Preferential synthesis of yeast mitochondrial DNA in the absence of protein synthesis

J. Mol. Biol. (1969) 46, 367-376

Preferential Synthesis of Yeast Mitochondrial Absence of Protein Synthesis

DNA in the

LAWREKCE I. GROSSMAN,ELIZABETH ~.GOLDRINGAND Jumus MARMUR Department

of Genetics (Division of Biological

Sciences)

and

Dejlartment of Biochemistry, Albert Einstein College of Medicine Bronx, New York 10461, U.S.A. (Received 5 May 1969, and in revised form 3 August 1969) Under normal growth conditions, mitochondrial DNA comprises 5 to 15% of the total yeast cell DNA. However, mitochondrial DNA is the major DNA species synthesized in Saccharomyces cerevisiae when protein synthesis is inhibited by cycloheximide, amino acid starvation or by using temperature-sensitive mutants. This DNA is synthesized for four to six hours after the cessation of protein synthesis ; during this period, the relative amount of mitochondrial DNA doubles and can be labeled to contain 90% of the total DNA radioactivity. Similar effects were observed in a cytoplasmic petite yeast strain when protein synthesis was inhibited by cycloheximide.

1. Introduction DNA is now accepted as a normal constituent of mitochondria. The additional demonstration of distinct components for its replication (Meyer & Simpson, 1968 ; Kalf & Ch’ih, 1968), transcription (reviewed by Neubert, Helge & Merker, 1968) and translation (reviewed by Work, 1968; Neupert, Brdiczka & Sebald, 1968) completes the requirements for a genetically independent organelle. However, considerable evidence exists for substantial mitochondrial dependence on nuclear-controlled functions. The demonstration that nuclear genes exist for at least some respiratory chain enzymes (Sherman, Stewart, Margoliash, Parker $ Campbell, 1966 ; Longo & Scandalios, 1969) implies that mitochondria are not wholly autonomous. Additional evidence comes from a study of respiratory-deficient mutants in yeast (reviewed by Sherman, 1965; Mounolou, Jakob & Slonimski, 1967). Such mutant,s cannot carry out oxidative respiration, and are usually deficient in several cytochromes and other respiratory chain enzymes. This “petite” phenotype can be genetically defined by loss of a cytoplasmically inherited factor (p) ; however, similar phenotypes are also associated with mutations in any of a number of nuclear genes (Sherman, 1963; Sherman & Slonimski, 1964) when the cytoplasmic factor is in the p+ state. Furthermore, return of respiratory function by complementation in diploids formed from a cytoplasmic petite and a chromosomal petite strain occurs at a time determined by the nuclear petite genotype (Jakob, 1965). Finally, the fact that mitochondrial DNA replicates at a unique time in synchronously dividing yeast cells has been taken to imply nuclear control over mitochondrial DNA synthesis (Smith, Tauro, Schweizer & Halvorson, 1968). 24

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A knowledge of the structure and mechanism of replication of mitochondrial DNA would help in understanding nucleocytoplasmic interrelationships, particularly with regard to the potential information content of mitochondrial DNA. Several workers have demonstrated the presence of two to six DNA molecules per mitochondrion (Nass, 1966; Avers, Billheimer, Hoffmann & Pauli, 1968), but its in situ form has been difficult to determine, particularly in yeast. In this organism the DNA is probably damaged during the isolation of highly purified mitochondria, possibly accounting for the conflicting reports of size and topology of the DNA. Circular DNA molecules from yeast mitochondria have been reported by various investigators. The proportion and, more significantly, the size estimate of these molecules varies considerably (Shapiro, Grossman, Marmur & Kleinschmidt, 1968; Avers et al., 1968; Guerineau, Grandchamp, Yotsuyanagi & Slonimski, 1968). Several investigators have found no evidence for circular mitochondrial DNA molecules (Sinclair, Stevens, Sanghavi $ Rabinowitz, 1967; Van Bruggen et al., 1968). By inhibiting protein synthesis we have been able to dissociate mitochondrial from nuclear DNA synthesis and allow specific synthesis of mitochondrial DNA. In addition, such synthesis in the presence of radioisotopes results in predominant labeling of mitochondrial DNA and provides a rapid and gentle method of obtaining mitochondrial DNA for physical analysis.

