ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
170,
392-399
(1975)
Chloramphenicol
Resistant Mitochondrial in Crithidia fascicu/atal
RUSSELL
CAROL
Department
C. HILL, of Microbiology,
St. Louis
A. MORRIS, University
Received
School
February
AND
Protein
MORTON
of Medicine,
St. Louis,
Synthesis
M. WEBER’ Missouri
63104
3, 1975
Growth of Crithidia fasciculata, a heme-requiring flagellated protozoan, was not affected by the presence of chloramphenicol in concentrations as high as 4 mg/ml. Amino acid incorporation by a mitochondrial fraction, containing both mitochondria and kinetoplasts, was resistant to chloramphenicol and cycloheximide, but sensitive to puromycin. The postmitochondrial supernatant (27,OOOg) also incorporated [‘%]leucine into trichloroacetic acid insoluble material, but this incorporation was inhibited by cycloheximide. Chloramphenicol did not affect this system. Treatment of the mitochondrial fraction with digitonin enhanced the rate of YClleucine incorporation, but did not render the preparation sensitive to chloramphenicol. Electron microscopic examination of the digitonin treated mitochondrial fraction revealed the presence of ghosts. Separation of kinetoplasts from mitochondrial vesicles was achieved by discontinuous sucrose gradient centrifugation. Both the kinetoplast and mitochondrial fractions incorporated [Wlleucine at about the same rate. No inhibition of incorporation by chloramphenicol was observed with either fraction.
Extensive research on the biogenesis of mitochondria in fungi and animal cells has revealed that biosynthesis of functional mitochondria involves proteins made by the mitochondrial as well as cytoplasmic ribosomes (1). Mitochondrial protein synthesis is sensitive to chloramphenicol, but insensitive to cycloheximide in all systems so far reported (l-3). Proteins which are synthesized outside the mitochondrion on cytoplasmic ribosomes are inhibited by cycloheximide but not chloramphenicol. This differential effect of antibiotics has allowed for the identification of products of the two different systems, both in vivo, in growing cells (4, 5) and in vitro, with isolated mitochondria (6, 7). By contrast, very little is known about mitochondrial biogenesis in protozoa. 1 This work was supported by Research Grant AI 03046 from the National Institutes of Health, U.S.P.H.S. 2 Author to whom reprint requests should be sent. 392 Copyright All rights
0
1975 by of reproduction
Academic in any
Press, Inc. form
reserved.
Since flagellates occupy an important comparative position in the evolutionary scheme, close to the extinct early protozoa from which fungi, protozoa, and animals evolved, Crithidia fasciculata, a heme-requiring, protozoan was selected as the experimental organism. The organism possesses a single flagellum and a single mitochondrial organelle (8). Situated within the inner matrix of the mitochondrion near the base of the flagellum is the kinetoplast which contains the DNA. Kinetoplast DNA appears to be necessary for normal mitochondrial development (8, 9). However, whether the DNA is equivalent to the mitochondrial DNA of yeast and animal cells remains to be determined. Mitochondrial protein synthesis in Tetrahymena, a ciliated protozoan, is inhibited by chloramphenicol and ethidium bromide (10). Also Crithidia Zuciliae appears to be susceptible to the differential effects of the antibiotics chloramphenicol and cycloheximide (11). Results presented
MITOCHONDRIAL
PROTEIN
here indicate that mitochondrial protein synthesis in C. fasciculatu is not sensitive to chloramphenicol; however, cytoplasmic protein synthesis is sensitive to cycloheximide. MATERIALS
AND
METHODS
Materials. Mannitol, MOPS,3 PVP, defatted BSA-Fraction V, digitonin, dimethylformamide, sorbitol, ATP, GTP, PEP, amino acids, pyruvate kinase, cycloheximide, and puromycin dihydrochloride were all purchased from Sigma International. Nutritional Biochemicals Corp. was the supplier of hemin. Silicon carbide, Crystalon grit #400B, was obtained from Norton Co. [14C]Leucine and Omnifluor were supplied by New England Nuclear Corp. Chloramphenicol was the product of Parke, Davis and Co. For the electron microscopy, Polysciences, Inc. was the supplier of osmium tetroxide, and uranylacetate was from Fisher Chemical Co. Cell growth. Crithidia fasciculata, ATCC No. 11745, was cultured in liquid medium (12) with the modification described by Toner and Weber (13). The organisms were grown to a density of 1.5 x 10s cells/ml, rapidly chilled in ice, and harvested by centrifugation for 8 min at 1600g. Preparation of mitochondria. The technique used was essentially that described by Kusel and Storey (14). All operations were performed at 0-4°C. Unwashed cells were resuspended to 5% w/v in isolation medium containing 0.25 M mannitol, 10 mM MOPS, 2.0 g/liter of PVP (average molecular weight lO,OOO), 3.0 g/liter of BSA, 4.5 mM ascorbate, 250 ELM MgCl,, and 250 PM EGTA. The pH was adjusted to 8.0 with potassium hydroxide. Digitonin in dimethylformamide (40 mg/ml) was added dropwise to the cell suspension with rapid stirring to a final concentration of 6.0 mg/g wet weight cells. After slow stirring for 3040 min, the cells were recovered by centrifugation at 16OOg for 8 min, and were resuspended to 10% w/v in isolation medium. Cells were disrupted with a Polytron homogenizer G’T-35 generator, Brinkmann Instruments Inc.). The suspension was subjected to a 20-s treatment at 6500 rpm before an equal volume of isolation medium was added and a further 10-s treatment was initiated. Approximately 75-90% of the cells were lysed. Unbroken cells and debris were removed by centrifugation at 68% for 10 min. Centrifugation of the supernatant at 12,000g for 10 min yielded a crude mitochondrial pellet. The pellet was suspended in 1 ml of isolation medium5 ml of the 3 Abbreviations used are: MOPS, morpholinopropane sulfonic acid; PVP, polyvinylpyrrolidone; BSA, bovine serum albumin; PEP, phosphoenolpyruvate; EGTA, ethylene glycol tetraacetic acid; TCA, trichloroacetic acid.
