Effect of proteolysis on the yeast mitochondrial deoxyribonucleases

252

Biochimwa et Biophvsica Acta. 786 (1984) 252-260 Elsevier

BBA31884

EFFECT OF PROTEOLYSIS ON THE YEAST MITOCHONDRIAL DEOXYRIBONUCLEASES HELleNE JACQUEMIN-SABLON and ALAIN JACQUEMIN-SABLON Unitb de Biochimie- Enzymologie, lnstitut Gustave - Roussy, 94800 Villejuif (France) (Received October 31st, 1983)

Key words: Proteolysis; Ethidium bromide," DNAase; (Yeast mitochondria)

One or two forms of deoxyribonuclease may be found when yeast mitochondrial membranes are extracted by treatment with Triton X-100 at high ionic strength. The two forms can be separated by chromatography on DEAE-cellulose. The forms differ from one another by their physical and catalytic properties: form I, retained on the column, degrades double-stranded and single-stranded DNA at neutral pH, and is not stimulated by ethidium bromide; form II (or ethidium bromide-activated DNAase), not retained on the column, degrades ss-DNA at neutral pH, and ds-DNA at pH 5.8. Form II is about 30-fold stimulated by ethidium bromide at pH 7.8. Most probably, form II derived from form I through a limited proteolytic process. This conversion depends on such factors as the storage of the mitochondrial extract or the physiological state of the cells. Form II is found when cells are harvested at stationary phase, coincident with the increment of the yeast proteinases; its formation is blocked by the addition of proteinase inhibitors to the extract. Conversion of form I to form II, which is associated with a change in the sedimentation coefficient of the enzymatic proteins from 5.4 S to 4.5 s, can be reproduced in vitro by treatment with c~-chymotrypsin.

Introduction Numerous studies, reviewed by Gillham [1], have been carried out to analyze the molecular events occurring during the induction of petite mutation by ethidium bromide in the yeast Saccharomyces cereoisiae. This mutagenesis is accompanied by both inhibition of mtDNA synthesis and breakdown of the pre-existing mtDNA. Studies aimed at the identification of the enzyme(s) involved in this degradation process led us to the description of an endonuclease active at pH 7.8 on the ethidium bromide-DNA complex, and designated as the ethidium bromide-activated DNAase [2]. Possible involvement of the ethidium bromide DNAase in the process of petite mutation induction by ethidium bromide was suggested by the Abbreviations: PMSF, phenylmethylsulfonyl fluoride; Mes, 4morpholineethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. 016%4838/84/$03.00 :~ 1984 Elsevier Science Publishers B.V.

correlation, using a series of phenanthridinium derivatives, between the ability of a drug to stimulate the enzyme activity and its mutagenic efficiency [3]. The most purified fraction of the ethidium bromide DNAase was also active on ss-DNA at neutral pH, and on ds-DNA at pH 5.8 [2]. Several other membrane-bound deoxyribonucleases have been isolated from yeast mitochondria [4-7]. They have some common characteristics such as solubilization with detergent in the presence of KCI, metal ion requirements, and greater activity on ss-DNA than on ds-DNA. They differ in other properties such as their optimum pH, molecular weights and activity on RNA. Because of their instability, which makes difficult any extensive purification, the relationships between these different activities remain unclear. During the purification of the ethidium bromide DNAase, the deoxyribonuclease activities in the

253

mitochondrial extract are fractionated by chromatography on DEAE-cellulose in two peaks. One is the ethidium bromide-activated DNAase. Enzyme in the other is active on ss-DNA and ds-DNA at neutral pH, and is inhibited by ethidium bromide. The properties of this enzyme were similar to that of the endonuclear purified by Rosamond [5]. It was also found that the mitochondrial extracts displayed a great variability in the levels of the ethidium bromide DNAase, depending in particular on the storage of the extract and the physiological state of the yeast culture. A further analysis showed that an increased activity in the peak of ethidium bromide-activated DNAase was always associated with a loss of activity in the other peak. This led us to examine what could be the relationship between the DNAase activities contained in each peak. For reasons detailed later, the peak of ethidium bromide-activated DNAase was designated as form II and the other as form I. We show in this paper that a limited proteolytic process is able to provoke the loss of form I concomitantly with the rise of form II. Both peaks would then represent two forms of the same enzyme, form II resulting from a partial proteolysis of form I. Our results also provide a possible explanation for the different properties of the mitochondrial deoxyribonucleases described by various authors.