2. Materials and Methods (a) Strains The Sacc?zuromycee cerewi&e strain A664a/18A ura,? and the double auxotroph A2103C his, trpz, were obtained from Dr B. Dorfman; the diploid petite strain D261-lpwas supplied by Dr F. Sherman. Strain AHS, which is sensitive to 50 rg chloramphenicol/ml., was kindly sent by Dr D. Wilkie. Strains 241, 275, 296 and 341 are multiply auxotrophic temperature-sensitive mutants in which protein synthesis is blocked at the non-permissive temperature. They were obtained from Dr L. Hartwell, who has described their properties (Hartwell & McLaughlin, 1968). (b) Cell growth Yeast cells were grown in yeast nitrogen base supplemented with amino acids (Difco) and containing either glucose or lactate (1 to 2%), as indicated in the text. Uridine at 20 pg/ml. was added for growth of strain A664a/18A and the following supplements for growth of the temperature-sensitive strains : adenine sulfate and uridine, 10 rg/ml.; lysine, tryptophan and histidine, 50 pg/ml. For amino acid starvation, strain A2103C was transferred to Difco yeast nitrogen base medium minus amino acids. These experiments were performed on cells in the log phase of growth (0.7 to 1.2 x lO’/ml., equivalent to 50 to 90 optical density units in the Klet&Summerson calorimeter using a no. 62 filter). Growth was at 30°C with aeration by shaking. For the temperature-sensitive mutants 23°C was used as the permissive temperature and 36°C was used for inhibition of protein synthesis. (0) Labeling of cells DNA was labeled by the addition of r3H]adenine (Schwarz, 20 c/m-mole, 5 to 15 &ml.). Cycloheximide (Sigma) and chloramphenicol (Parke, Davis & Co.) were added to final concentrations of 200 pg/ml. Pretreatment of the appropriate strain with antibiotics or high temperature was carried out for 15 min before the addition of label unless otherwise stated. t Abbreviations

used: um, urmil;

hia, histidine;

trp, tryptophan;

CH, oycloheximide.

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(d) Spheroplaatformabion and preparative churn chlorirEe density-gradient centrifugation Cells were harvested by centrifugation at 4°C and washed once with distilled weter. Spheroplast formation, using the Stwptomyces lytic enzyme preparation has already been described (Shapiro et al., 1968). Cells from 5 to 10 ml. of growing culture were converted to spheroplasts at a concentration of 6 to 10 x lO’/ml. The spheroplasts were collected by oentrifugation at 4°C in an angle rotor for 10 min at 3000 g. The spheroplast pellet was transferred to a nitrocellulose centrifuge tube with 1.4 ml. 0.1 x SSC (SSC is 0.15 M-N&I, 0.016 M-sodium citrate, pH 7) and lysed by the addition of the detergent Sarkosyl (Geigy Chemical Co.) to 1.3%. A saturated CsCl solution (4.2 ml.) was used to bring the density to I.676 to 1.690 g/ml. The samples were overlayed with mineral oil and centrifuged in the Spinco no. 40 rotor at WC, 31,000 rev./min for at least 55 hr. Frections (O-16 ml.) were collected from the bottom of the tube punctured with a no. 23 needle. All fractions were adjusted to 0.6 M-NaOH and incubated overnight at 37°C to digest RNA before being precipitated by the addition of bovine serum albumin (100 pg) and 0.8 ml. of 20% trichloroacetic acid. Fractions were chilled, collected by filtration on nitrocellulose membranes, washed with 5% triohloroacetic acid, dried and counted in a toluene-based scintillant (Liquiflor, New England Nuclear Corp.). (e) Analytical density-gradient centrqugation Growth was stopped by addition of sodium azide and sodium fluoride @aI concentration 0.02 M each). Cells were then harvested at 4”C, washed once with distilled water and converted to spheroplssts. The spheroplasts were collected, washed once with 1 M-sorbitol and resuspended in 0.15 M-NaCl-0.1 M-EDTA (pH 8). Lysis was carried out by the addition of Sarkosyl to 1.3%. The lysates were adjusted to 1 M-sodium perchlorate and deproteinized once by shaking with chloroform. After centrifugation to separate the phases, pronase (Enzyme Development Corp. or Calbiochem) (predigested 1 hr at 37°C) was added to a final concentration of 1 mg/ml. and the lysate was dialyzed against SSC for several hours at room temperature, then overnight at 4°C. The solutions were treated with RNasc (pancreatic and T,) and again dialyzed against SSC. Centrifugation was carried out in a Spinco model E ultracentrifuge at 44,770 rev./min for 16 to 18 hr at 25’C as described by Meselson, Stahl & Vinograd (1957). Ultraviolet photographs were scanned with a Joyce-Loebl microdensitometer. Bacillus subtilis phage PBS2 DNA (p = 1.723 g/ml. when Escherichia coli DNA was taken to be 1.710 g/ml.) was added as a density marker. Amounts of nuclear and mitochondrial DNA were obtained by summation of areas under the peaks of microdensitometer tracings.

