13,000 to 50,000 daltons contain label when the electrophoresis is performed in gels containing different concentrations of acrylamide. Analysis of gel patterns does not permit the quantitative estimation of the number of individual polypeptides present but only allows a measure of the range of molecular weights present. Nevertheless, the limited number of polypeptide bands observed after electrophoresis of mitochondrial membranes is consistent with the low

700,, / , , , , , ,,

650 o..... Control

600 .- CAP

300

250

200

150

100

50

0 IO 20 30 40 50 60 70 GEL SLICE (mm)

FIG. 8. Electrophoretic profile of chloroform: methanol extracts of submitochondrial particles obtained from yeast cells grown as described in the legend to Table III. (04) extracts from control cells; (04) extracts from cells preincubated in chloramphenicol.

informational content of mitochondrial DNA. Previous calculations (32) have suggested that mitochondrial DNA has sufficient information to code for at most 15-20 proteins with an average M, of 20,000.

Electrophoretic profiles of mitochondrial membrane proteins labeled in uiuo in the presence of cycloheximide by yeast (2, 9, 17, 29, 30), Neurospora (15, 28) HeLa cells (13, 33), and BHK cells (12) or labeled in vitro by rat liver mitochondria (12) appear very similar. The presence of 8-10 labeled bands in the same molecular-weight range has been reported for mitochondria of most species. It is of some interest that mammalian mitochondria appear to have the ability to synthesize the same number of polypeptides as mitochondria from yeast or Neurospora, despite the fact that the latter mitochondria contain significantly more DNA.

When liver mitochondria were incubated for short times, i.e., 5 or 10 min, with [3H]leucine followed by addition of a chase of unlabeled leucine for 25 min, no loss of radioactivity in the mitochondrial membranes was observed. Wheeldon (34) has recently reported a 30% decay in the acid- insoluble counts present in the membranes of rat liver mitochondria incubated under identical conditions. No explanation for this discrepancy is apparent; however, our mitochondrial preparation is almost completely free of contamination by lysosomes which may be a source of proteolytic en- zymes.

The gel patterns of the membranes obtained from rat liver mitochondria incubated for only 5 min indicated that significant synthesis of polypeptides in the M, 40,000 range had occurred during this short incubation, while little synthesis of polypeptides in the low molecular-weight range had occurred. The observation that no counts in the low molecular-weight regions of the gel were lost after addition of the chase suggests that rat liver mitochondria do not synthesize a low molecular-weight polypeptide which is subsequently modified to higher molecular weight as has been suggested for Neurospora mitochondria (15). The possibility that ribosomes containing nascent chains of lower molecular

10 BURKE AND BEATTIE

weight are present in our sonicated sub- phenicol. In our laboratory (17), we ob- mitochondrial particles, has not been ex- served that the actual rate of mitochon- plored; however, Coote and Work (12) had drial protein synthesis measured both in observed that nascent chains were present vitro and in vivo was stimulated under on liver mitochondrial ribosomes during 10 these conditions. This stimulation of pro- min of incubation. It has been reported ein synthesis did not result from increased that Neurospora mitochondria, incubated synthesis of all polypeptides in the mem- in vitro, have the ability to synthesize only brane but only from increased synthesis of polypeptides of molecular weight less than polypeptides of low molecular weight. 17,000 daltons. The lack of synthesis of The increased labeling of these mem- other proteins may be a reflection of the brane proteins is also apparently reflected conditions used for the in vitro incubation. in an increased synthesis of the proteolip- Numerous investigators have reported that ids of low molecular weight. The polypep- the products of mitochondrial protein syn- tide of M, 10,000 which contains signifi- thesis labeled either in vitro or in vivo are cant radioactivity when products of identical in HeLa cells (13), rat liver (12, cytoplasmic protein synthesis have ac- 14), and yeast. cumulated possibly correspond to the pro-

The results of the present study confirm teolipid observed in yeast mitochondria by previous reports that mitochondria synthe- Tzagoloff and Akai (9). Our results, how- size proteins which are soluble in chloro- ever, indicate that yeast mitochondria form : methanol (9-11). In addition, the may also synthesize a wide range of poly- data suggest that the procedures used for peptides similar to those observed in rat preparation of submitochondrial particles liver mitochondria. and for extraction with organic solvents may affect the labeling pattern observed ACKNOWLEDGMENTS

after gel electrophoresis (Figs. 6 and 7). This work was supported in part by grants from the Hence, the discrepancies which have been National Institutes of Health (HD-04007) and the reported concerning the molecular weight National Science Foundation (GB-23357). The au- of the pro&lipids synthesized by mite- thors thank Dr. Robert N. Stuchell for his help in the

chondria may reflect significant differences g rowth and preparation of yeast cells.

in the methods used for their preparation. One conclusion from all these studies is REFERENCES

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solvents. W., WHARTON, D. C., AND SALTZGABER, J. (1972)

The proteolipids synthesized by yeast Fed. Proc. 31, 21-29.

mitochondria in vivo also vary with the 3. WEISS, H. (1972) Eur. J. Biochem. 30, 469-478.

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products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and...

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