SYNTHESIS
393
original crude homogenate using a glass-Teflon homogenizer. Upon centrifugation at 12,OOOg for 10 min, a three-layered sediment was obtained. The supernatant and upper layer were removed by aspiration. The brown mitochondrial layer was resuspended in half the previous volume of isolation medium, avoiding the bottom viscous white layer. Particulate aggregrates were removed by centrifugation at 48Og for 5 min. The supernatant was then centrifuged at 12,00(% for 10 min. The supematant and upper layer of the pellet were again removed by aspiration, and the brown mitochondrial layer was resuspended in the same volume of isolation medium used in the previous step. The last two centrifugation steps were repeated. The final mitochondrial pellet was resuspended in a minimal volume of isolation medium, usually to 92 mglml wet weight. Although this fraction is referred to as the mitochondrial pellet, it also contained kinetoplasts. However, the presence of succinic dehydrogenase activity has been demonstrated in kinetoplasts. Preparation of cytoplasmic supernatant. Cells were washed twice in isolation medium and collected by centrifuging 8 min at 1600g. The wash medium was carefully poured off and the cells were broken by grinding in the cold with 2 g silicon carbide/g wet weight of cells. The cell paste was extracted with 1 ml of isolation medium/g wet weight of cells and centrifuged at 5,000g for 10 min to remove abrasive and cell debris. The supernatant was centrifuged at 27,OOO.g for 20 min and the new supernatant was immediately assayed for amino acid incorporation. Amino acid incorporation assay. The system used was essentially that of Lamb et al. (15) for yeast mitochondrial protein synthesis, but with a number of modifications. The standard incubation medium contained 45 mM Tris-HCl, pH 7.4, 5 mM KH,PO,, 20 mM KCl, 15 mM MgCl,, 100 mM sorbitol, 1 mM ATP, 0.2 mM GTP, 5 mM PEP, 30 wg of pyruvate kinase, 1.5 pg/ml of each of the 20 amino acids 1 &i of uniformly labeled except leucine, [“‘Clleucine (280 &i/fimol), and approximately 5 mg of mitochondrial or cytoplasmic protein. The total volume was 1.5 ml. Reactions were initiated by the addition of the mitochondrial suspension or cytoplasmic supernatant. Antibiotics, when included, were added prior to the addition of mitochondrial suspension or cytoplasmic supernatant. Incubations were mostly carried out at 20°C in a New Brunswick gyrotory water bath. Exceptions are described in the text. 100 ~1 samples were withdrawn at various times and spotted on 2.3 cm Whatman #3 MM filter discs. The reactions were stopped by placing the discs in cold 10% (w/v) TCA. Discs were transferred to 5% TCA and incubated at 90°C for 10 minutes, washed twice with 5% TCA, dehydrated by rinsing in ethanol, ethanol-ether (l:l, v/v), and diethyl
394
HILL,
MORRIS
ether, and then air dried. The dried discs were placed in glass vials in 5 ml nonaqueous scintillation fluid (4 g Omniflour/liter scintillation grade toluene), and radioactivity determined in a Nuclear Chicago model Isocap1300 scintillation counter. Under the conditions employed, counting efficiency was about 35%. Sucrose gradient centrifugation. Sucrose solutions were prepared in isolation medium excluding mannitol and BSA. The solutions were layered in a cellulose nitrate centrifuge tube (1 x 3 in.) as follows: 6 ml 75% sucrose, 6 ml 65% sucrose, 6 ml 55% sucrose, 6 ml 45% sucrose, and 5 ml 35% sucrose. The discontinuous gradients were equilibrated at 4°C for 1 h. Mitochondrial suspensions (3 ml containing 2575 mg protein) were layered on the top of each gradient. The samples were centrifuged at 5°C for 15 h at 24,000 rpm in a Beckman SW 27 rotor. After centrifugation the Buchler Auto Densi flow apparatus was used to draw the gradients from top to bottom of the tube. Electron microscopy. Preparations were fixed at 0°C for 45 min in a solution containing 30 mhf glucose, 15 mM sodium phosphate buffer, pH 7.3, 0.45 mM CaCI,, and 5% glutaraldehyde. The samples were washed four times with a solution containing 30 mM glucose, 15 mM sodium phosphate buffer, pH 7.3, and 0.45 mM CaCI, before a post-fixing procedure at 4°C for 45 min in the same solution containing 2% osmium tetroxide. After washing once more with the glucose/phosphate/CaC1, solution, the fixed preparations were embedded in Epon-araldite, sectioned on an LKB-Huxley ultramicrotome, and stained with uranylacetate and then lead citrate. Micrographs were taken using a Phillips 300 electron microscope at 60 kv. RESULTS