3. Experimental (a) SpeciJic incorporation

of radioactive

material

Results into mitochondrial

DNA

in the presence

of cycloheximide The normal CsCl profile of total yeast DNA labeled for several generations is shown in Figure l(a). Nuclear DNA has a buoyant density of 1.699 g/ml. and mitochondrial DNA bands at 1.683 g/ml. The proportion of mitochondrial DNA varies from about 5 to 15% of the total, depending on the carbon source used, the nuclear genotype (Mounolou, Jakob & Slonimski, 1966) and the phase of growth cycle examined (Moustacchi & Williamson, 1966). This proportion can be altered strikingly by growth in the presence of 2OOpg CH/ml. (an amount which stops further incorporation of amino acids within 10 min). The inhibition of protein synthesis in yeast by CH has been described (Siegel & Sisler, 1964,1965; deKloet, 1966). Cells pretreated with antibiotic for 15 minutes and then labeled for 5 hours in the continued presence of CR now contain 90% of the DNA radioactivity in mitochondrial DNA (Fig. l(c)). After 60 minutes of labeling, nuclear and mitochondrial DNA are approximately equally labeled (Fig. l(b)). No further increase of radioactivity in the nuclear DNA regionisseenafter 6Ominutes (Fig. 1 (b)and (c)).Percentagesbasedonradioactivityhave

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600-

Fraction

no

FIG. 1. Equilibrium density-gradient centrifugation of total DNA extracted from S. cerevisiue A664a/lSA grown with lactate as a carbon source. Cells were labeled with r3H]adenine (10 PC/ml.) and total spheroplast lysates were centrifuged to equilibrium as described in Materials and Methods. (a) CsCl density-gradient fractionation pattern obtained after labeling for several generations in the absence of CH. (b) and (c) Pattern obtained after 15 min pretreatment with CH followed by labeling for 60 min (b) and 214 min (c). N, nuclear DNA; M, mitochondrial DNA, in all Figures.

not been corrected for the different adenine content of nuclear and mitochondrial DNA’s. When CH was added to the petite strain D261-lp-, mitochondrial DNA was synthesized selectively (Fig. 5(a)) in a similar manner to that described for the wildtype respiratory strain A664a/lSA. Exposure of the petite to CH did not alter the buoyant density of the mitochondrial DNA as judged by preparative CsCl densitygradient centrifugation. The density of the mitochondrial DNA from this petite strain was determined to be I.677 g/ml. (b) Net synthesis of mitochondrial

DNA

in the presence of cycloheximide

To determine whether radioactivity appearing in mitochondrial DNA represents net synthesis, a culture was treated with CH and samples were removed at various times for analysis of their DNA in the analytical ultracentrifuge. The results are presented in Figure 2. A control culture (Fig. 2(a)) contains about 15% mitochondrial DNA, as estimated from the absorbancy areas under the peaks. Samples taken at 45 and 225 minutes after the addition of CH show, respectively, 21 and 29% of