Experimental procedures Yeast strains and growth conditions. The haploid strain of Saccharomyces cerevisiae MH 41-7B (nuclear genotype: a, ade 2, his 1; mitochondrial genotype : w +, C R, E R O1 R p R) was kindly supplied by Dr. H. Fukuhara. The yeast growth medium has been previously described [2]. As specified for each experiment, the cells were harvested at exponential phase (A650 = 3.5; about 2- 107 cells/ml), or at early stationary phase (A650 = 15; about 3 . 1 0 8 cells/ml), or at stationary phase (3 h after reaching the early stationary phase). Nucleic acids. Tritium-labeled phage T7 DNA was prepared as previously described [2]. Assays of the nuclease activities. The assay measured the release of acid soluble products from double-stranded T7 [3H]DNA either at pH 7.8,

without or with ethidium bromide (final concentration = 1 • 10 -5 M), or at pH 5.8, under the conditions previously described [2]. The activity on ss-DNA was also measured as described in the latter reference, except that heat-denatured T7 [3H]DNA was used as substrate. One unit of enzyme activity converts 1 nmol DNA nucleotide to acid-soluble product in 30 min at 28°C. DEAE-cellulose fractionation of mitochondrial crude extract. This fractionation corresponds to step II in the ethidium bromide-activated DNAase purification procedure previously published [2]. 3 1 of medium were inoculated with 20 ml of a stationary phase preculture. After reaching the appropriate optical absorbance, corresponding to the different growth phases, the cells were harvested and mitochondria were isolated after spheroplast formation and cell breakage [2]. The mitochondrial crude extract was obtained by lysis of the isolated mitochondria with Triton X-100 in 1 M KCI. After dialysis against buffer A, comprising 0.1% Triton X-100/1 mM fl-mercaptoethanol/0.01 M Tris HC1 (pH 7.4), the extract was stored at 4°C [2]. For DEAE-chromatography, an aliquot (1 ml containing about 1 mg protein) was applied to the column (2 cm × 0.2 cm 2), previously equilibrated with buffer A. The column was washed with 2 ml buffer A and 0.5 ml fractions were collected. The retained proteins were then eluted with 2 ml of buffer A/0.2 M NaC1. a-Chymotrypsin treatment of the mitochondrial crude extract, a-Chymotrypsin treatment was carried out on a mitochondrial extract prepared from cells grown to exponential phase. After dialysis against buffer A, aliquots of the extract (0.5 ml containing about 0.5 mg protein) were incubated with a-chymotrypsin at different concentrations for 18 h at 2°C. The proteinase concentrations were adjusted to reach the desired value of the ratio R = mg mitochondrial protein/mg added chymotrypsin. The incubation was arrested by adding PMSF at a final concentration of 1 mM. Sucrose gradient sedimentation analysis. The sedimentation coefficient of the protein-Triton X100 complex was determined by density gradient sedimentation as described by Martin and Ames [8]. The proteins (about 250/~g) were centrifuged through a linear 4-25% sucrose gradient in 0.01 M

254

Tris-HC1 (pH 7.5)/0.1% Triton X-100. Centrifugation was carried out for 18 h at 2°C, at 50000 rpm, in a Spinco SW 65 rotor. Fractions (0.12 ml) were collected from the bottom, and assayed for enzyme activities. Standard deviations on the S values were calculated on six or seven independent experiments. Other materials. Ethidium bromide was purchased from Boots Pure Drug Co.; achymotrypsin (from bovine pancreas type IV, 37 U/mg), PMSF, 1,10-phenanthroline, Mes and Hepes were from Sigma; Triton X-100 from Calbiochem, and lyophylized helicase from Industrie Biologique Franqaise. DEAE-cellulose (Cellex-D) was obtained from Bio-Rad; [methyl14C]methylated bovine serum albumin, and [methylJ4C]methylated carbonic anhydrase were purchased from New England Nuclear. Antipain, leupeptin and pepstatin were from Protein Research Foundation. Bacitracin was from Sigma. The proteinase inhibitors were used under the following conditions. Three separate solutions were prepared, which were 1/100 diluted in the mitochondrial extract. Solution A:0.1 M PMSF, 0.5 M 1,10-phenanthroline, 0.5 m g / m l pepstatin in ethanol; solution B: 0.2 m g / m l leupeptin, 0.2 m g / m l antipain in water; solution C : 0.1 M EDTA (pH 6.5). When used, bacitracin was added at a final concentration of 1 mM. Results Fractionation of the mitochondrial nuclease activities on DEAE-cellulose Purification of the ethidium bromide DNAase was carried out as previously described [2] from cells harvested at stationary phase, which were found to contain higher enzyme levels. The second step in the purification procedure was a fractionation of the mitochondrial crude extract on DEAE-cellulose. Fig. 1 shows the result of such chromatography, where the enzyme activity in the different fractions was determined by the procedure defined for the ethidium bromide-activated DNAase (activity on ds-DNA at pH 7.8, in presence or absence of ethidium bromide). The activity was distributed in two peaks: one peak (which we now designate as form I), retained on the column and eluted with 0.2 M NaC1, was equally active, or