Effects of inhibitors on growth of C. fasciculata. Addition of chloramphenicol in concentrations as high as 4 mg/ml did not affect the growth rate of C. fasciculata. This organism had experienced no previous exposure to chloramphenicol. Other bacterial protein synthesis inhibitors such as erythromycin, lincomycin, and tetracycline also had no effect. By contrast, the presence of cycloheximide at 10 pg/ml completely prevented the growth of C. fasciculata. Electron micrographs of cells grown in the presence of chloramphenicol showed the presence of normal appearing mitochondria. The antibiotic was not destroyed during the growth period. Dilutions of the medium containing chloramphenicol at 0 h
AND
WEBER
or following 24 h of C. fasciculata growth were able to prevent the growth of a culture of Escherichia coli at the same dilution. Effects of inhibitors on incorporation of [‘*ClZeucine into isolated mitochondrial fractions. Since chloramphenicol had no effect on the rate of growth of C. fascicuZata, it was of interest to determine whether the organism was impermeable to the antibiotic or whether indeed it did not inhibit mitochondrial directed protein synthesis. Figure 1 contrasts the effect of inhibitors of protein synthesis on the incorporation of [14Clleucine by the mitochondrial and cytoplasmic fractions. In the mitochondrial fraction (Fig. IA), incorporation increased at a linear rate for 12 min, then at a decreased rate, until the reaction ceased at about 25 min. Neither chloramphenicol nor cycloheximide had any affect on the incorporation, whereas puromycin inhibited the reaction by 60%. Cytoplasmic protein synthesis (Fig. 1B) was inhibited 90% by cycloheximide, 50% by puromycin, and was unaffected by chloramphenicol. Hence, mitochondrial protein synthesis can be distinguished from cytoplasmic based on the resistance of the former to cycloheximide. However, in contrast to all other systems studied so far, the mitochondria also appear to be resistant to chloramphenicol, even at concentrations as high as 2 mglml. Since a nutrient agar plate count revealed less than lo3 cells/ml of incubation mixture, bacterial contamination could have accounted for only a small fraction of the incorporation observed (16). The incorporation of amino acids by bacteria would usually be sensitive to chloramphenicol. Sensitivity of E. coli protein synthesis to chloramphenicol after incubation with mitochondria. In order to determine whether chloramphenicol was inactivated by the mitochondrial preparation, an experiment was performed to assay for chloramphenico1 using an E. coli protein synthesizing system. After 30 min, 75 pmol of [ 14C]leucine was incorporated/mg protein x 10e2 into TCA insoluble counts in mitochondria both in the presence and absence of chloramphenicol (250 pg/ml). At this
MITOCHONDRIAL
PROTEIN
395
SYNTHESIS
3
0
6
12
16
24
30
MINUTES
0
6
12
18
24
30
INCUBATION
FIG. 1. Effect of inhibitors on [‘Qleucine incorporation by isolated mitochondria and cytoplasmic supernatant. Preparation of (A) isolated mitochondria and (B) cytoplasmic supernatant and amino acid incorporation assay are described in Methods, with the one exception that the incorporation by cytoplasmic supernatant was carried out at 30°C. O--O, no addition (control); A--A, chloramphenicol (CAP) at 250 pg/ml; X--X, cycloheximide (CH) at 5 pg/ml; M--m, puromycin (PM) at 300 pg/ml.