YEAST

MITOCHONDRIAL

DNA

I683 1,699 --___I 723 Buoyant density-w

FIG. 2. Increase of proportion of yeast mitochondrial DNA by treatment with CH. Microdensitometer tracings of DNA centrifuged to equilibrium in C&l. Gradients were formed at 25”C, 44,770 rev./min and contained DNA extracted from A664e/lSA following treatment with CH. Purification is described in Materials and Methods. The density marker was B. mbtilia phage PBS2 DNA (p= 1.723 g/ml.). Lactate was used as carbon source. (a) No CH. (b) 45 min after addition of CH. (0) 225 min after addition of CH.

the total DNA in the mitochondrial region (Fig. 2(b) and (c)). The latter figure represents a doubling of the amount of mitochondrial DNA relative to nuclear DNA. (c) Kinetics of mitochondria.? DNA synthesis in the presence of cycloheximide When [3H]adenine is added to a yeast culture 15 minutes after the addition of CH, the radioactivity entering the mitochondrial DNA increases with time both in amount and as the fraction of total DNA radioactivity. Radioactive material enters nuclear DNA at a greatly reduced rate for about five hours (insert, Fig. 3) and thereafter remains constant in amount. The mitochondrial DNA incorporates label for about 6-5 hours (insert, Fig. 3) ; the rate is approximately that of uninhibited cultures. Curve A in Figure 3 shows as a percentage the ratio of mitochondrial DNA to nuclear plus mitochondrial DNA. This was determined by radioactive incorporation in the presence of CH and analyzed by preparative density-gradient centrifugation. By five to six hours, 90% of the radioactivity of the total cellular DNA is found in the mitochondrial region of a CsCl gradient. Longer labeling times do not significantly raise either this ratio or the net amount of radioactivity. When pretreatment with cycloheximide is extended from 15 to 45 minutes, no change is seen in either the ratio of mitochondrial to total DNA (open circles, Fig. 3 curve A) or in the radioactive material entering the nuclear DNA region (data not shown). The net increase in mitochondrial DNA relative to nuclear DNA ia also shown in Figure 3 (curve B) ; open and filled squares indicate separate experiments. Amounts of each DNA were determined as described in Materials and Methods. The initial proportion of mitochondrial DNA approximately doubles in 100 minutes, indicating that net synthesis is taking place, and thereafter remains constant. In the absence of

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(min)

FICA 3. Kinetics of synthesis of yeast DNA in the presence of 200 pg CH/ml. Cells were grown using lactate as carbon source. Curve A. Proportion of radioactive material in mitochondrial DNA. DNA from 8. cerevieiae AtX34@8A was labeled with [sH]edenine (10 p/ml.) for various times after the pretreatments indicated. The pementage of counts in mitochondrial DNA was estimated by summation of radioactive material contained in the peaks of nuclear and mitochondrial DNA separated by preperative equilibrium density-gradient centrifugation. (0, A) Independent experiments following 15min pretreatment with CH; (0) 45-min pretreatment with CH. Curue B. Synthesis of DNA following treatment with CH. The percentage of mitochondrial DNA was measured by summetion of arems under nuclear and mitochondrial DNA absorbancy peaks obtained from analytical equilibrium density-gradient centrifugation. ( n , 0) Independent experiments. 1n.sert. Time-course of incorporation of [3H]adenine (10 &ml.) into nuclear and mitochondriel DNA following pretreatment (15 min) with CH. Points represent total radioactive materiel in nuclear and mitochondrial DNA peaks obtained by preparative equilibrium density-gradient centrifugation in CsCl. (0) Nuclear DNA; (0) mitoohondrial DNA.