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Fig. 1. DEAE-cellulose chromatography of yeast mitochondrial extract. 1 ml of mitochondrial crude extract (2 mg protein), from cells harvested at stationary phase, was fractionated by chromatography on DEAE-cellulose, and the fraction were assayed for enzyme activity on ds-DNA at pH 7.8, either in the absence (e) or in the presence ((3) of ethidium bromide. The arrow indicates where 0.2 M NaCI was added.

sometimes inhibited, in the presence of ethi:lium bromide; another peak (form II, previously called ethidium bromide DNAase) recovered in the washed fractions of the column, displayed a very low activity in the absence of ethidium bromide, but, as previously shown [2], was greatly stimulated in the presence of this drug. The other nuclease activities, present in the extract (hydrolysis of ss-DNA at neutral pH, and ds-DNA at pH 5.8) were about equally distributed in both peaks. Designation of these peaks as forms I and II, which indicates a relationship between them, was suggested by the results presented in this paper. The levels of the different nuclease activities in the mitochondrial extracts were variable from one preparation to another. We will show that this variability depends on the growth phase, storage, and proteinase concentration. Growth phase effect on the variations of the nuclease activities Levels of the different nuclease activities were

255 measured in mitochondrial extracts from cells harvested at different growth phases. Important variations in the specific activities of the different mitochondrial nuclease activities were observed. First, in extract from stationary phase cells, the activity on ds-DNA at pH 7.8 is only about 10% of that in exponential phase cells. However this activity is about 2-fold increased in the presence of ethidium bromide, whereas it is partially inhibited in extracts from exponential cells. Second, in contrast to the previous one, both activities on ss-DNA and ds-DNA at p H 5.8 are 5-6-fold higher in stationary-phase cells than in exponential-phas~ cells. The nuclease activities, in the different extracts, were then fractionated by chromatography on DEAE-cellulose. Fig. 2 shows that the quantitative variations observed in the extracts are associated with changes in the chromatographic behavior of the enzyme activities. In extracts from exponentially growing cells, the enzyme activities are retained on the column (form I), while a peak of =

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Fig. 2. DEAE-cellulosechromatography of yeast mitochondrial extracts from cells harvested at different growth phases. Mitochondrial extracts prepared from cells collected at different growth phases as described in legend to Table I, were fractionated on DEAE-cellulose as described in the legend to Fig. 1. (A) Exponential phase; (B) early stationary phase; (C) stationary phase. Upper part: activitieson ds-DNA, pH 5.8 (11) and ss-DNA at pH 7.4 (zx); lower part: activity on ds-DNA, pH 7.8, in the absence (e) or in the presence (C)) of ethidium bromide.

unretained activities (form II) only appears in extracts from early stationary-phase cells (Fig. 2A and B). At stationary phase, the activities on ssD N A and ds-DNA at p H 5.8 are recovered as both forms I and II. In contrast, at this phase, the activity on ds-DNA at p H 7.8 is detected only in form II and in the presence of ethidium bromide (Fig. 2C)