time a 27,000g supernatant fraction from E. coli was added. This resulted in an additional incorporation of 19 and 170 pmol of [14C]leucine/mg protein x lop2 in the presence and absence of chloramphenicol, respectively, in a subsequent 30-min incubation. The additional incorporation of 19 pmol of [‘4Clleucine/mg protein x lo-’ observed in the presence of chloramphenicol was that obtained by the increase in mitochondrial protein synthesis during that period. Effects of inhibitors on incorporation of [“Clleucine into mitochondrial fractions which were treated with digitonin. The possibility still existed that chloramphenicol-insensitive amino acid incorporation was the result of exclusion of chloramphenicol from the mitochondrial ribosomes by the mitochondrial membrane. Digitonin treatment disrupts mitochondria without affecting their capacity for oxidative phosphorylation (17). Submitochondrial fragments obtained by digitonin treatment of mitochondria from beef heart and rat liver were shown to incorporate amino acids into protein at a greater rate than untreated mitochondria, and this incorporation could be inhibited by chloramphenicol (18). Figure 2 shows the effect of digitonin treatment on amino acid incorporation by
0.2
0.4
m g DlGlTONlNmg
a6
ae
14
MT PROTEIN
FIG. 2. Effect of digitonin on [‘Qleucine incorporation by isolated mitochondria (mtj. Incubations at each digitonin concentration were carried out for 30 min before treatment with cold 10% TCA.
isolated mitochondria. Treatment with low levels of digitonin (~0.2 mg digitonimmg mitochondrial protein) caused a stimulation of incorporation of [14C]leucine after 30 min incubation over that of untreated mitochondria. At levels of digitonin greater than 0.2 mg/mg of mitochondrial protein, the increase in amino acid incorporation was diminished. Maximum
396
HILL,
MORRIS
incorporation resulted when the mitochondrial suspension was treated with approximately 0.2 mg digitonimmg of mitochondrial protein. Figure 3 shows electron micrographs of an untreated mitochondrial suspension and a suspension after incubation with 0.2 mg digitonin/mg of mitochondrial protein for 5 min at 4°C. During isolation, the mitochondrion suffered extensive comminution and in the final preparation appeared as small vesicles, ranging in size from 0.3 to 0.7 pm in diameter. No tubular structures similar to those appearing in mitochondria of whole cells were seen. By comparison, mitochondria treated with digitonin were swollen and appeared to have lost most of the electron dense material which filled untreated mitochondria. Two distinct membranes were visible in the untreated preparation, whereas the membranes of digitonin-treated mitochondria were variable. In some places two membranes were visible, while in other places only one membrane or a discontinuous membrane was seen. Thus it appears that digitonin treatment causes extensive damage to the membranes, probably rendering them more permeable. It has also been shown in our laboratory4 that digitonin treatment of whole cells of C. fusciculatu eliminated endogenous respiration. Such respiration was now dependent on the addition of mitochondrial utilizing substrates such as succinate. The rate and extent of amino acid incorporation by digitonin-treated mitochondria is illustrated in Fig. 4. Once again, chloramphenicol (Fig. 4A) and cycloheximide (Fig. 4B) had no effect on the incorporation of [14Clleucine. Puromycin (Fig. 40 did inhibit the incorporation, although, only by about 30%. The effect of each antibiotic was tested over a range of digitonin concentrations. The same results were obtained at each detergent to mitochondrial protein ratio. Separation of mitochondria and kinetoplasts and incorporation of [‘4C]leucine by the isolated fractions. Since mitochondrial 4 M. M. Unpublished
Weber, J. J. Toner, observation.
and
M.
Kincaid.
AND
WEBER
suspensions were shown by electron microscopy to consist of a mixture of mitochondrial vesicles and vesicles containing kinetoplasts (see Fig. 3A), it was of interest to determine if both structures were involved in the amino acid incorporation activity of the mixture. Separation of kinetoplasts and mitochondria was achieved by a single discontinuous sucrose gradient centrifugation as described in Materials and Methods. This method was developed using acriflavin, a yellow dye which fluoresces under ultraviolet illumination. When cells were grown for 12 h in the presence of 0.5 pg/ml acriflavin and viewed under a fluorescent light microscope, a single fluorescent spot was seen inside each cell at the position of the kinetoplast, indicating that acriflavin was concentrated in the kinetoplast. During subsequent cell lysis and isolation procedures, the dye remained associated with the kinetoplast. Sucrose density gradient centrifugation separated the final mitochondrial suspension into two bands. The more rapidly migrating band at the 55-65% sucrose interface appeared yellow and was therefore identified as containing the kinetoplasts. The second band at the 45-55% interface appeared white. Fluorescent light microscopic examination showed that all of the particles in the yellow band fluoresced while no fluorescing particles were seen in the white band. Centrifugation of a similar mitochondrial suspension, but prepared from cells grown without acriflavin, separated the preparation into two white bands in positions identical to those of the white and yellow bands from acriflavingrown cells. Electron micrographs were prepared from samples taken from the two bands. Vesicles with cristae very similar to those in Fig. 3A were seen in both fractions. However, no kinetoplasts appeared in the vesicles from the more slowly migrating band. In contrast, kinetoplasts appeared in many, but not all, of the vesicles from the more rapidly migrating band. Vesicles under the electron microscope may have appeared to lack the DNA structure if the kinetoplast was not in the plane of the thin section. Since every particle in the kinetoplast band from acriflavin-