inhibitor, the cell doubling time is approximately two hours. The earlier plateau seen in the optical density measurements (Fig. 3, curve B Zter8u.scurve A) may be a reflection of the less sensitive type of measurement being made. (d) Reversal of the effects of cycloheximide In order to examine whether the effect of CH was reversible, a lo-ml. culture was treated with CH for 20 minutes, then labeled for 120 minutes with 2 PC [14C]adenine/ ml. in the presence of the antibiotic. The cells were collected by centrifugation at room temperature, washed once with warm Difco yeast nitrogen base medium and resuspended in that medium containing 5 pc [3H]adenine/ml. in the absence of CH. After 160 minutes cells were collected and analyzed by preparative CsCl centrifugation. Figure 4 shows that during recovery from CH inhibition, nuclear DNA synthesis recommences. The time necessary for complete recovery was not determined. (e) Effects on. mitochondriul DNA synthesis using other methods of inhibiting synthesis

protein

To check the generality of the effect obtained with cycloheximide, several other conditions for inhibiting protein synthesis were tried. An amino acid double auxotroph was transferred to a medium lacking amino acids, and portions were removed at 20, 45 and 90 minutes after transfer for labeling with [3H]adenine for 150 minutes.

YEAST

MITOCHONDRIAL

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DNA

1 I

-L-L

0

IO

20 Fraction

30

40

no.

Fra. 4. Reverse1 of CH inhibition. 10 ml. of 8 culture of strain A6648/18A were babeled with 2 pc [14C]adenine/ml. for 2 hr in the presence of CH. Cells were weshed, resuspended in the absence Spheroplast lysates were aentrifuged to of CH and labeled 2.6 hr with 5 PC [3H]adenine/ml. equilibrium in CsCl and redioactivity analyzed. Glucose was provided 8s carbon source. (--O--O--) [“C]Adenine + CR; (-•--e.--:) [3H]adenine:CH.

(c)

-AA M

20

30

40

Fraction no

FIQ. 5. Equilibrium density-grrtdient centrifugation of spheropl8st lysates. (a) From petite str8in D261.lp-. Cells were treated with CR for 20 min and then labeled with [3H]8denine (10 @/ml.) for 210 min in the presence of CH. (b) From temperature-sensitive strain 341. This culture was first grown at 23’C, then transferred to 3@C. 20 min after the transfer [3H]adenine (5 &ml.) was added and incubation oontinued for 2.5 hr. (o) From amino aoid-requiring strain A2103C his, tr&. Cells were deprived of amino acids for 90 min, and the DNA was labeled with 7.6 PC [3H]adenine/ml. for 150 min. Glucose W&S used as cerbon source in these experiments.

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The samples were then analyzed by preparative CsCl centrifugation. The prop&ion of radioactivity in the mitochondrial DNA region increased with time of starvation, and presumably reflects the delayed inhibition of protein synthesis due to the presence of residual amino acid pools (C. T. Wehr & L. W. Parks, Bact. Proc. p. 28, 1969). Labeling for 150 minutes following 90 minutes of starvation (Fig. 5(c)) showed about 70% of label in mitochondrial DNA. Four independently isolated temperature-sensitive mutants blocked for protein synthesis were grown at 23°C and then transferred to 36°C. After 20 minutes, [3H]adenine was added and incorporation for 150 minutes was allowed. All contained more than 90% of label in the mitoohondrial region. The results for mutant 341 are shown (Fig. 5(b)). Growth of strain A664a/lSA at 36°C did not alter the CsCl pattern normally seen at 30°C. Several workers have shown that chloramphenicol specifically inhibits mitochondrial, but not cytoplasmic, protein synthesis in yeast (Kroon, 1965; Wintersberger, 1965; Wilkie, Saunders & Linnane, 1967; Lamb, Clark-Walker & Linnane, 1968). It was thus of interest to determine the effects of this antibiotic on mitochondrial DNA synthesis. Our strain AHS was sensitive to this antibiotic by two criteria: it was unable to grow on plates containing chloramphenicol (100 pg/ml.) when glycerol was the carbon source, and its growth was inhibited in liquid culture in the presence of the drug (200 pg/ml.) when lactate was provided. In both cases the use of glucose as the carbon source allowed normal growth. This demonstrated the expected dependence on mitochondrial function in the presence of a non-fermentable carbon source. Addition of [3H]adenine to a culture containing glucose as a carbon source up to 120 minutes after the addition of 200 pg chloramphenicol/ml., followed by labeling for 150 minutes, resulted in a CsCl pattern resembling uninhibited cells; the bulk of the radioactivity was found in nuclear DNA. Furthermore, even after 180 minutes in the presence of chloramphenicol, CH could be used to induce the preferential labeling of mitochondrial DNA.