Changes in the nuclease activities during storage of the mitochondrial crude extract We observed that changes occurred in the levels of the different nuclease activities during storage of the mitochondrial extract at 4°C. In order to analyze these changes, a mitochondrial extract, prepared from cells harvested at early stationary phase, was stored for 30 days at 4°C, either in the absence or in the presence of a mixture of proteinase inhibitors, including antipain, leupeptin, pepstatin, 1,10-phenanthroline, PMSF and EDTA. These inhibitors, which are known to inhibit most of the yeast proteinases [9,10], were added during the preparation of the extract, after lysis of the mitochondria with Triton X-100. The nuclease activities were then assayed after storage at 4°C for 1 and 30 days, and their chromatographic properties were examined. The results are gathered in Table I. In absence of proteinase inhibitors, at day 1, all the activities were recovered in peak 1, retained on the column. At day 30, all of them were markedly decreased, and mostly recovered in peak 2. The most affected was the activity on ds-DNA at pH 7.8, which was undetectable when assayed in absence of ethidium bromide. However, in the presence of ethidium bromide, about 10% of the activity, measured under the same conditions as at day 1, was recovered at day 30, but now in form II. Addition of proteinase inhibitors to the mitochondrial extract resulted in a stabilizing effect on the enzyme activities, and partially prevented the development of form II. Again, the most striking effect was observed on the activity on ds-DNA at p H 7.8, which, at day 1, is about 3-4-fold higher in the presence of the proteinase inhibitors. At day 30, 30% of the activity was recovered exclusively in peak 1, and was about 60% inhibited in the presence of ethidium bromide. This indicated that ethidium bromide stimulation

256 TABLE I CHANGES IN THE NUCLEASE ACTIVITIES IN THE M I T O C H O N D R I A L EXTRACT AFTER STORAGE AT 4 ° (" WITH OR W I T H O U T PROTEINASE INHIBITORS A mitochondrial extract prepared from cells at early stationary phase was stored at 4 o C for 30 days either in the absence or in the presence of proteinase inhibitors. Enzyme activities ( U / m g ) were measured in the extract, and percentages of forms I and I1 refer to the relative amounts of these activities which were recovered in peaks 1 and 2 after DEAE-chromatography. Enzyme activity

ds-DNA

ss-DNA + Proteinase inhibitors ds-DNA

ss-DNA

Day 1

pH 7.8

pH 5.8 pH 7.4

% form I

% form 1I

106 52 227 175

98 96 94 97

2 4 6 3

0 5 47 35

0 15 l0

100 85 90

- ethidium bromide - ethidium bromide

394 131 328 210

99 90 93 94

1 10 7 6

131 45 218 66

100 85 50 50

0 15 50 50

of this activity can be observed only in form II. Both activities on ss-DNA and ds-DNA at pH 5.8 were almost equally distributed between forms I and II. These data indicate that during storage of the extract at 4 ° C, the enzyme activities undergo some proteolytic degradation, which is associated with the disappearance of form I and the development of form II. When the same experiment was carried out with an extract prepared from exponentially growing cells, all the activities were almost stable and were recovered in form I, even after storage at 4 ° C for 2 months. This correlates with the results presented in Fig. 2, showing that form II appears only at stationary phase. This observation should then be related to the enhancement of the yeast proteinases, which occurs during the transition from logarithmic to stationary phase of growth [11]. Indeed, when proteinase inhibitors were added to stationary phase cell mitochondria prior to lysis, apparition of DNAase form II was prevented, and only form I was obtained.

Treatment with c~-chymotrypsin The previous conclusions were further supported by experiments in which a mitochondrial extract, prepared from cells harvested during the exponential phase of growth, was treated at 2°C for 17 h with a-chymotrypsin at different concentrations. Protein concentrations were such that

U/rag

% form I

% form I1

U/mg - ethidium bromide + ethidium bromide

pH 5.8 pH 7.4

pH 7.8

Day 30

the ratio R of the protein weights (extract proteins to c~-chymotrypsin) were adjusted to the following values: 4, 2, 1 and 0.5. Fig. 3 confirms that the activity on ds-DNA at pH 7.8 is very sensitive to proteolytic degradation, and, under these conditions, is completely lost at the lowest proteinase concentration. However, up to the c~-chymotrypsin concentration corresponding to R = 2, an important part of this activity was retained in the presence of ethidium bromide. At all proteinase concentrations, activity on ss-DNA remained about stable, while the activity on ds-DNA at pH 5.8 was increasing up to the maximum c~chymotrypsin concentration used in this experiment (R = 0.5). Thus, in agreement with storage experiments, proteinase digestion is able to increase the activity at pH 5.8, and to reveal the stimulatory effect of ethidium bromide on the partially proteolysed activity on ds-DNA at pH 7.8. We then examined the chromatographic behavior of the nuclease activities on DEAE-cellulose after treatment of a mitochondrial extract for 17 h at 2°C, with a-chymotrypsin at a concentration corresponding to R = 2, which allows one to measure the activity at pH 7.8 with or without ethidium bromide. As expected, the proteinase treatment provoked the transfer of the major part of the three activities from the bound form to the unbound form. Again, the stimulatory effect of