1 v B c
-.i $
ka*
OF
BIOCHEMISTRY
AND
BIOPHYSICS
170,
392-399
(1975)
Chloramphenicol
Resistant Mitochondrial in Crithidia fascicu/atal
RUSSELL
CAROL
Department
C. HILL, of Microbiology,
St. Louis
A. MORRIS, University
Received
School
February
AND
Protein
MORTON
of Medicine,
St. Louis,
Synthesis
M. WEBER’ Missouri
63104
3, 1975
Growth of Crithidia fasciculata, a heme-requiring flagellated protozoan, was not affected by the presence of chloramphenicol in concentrations as high as 4 mg/ml. Amino acid incorporation by a mitochondrial fraction, containing both mitochondria and kinetoplasts, was resistant to chloramphenicol and cycloheximide, but sensitive to puromycin. The postmitochondrial supernatant (27,OOOg) also incorporated [‘%]leucine into trichloroacetic acid insoluble material, but this incorporation was inhibited by cycloheximide. Chloramphenicol did not affect this system. Treatment of the mitochondrial fraction with digitonin enhanced the rate of YClleucine incorporation, but did not render the preparation sensitive to chloramphenicol. Electron microscopic examination of the digitonin treated mitochondrial fraction revealed the presence of ghosts. Separation of kinetoplasts from mitochondrial vesicles was achieved by discontinuous sucrose gradient centrifugation. Both the kinetoplast and mitochondrial fractions incorporated [Wlleucine at about the same rate. No inhibition of incorporation by chloramphenicol was observed with either fraction.
Extensive research on the biogenesis of mitochondria in fungi and animal cells has revealed that biosynthesis of functional mitochondria involves proteins made by the mitochondrial as well as cytoplasmic ribosomes (1). Mitochondrial protein synthesis is sensitive to chloramphenicol, but insensitive to cycloheximide in all systems so far reported (l-3). Proteins which are synthesized outside the mitochondrion on cytoplasmic ribosomes are inhibited by cycloheximide but not chloramphenicol. This differential effect of antibiotics has allowed for the identification of products of the two different systems, both in vivo, in growing cells (4, 5) and in vitro, with isolated mitochondria (6, 7). By contrast, very little is known about mitochondrial biogenesis in protozoa. 1 This work was supported by Research Grant AI 03046 from the National Institutes of Health, U.S.P.H.S. 2 Author to whom reprint requests should be sent. 392 Copyright All rights
0
1975 by of reproduction
Academic in any
Press, Inc. form
reserved.
Since flagellates occupy an important comparative position in the evolutionary scheme, close to the extinct early protozoa from which fungi, protozoa, and animals evolved, Crithidia fasciculata, a heme-requiring, protozoan was selected as the experimental organism. The organism possesses a single flagellum and a single mitochondrial organelle (8). Situated within the inner matrix of the mitochondrion near the base of the flagellum is the kinetoplast which contains the DNA. Kinetoplast DNA appears to be necessary for normal mitochondrial development (8, 9). However, whether the DNA is equivalent to the mitochondrial DNA of yeast and animal cells remains to be determined. Mitochondrial protein synthesis in Tetrahymena, a ciliated protozoan, is inhibited by chloramphenicol and ethidium bromide (10). Also Crithidia Zuciliae appears to be susceptible to the differential effects of the antibiotics chloramphenicol and cycloheximide (11). Results presented
MITOCHONDRIAL
PROTEIN
here indicate that mitochondrial protein synthesis in C. fasciculatu is not sensitive to chloramphenicol; however, cytoplasmic protein synthesis is sensitive to cycloheximide. MATERIALS
AND
METHODS
Materials. Mannitol, MOPS,3 PVP, defatted BSA-Fraction V, digitonin, dimethylformamide, sorbitol, ATP, GTP, PEP, amino acids, pyruvate kinase, cycloheximide, and puromycin dihydrochloride were all purchased from Sigma International. Nutritional Biochemicals Corp. was the supplier of hemin. Silicon carbide, Crystalon grit #400B, was obtained from Norton Co. [14C]Leucine and Omnifluor were supplied by New England Nuclear Corp. Chloramphenicol was the product of Parke, Davis and Co. For the electron microscopy, Polysciences, Inc. was the supplier of osmium tetroxide, and uranylacetate was from Fisher Chemical Co. Cell growth. Crithidia fasciculata, ATCC No. 11745, was cultured in liquid medium (12) with the modification described by Toner and Weber (13). The organisms were grown to a density of 1.5 x 10s cells/ml, rapidly chilled in ice, and harvested by centrifugation for 8 min at 1600g. Preparation of mitochondria. The technique used was essentially that described by Kusel and Storey (14). All operations were performed at 0-4°C. Unwashed cells were resuspended to 5% w/v in isolation medium containing 0.25 M mannitol, 10 mM MOPS, 2.0 g/liter of PVP (average molecular weight lO,OOO), 3.0 g/liter of BSA, 4.5 mM ascorbate, 250 ELM MgCl,, and 250 PM EGTA. The pH was adjusted to 8.0 with potassium hydroxide. Digitonin in dimethylformamide (40 mg/ml) was added dropwise to the cell suspension with rapid stirring to a final concentration of 6.0 mg/g wet weight cells. After slow stirring for 3040 min, the cells were recovered by centrifugation at 16OOg for 8 min, and were resuspended to 10% w/v in isolation medium. Cells were disrupted with a Polytron homogenizer G’T-35 generator, Brinkmann Instruments Inc.). The suspension was subjected to a 20-s treatment at 6500 rpm before an equal volume of isolation medium was added and a further 10-s treatment was initiated. Approximately 75-90% of the cells were lysed. Unbroken cells and debris were removed by centrifugation at 68% for 10 min. Centrifugation of the supernatant at 12,000g for 10 min yielded a crude mitochondrial pellet. The pellet was suspended in 1 ml of isolation medium5 ml of the 3 Abbreviations used are: MOPS, morpholinopropane sulfonic acid; PVP, polyvinylpyrrolidone; BSA, bovine serum albumin; PEP, phosphoenolpyruvate; EGTA, ethylene glycol tetraacetic acid; TCA, trichloroacetic acid.