4. Discussion We have shown that inhibition of protein synthesis in yeast by three separate methods leads to a selective synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis. The labeling of nuclear DNA in the presence of cycloheximide is about 1% that of uninhibited cultures. Thus, the incorporation of radioactive material into nuclear DNA in inhibited cultures may represent primarily repair synthesis, or the replication of a small, special component of nuclear DNA, such as nucleolar DNA. In bacteria, it is believed that chromosome replication is controlled through initiation by a specific protein of new rounds of replication (reviewed by Kjeldgaard, 1967). If protein synthesis is inhibited, no new rounds of replication are initiated. When E. wli cells growing in a glucose-minimal medium are exposed to a protein inhibitor, the average increase of the DNA of the cells is approximately 40%, resulting from completion of replicating chromosomes (Maabe & Hanawalt, 1961). Nuclear DNA synthesis, however, stops almost immediately in yeast following treatment with CH; this suggests the need for continuous synthesis of proteins during nuclear DNA replication. In HeLa cells, it is known that histones are made continuously and exclusively during the DNA synthesis period and are an adjunct to DNA synthesis (Robbins & Borun, 1967). The finding of histone-like proteins in yeast

YEAST

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nuclei (Tonino & Rozijn, 1966u,b) suggests that a similar type of mechanism may also be operative in yeast cells. The continuing synthesis of mitochondrial DNA in the presence of inhibitors of protein synthesis differentiates it from nuclear DNA synthesis. Clearly, the synthesis of this DNA is not dependent upon concomitant cytoplasmic protein synthesis. The finding that chloramphenicol does not alter the amount of yeast mitochondrial DNA is surprising. If mitochondrial protein synthesis were required for initiation of DNA replication, the action of chloramphenicol should cause a reduced amount of mitochondrial DNA. Thus, the application of the bacterial initiator concept to mitochondria would require that a mitochondrial DNA-coded initiator be synthesized on cytoplasmic ribosomes in order to remain insensitive to chloramphenicol. Some evidence for the translation of mitochondrial messenger RNA on cytoplasmic polysomes has been presented (Attardi & Attardi, 1967). However, such a protein would be sensitive to CH, a possibility not consistent with our findings. A tentative interpretation may be that a pool of protein exists which is sufficient to initiate one or more rounds of replication in mitochondria. The eventual cessation of mitochondrial DNA synthesis in the presence of protein inhibitors cannot be used to draw conclusions about nuclear-cytoplasmic relationships at the present time. Synthesis may stop as a result of the decay of initiator protein coded for by nuclear DNA and synthesized on cytoplasmic ribosomes, or merely through the exhaustion of a DNA precursor the synthesis of which in some way involves a nuclear DNA-coded enzyme. Mitochondrial DNA synthesis is clearly not autonomous for very long periods ; however, as we have shown, it can be dissociated from nuclear DNA synthesis; this implies a considerable degree of independent control. Moreover, this dissociation can be used for rapid analysis of specifically labeled mitochondrial DNA by use of whole-cell lysates, and such experiments are in progress. We are grateful to Miss Deborah Krupnick for competent technical assistance. One of us (L.I.G.) is a predoctoral fellow of the National Institutes of Health (1 FOl GM 34806-OlA3) and was a trainee in genetics in the early phases of this work (2 TO1 GM 00110). Another (E.S.G.) is the recipient of a postdoctoral fellowship from the Anna Fuller Fund. Another (J.M.) is supported by the Health Research Council of the City of New York (l-322). This work w&s supported by grants from the National Institutes of Health (GM-11946 07) and the National Science Foundation (GB-4686). REFERENCES Attardi, B. & Attardi, G. (1967). Proc. Nat. Acad. Sci., Wash. 58, 1061. Avers, C. J., Billheimer, F. E., Hoffmann, H-P. & Pauli, R. M. (1968). Proc. Nat. Acad. Sci., Wash. 61, 90. Guerineau, M., Grandchamp, C., Yotsuyanagi, Y. & Slonimski, P. P. (1968). C. R. Acad. Sci. Paris,

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