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Fig. 3. Effect of a-chymotrypsin on mitochondrial DNAase activities. After proteinase treatment, DNAase activities were determined on ds-DNA at pH 7.8, either in absence (e) or in presence of ethidium bromide (©), on ds-DNA at pH 5.8 (m), and on ss-DNA at pH 7.4 (zx).

ethidium bromide is observed only on the unbound form (proteolysed form). These results show that treatment with achymotrypsin can mimic the different changes observed in the mitochondrial extract either during storage or depending on the growth phase of the cells.

prior to lysis with the detergent. After chromatography on DEAE-cellulose, only DNAase form I was present. By elution with a linear NaC1 gradient, a single peak of activity at pH 7.8 was recovered. Some activity at p H 5.8 was also found in the same fractions, which may be due to the residual activity of form I at this p H (Fig. 7). There was no detectable activity at p H 5.8 in the other fractions of the gradient. All the fractions were then individually incubated with a-chymotrypsin at a concentration such that the ratio R was approximately equal to 1. After 18 h at 2°C, the enzyme activities were measured. Activity at p H 7.8 was completely lost in all the fractions, whereas the activity at p H 5.8 remained almost unchanged. Therefore, this experiment did not reveal the presence, in some gradient fractions, of an inactive form II precursor which could have been separated from form I in a higher-resolving elution system. However, the following limitations should be noted: (a) after treatment with a-chymotrypsin under these conditions, activity at pH 5.8 was very unstable, and it was not possible to rechromatograph the proteolyzed protein to check its conversion to the DEAE-unbound form II; (b) proteolysis eliminates the activity at pH 7.8, but does not provoke a significant increase of the activity at p H 5.8. This may result from a balance between the increase expected from the conversion of form I to form II, and the decrease resulting from the proteolysis a n d / o r the physical instability of form II.

Further analysis of DEAE-retained enzyme activities.

Sedimentation analysis of forms I and II

The simplest interpretation of the results presented so far is that form II enzyme arises via proteolysis of form I. However, the possibility of an independent origin of form II has also to be considered. For example, non-proteolysed preparations could contain a DEAE-binding form of the form II enzyme which would not have been detected in a stepwise elution procedure. Alternatively, form II could also be generated via proteolysis of an inactive precursor, different from form I. To test these possibilities, a mitochondrial extract was prepared from exponentially growing cells, in the presence of proteinase inhibitors (1,10-phenanthroline, antipain and bacitracin) which were added to the mitochondrial suspension

Since the previous experiments indicated that form II was generated from form I, through a proteolytic process, we then examined whether this proteolysis would also modify the molecular weight of the enzymatic protein(s). For this purpose, we measured the sedimentation velocities of forms I and II either in mitochondrial extracts from cells at exponential and stationary phase, or after in vitro proteolytic digestion of form I with achymotrypsin. A mitochondrial extract from yeast cells at exponential phase, containing mainly form I, was centrifuged through a 4-25% sucrose gradient. The activity on ds-DNA at pH 7.8 (characteristic of form I) sedimented in a single zone, at 5.4 S

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Fig. 4. Sucrose gradient sedimentation of mitochondrial DNAase forms I and II. Left part: a mitochondrial extract was prepared either from exponentially growing cell~ (Exp.) or from cells at stationary phase (Stat.), and centrifuged through a 4-25% sucrose gradient. Activities on d s - D N A at pH 7.8 (O) and 5.8 (11) were determined using 30-/LI aliquots (Exp.) or 10-#1 aliquots (Stat.) of the fractions (0.12 ml). Right part: a mitochondria] extract was prepared from exponentially growing cells and centrifuged through a 4-25% sucrose gradient, after treatment with a-chymotrypsin. ((A) control; (B) R = 6; (C) R = 3). Bovine serum albumin (arrow b) and carbonic anhydrase (arrow c) were used as reference markers. Activity on ds-DNA at pH 5.8 (11) was determined using 10 ~tl of each fraction (0.12 ml).