SYNTHESIS
393
original crude homogenate using a glass-Teflon homogenizer. Upon centrifugation at 12,OOOg for 10 min, a three-layered sediment was obtained. The supernatant and upper layer were removed by aspiration. The brown mitochondrial layer was resuspended in half the previous volume of isolation medium, avoiding the bottom viscous white layer. Particulate aggregrates were removed by centrifugation at 48Og for 5 min. The supernatant was then centrifuged at 12,00(% for 10 min. The supematant and upper layer of the pellet were again removed by aspiration, and the brown mitochondrial layer was resuspended in the same volume of isolation medium used in the previous step. The last two centrifugation steps were repeated. The final mitochondrial pellet was resuspended in a minimal volume of isolation medium, usually to 92 mglml wet weight. Although this fraction is referred to as the mitochondrial pellet, it also contained kinetoplasts. However, the presence of succinic dehydrogenase activity has been demonstrated in kinetoplasts. Preparation of cytoplasmic supernatant. Cells were washed twice in isolation medium and collected by centrifuging 8 min at 1600g. The wash medium was carefully poured off and the cells were broken by grinding in the cold with 2 g silicon carbide/g wet weight of cells. The cell paste was extracted with 1 ml of isolation medium/g wet weight of cells and centrifuged at 5,000g for 10 min to remove abrasive and cell debris. The supernatant was centrifuged at 27,OOO.g for 20 min and the new supernatant was immediately assayed for amino acid incorporation. Amino acid incorporation assay. The system used was essentially that of Lamb et al. (15) for yeast mitochondrial protein synthesis, but with a number of modifications. The standard incubation medium contained 45 mM Tris-HCl, pH 7.4, 5 mM KH,PO,, 20 mM KCl, 15 mM MgCl,, 100 mM sorbitol, 1 mM ATP, 0.2 mM GTP, 5 mM PEP, 30 wg of pyruvate kinase, 1.5 pg/ml of each of the 20 amino acids 1 &i of uniformly labeled except leucine, [“‘Clleucine (280 &i/fimol), and approximately 5 mg of mitochondrial or cytoplasmic protein. The total volume was 1.5 ml. Reactions were initiated by the addition of the mitochondrial suspension or cytoplasmic supernatant. Antibiotics, when included, were added prior to the addition of mitochondrial suspension or cytoplasmic supernatant. Incubations were mostly carried out at 20°C in a New Brunswick gyrotory water bath. Exceptions are described in the text. 100 ~1 samples were withdrawn at various times and spotted on 2.3 cm Whatman #3 MM filter discs. The reactions were stopped by placing the discs in cold 10% (w/v) TCA. Discs were transferred to 5% TCA and incubated at 90°C for 10 minutes, washed twice with 5% TCA, dehydrated by rinsing in ethanol, ethanol-ether (l:l, v/v), and diethyl
394
HILL,
MORRIS
ether, and then air dried. The dried discs were placed in glass vials in 5 ml nonaqueous scintillation fluid (4 g Omniflour/liter scintillation grade toluene), and radioactivity determined in a Nuclear Chicago model Isocap1300 scintillation counter. Under the conditions employed, counting efficiency was about 35%. Sucrose gradient centrifugation. Sucrose solutions were prepared in isolation medium excluding mannitol and BSA. The solutions were layered in a cellulose nitrate centrifuge tube (1 x 3 in.) as follows: 6 ml 75% sucrose, 6 ml 65% sucrose, 6 ml 55% sucrose, 6 ml 45% sucrose, and 5 ml 35% sucrose. The discontinuous gradients were equilibrated at 4°C for 1 h. Mitochondrial suspensions (3 ml containing 2575 mg protein) were layered on the top of each gradient. The samples were centrifuged at 5°C for 15 h at 24,000 rpm in a Beckman SW 27 rotor. After centrifugation the Buchler Auto Densi flow apparatus was used to draw the gradients from top to bottom of the tube. Electron microscopy. Preparations were fixed at 0°C for 45 min in a solution containing 30 mhf glucose, 15 mM sodium phosphate buffer, pH 7.3, 0.45 mM CaCI,, and 5% glutaraldehyde. The samples were washed four times with a solution containing 30 mM glucose, 15 mM sodium phosphate buffer, pH 7.3, and 0.45 mM CaCI, before a post-fixing procedure at 4°C for 45 min in the same solution containing 2% osmium tetroxide. After washing once more with the glucose/phosphate/CaC1, solution, the fixed preparations were embedded in Epon-araldite, sectioned on an LKB-Huxley ultramicrotome, and stained with uranylacetate and then lead citrate. Micrographs were taken using a Phillips 300 electron microscope at 60 kv. RESULTS
Effects of inhibitors on growth of C. fasciculata. Addition of chloramphenicol in concentrations as high as 4 mg/ml did not affect the growth rate of C. fasciculata. This organism had experienced no previous exposure to chloramphenicol. Other bacterial protein synthesis inhibitors such as erythromycin, lincomycin, and tetracycline also had no effect. By contrast, the presence of cycloheximide at 10 pg/ml completely prevented the growth of C. fasciculata. Electron micrographs of cells grown in the presence of chloramphenicol showed the presence of normal appearing mitochondria. The antibiotic was not destroyed during the growth period. Dilutions of the medium containing chloramphenicol at 0 h
AND
WEBER
or following 24 h of C. fasciculata growth were able to prevent the growth of a culture of Escherichia coli at the same dilution. Effects of inhibitors on incorporation of [‘*ClZeucine into isolated mitochondrial fractions. Since chloramphenicol had no effect on the rate of growth of C. fascicuZata, it was of interest to determine whether the organism was impermeable to the antibiotic or whether indeed it did not inhibit mitochondrial directed protein synthesis. Figure 1 contrasts the effect of inhibitors of protein synthesis on the incorporation of [14Clleucine by the mitochondrial and cytoplasmic fractions. In the mitochondrial fraction (Fig. IA), incorporation increased at a linear rate for 12 min, then at a decreased rate, until the reaction ceased at about 25 min. Neither chloramphenicol nor cycloheximide had any affect on the incorporation, whereas puromycin inhibited the reaction by 60%. Cytoplasmic protein synthesis (Fig. 1B) was inhibited 90% by cycloheximide, 50% by puromycin, and was unaffected by chloramphenicol. Hence, mitochondrial protein synthesis can be distinguished from cytoplasmic based on the resistance of the former to cycloheximide. However, in contrast to all other systems studied so far, the mitochondria also appear to be resistant to chloramphenicol, even at concentrations as high as 2 mglml. Since a nutrient agar plate count revealed less than lo3 cells/ml of incubation mixture, bacterial contamination could have accounted for only a small fraction of the incorporation observed (16). The incorporation of amino acids by bacteria would usually be sensitive to chloramphenicol. Sensitivity of E. coli protein synthesis to chloramphenicol after incubation with mitochondria. In order to determine whether chloramphenicol was inactivated by the mitochondrial preparation, an experiment was performed to assay for chloramphenico1 using an E. coli protein synthesizing system. After 30 min, 75 pmol of [ 14C]leucine was incorporated/mg protein x 10e2 into TCA insoluble counts in mitochondria both in the presence and absence of chloramphenicol (250 pg/ml). At this
MITOCHONDRIAL
PROTEIN
395
SYNTHESIS
3
0
6
12
16
24
30
MINUTES
0
6
12
18
24
30
INCUBATION
FIG. 1. Effect of inhibitors on [‘Qleucine incorporation by isolated mitochondria and cytoplasmic supernatant. Preparation of (A) isolated mitochondria and (B) cytoplasmic supernatant and amino acid incorporation assay are described in Methods, with the one exception that the incorporation by cytoplasmic supernatant was carried out at 30°C. O--O, no addition (control); A--A, chloramphenicol (CAP) at 250 pg/ml; X--X, cycloheximide (CH) at 5 pg/ml; M--m, puromycin (PM) at 300 pg/ml.
time a 27,000g supernatant fraction from E. coli was added. This resulted in an additional incorporation of 19 and 170 pmol of [14C]leucine/mg protein x lop2 in the presence and absence of chloramphenicol, respectively, in a subsequent 30-min incubation. The additional incorporation of 19 pmol of [‘4Clleucine/mg protein x lo-’ observed in the presence of chloramphenicol was that obtained by the increase in mitochondrial protein synthesis during that period. Effects of inhibitors on incorporation of [“Clleucine into mitochondrial fractions which were treated with digitonin. The possibility still existed that chloramphenicol-insensitive amino acid incorporation was the result of exclusion of chloramphenicol from the mitochondrial ribosomes by the mitochondrial membrane. Digitonin treatment disrupts mitochondria without affecting their capacity for oxidative phosphorylation (17). Submitochondrial fragments obtained by digitonin treatment of mitochondria from beef heart and rat liver were shown to incorporate amino acids into protein at a greater rate than untreated mitochondria, and this incorporation could be inhibited by chloramphenicol (18). Figure 2 shows the effect of digitonin treatment on amino acid incorporation by
0.2
0.4
m g DlGlTONlNmg
a6
ae
14
MT PROTEIN
FIG. 2. Effect of digitonin on [‘Qleucine incorporation by isolated mitochondria (mtj. Incubations at each digitonin concentration were carried out for 30 min before treatment with cold 10% TCA.
isolated mitochondria. Treatment with low levels of digitonin (~0.2 mg digitonimmg mitochondrial protein) caused a stimulation of incorporation of [14C]leucine after 30 min incubation over that of untreated mitochondria. At levels of digitonin greater than 0.2 mg/mg of mitochondrial protein, the increase in amino acid incorporation was diminished. Maximum
396
HILL,
MORRIS
incorporation resulted when the mitochondrial suspension was treated with approximately 0.2 mg digitonimmg of mitochondrial protein. Figure 3 shows electron micrographs of an untreated mitochondrial suspension and a suspension after incubation with 0.2 mg digitonin/mg of mitochondrial protein for 5 min at 4°C. During isolation, the mitochondrion suffered extensive comminution and in the final preparation appeared as small vesicles, ranging in size from 0.3 to 0.7 pm in diameter. No tubular structures similar to those appearing in mitochondria of whole cells were seen. By comparison, mitochondria treated with digitonin were swollen and appeared to have lost most of the electron dense material which filled untreated mitochondria. Two distinct membranes were visible in the untreated preparation, whereas the membranes of digitonin-treated mitochondria were variable. In some places two membranes were visible, while in other places only one membrane or a discontinuous membrane was seen. Thus it appears that digitonin treatment causes extensive damage to the membranes, probably rendering them more permeable. It has also been shown in our laboratory4 that digitonin treatment of whole cells of C. fusciculatu eliminated endogenous respiration. Such respiration was now dependent on the addition of mitochondrial utilizing substrates such as succinate. The rate and extent of amino acid incorporation by digitonin-treated mitochondria is illustrated in Fig. 4. Once again, chloramphenicol (Fig. 4A) and cycloheximide (Fig. 4B) had no effect on the incorporation of [14Clleucine. Puromycin (Fig. 40 did inhibit the incorporation, although, only by about 30%. The effect of each antibiotic was tested over a range of digitonin concentrations. The same results were obtained at each detergent to mitochondrial protein ratio. Separation of mitochondria and kinetoplasts and incorporation of [‘4C]leucine by the isolated fractions. Since mitochondrial 4 M. M. Unpublished
Weber, J. J. Toner, observation.
and
M.
Kincaid.
AND
WEBER
suspensions were shown by electron microscopy to consist of a mixture of mitochondrial vesicles and vesicles containing kinetoplasts (see Fig. 3A), it was of interest to determine if both structures were involved in the amino acid incorporation activity of the mixture. Separation of kinetoplasts and mitochondria was achieved by a single discontinuous sucrose gradient centrifugation as described in Materials and Methods. This method was developed using acriflavin, a yellow dye which fluoresces under ultraviolet illumination. When cells were grown for 12 h in the presence of 0.5 pg/ml acriflavin and viewed under a fluorescent light microscope, a single fluorescent spot was seen inside each cell at the position of the kinetoplast, indicating that acriflavin was concentrated in the kinetoplast. During subsequent cell lysis and isolation procedures, the dye remained associated with the kinetoplast. Sucrose density gradient centrifugation separated the final mitochondrial suspension into two bands. The more rapidly migrating band at the 55-65% sucrose interface appeared yellow and was therefore identified as containing the kinetoplasts. The second band at the 45-55% interface appeared white. Fluorescent light microscopic examination showed that all of the particles in the yellow band fluoresced while no fluorescing particles were seen in the white band. Centrifugation of a similar mitochondrial suspension, but prepared from cells grown without acriflavin, separated the preparation into two white bands in positions identical to those of the white and yellow bands from acriflavingrown cells. Electron micrographs were prepared from samples taken from the two bands. Vesicles with cristae very similar to those in Fig. 3A were seen in both fractions. However, no kinetoplasts appeared in the vesicles from the more slowly migrating band. In contrast, kinetoplasts appeared in many, but not all, of the vesicles from the more rapidly migrating band. Vesicles under the electron microscope may have appeared to lack the DNA structure if the kinetoplast was not in the plane of the thin section. Since every particle in the kinetoplast band from acriflavin-
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