( + 0.2) (Fig. 4). Some activity at pH 5.8 was also recovered in the same zone. However, the recoveries of the enzyme activities, after centrifugation, were very low: less than 10% for both. Reasons for this loss of activity (denaturation, dissociation from a catalytically required subunit, or cofactor, e t c . . . ) are as yet unknown. After centrifugation of an extract from cells at stationary phase, containing form II, the activity at pH 5.8 was again recovered in a single zone, but now sedimenting at 4.2 ( + 0 . 5 ) S. There was no remaining activity at pH 7.8. Under these conditions, the recovery of the activity at pH 5.8 was about 50%.

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Fig. 5. pH activity curves of DNAase forms I and II. Forms I and II were obtained by chromatography on DEAE-cellulose of a mitochondrial extract from yeast cells harvested either at exponential or at stationary phases. The following buffers were used at a final concentration of 0.02 M: sodium acetate buffer (v) pH 4.8 to 5.2, Mes-NaOH buffer (I) pH 5.6 to 6.8, Hepes-NaOH buffer ( - ) pH 6.8 to 8.2. Insets show the effect of ethidium bromide on the enzyme activities at pH 7.7. (A) Form I: (B) form II.

Fig. 4 also shows that in vitro treatment of form I with a-chymotrypsin at different concentrations led to comparable modifications of the sedimentation velocity. At R = 6 and R = 3, the sedimentation zone was progressively shifted from 5.4 S to 4.7 and 4.5 S. These experiments show that, whether the change from form I to form II is caused by yeast proteinases or a-chymotrypsin, similar molecular weight modifications are observed.

pH-activity curves of forms I and II The opposite effects on the activities toward d s - D N A at pH 5.8 and 7.8, observed during the conversion of form I to form II, led us to investigate in more detail the pH-activity curves of the enzymatic forms present in extracts prepared either from exponential- or from stationary-phase cells.

259 Fig. 5A shows that form I activity on ds-DNA is progressively increasing with pH up to a maximum at 7.2. In contrast, form II displays a maximum at pH 5.8, with almost no residual activity at pH 7.8 (Fig. 5). However, activity at the latter pH is about 25-30-fold increased in the presence of ethidium bromide, which has no effect on form I under the same conditions (insets in Fig. 5). After the DEAE step in the purification, the pH activity curve of form II is similar to that observed with the most purified preparation [2]. Discussion The deoxyribonuclease activities, extracted from the yeast mitochondrial membrane by treatment with Triton X-IO0 at high ionic strength, are present in one form which can be transformed in a second form by limited proteolytic activities. Variations in the relative amounts of forms I and II, resulting from this proteolysis, are observed in different conditions, and are associated with quantitative variations of the different activities which characterize each form. These results can be brought together with the previous studies on yeast mitochondrial nucleases [2,4-6], and, at least partially, explain the apparent diversity of the enzyme activities described in these reports. It is clear that DNAase form I, described in this work, is very close to the enzyme purified by Rosamond [5]. Both activities display similar chromatographic behavior, sedimentation coefficient, and catalytic properties. As already pointed out, form II corresponds to the ethidium bromide-activated DNAase which we previously purified [2]. The Mg2+-dependent nuclease recently purified in absence of proteinase inhibitors by Tigerstrom [7] has a maximum activity on ds-DNA at pH 6 to 6.5, close to that of the ethidium bromide-activated DNAase. However, the effect of ethidium bromide on this enzyme was not reported. In addition to these activities, other nucleases are present in the yeast mitochondrial membrane. Apurinic and manganese-stimulated deoxyribonucleases have been identified by Foury [12]. Another mitochondrial, membrane-bound nuclease has been extracted by Morosoli and Lusena [4], which is very similar to form I isolated in the presence of inhibitors. However, its known

properties do not allow one to establish a clear relationship between this enzyme and the other described activities. The simplest interpretation of the results presented in this paper is that form II enzyme arises via a proteolysis of form I. The major argument in favor of this interpretation is that, in all the experimental conditions examined, the rise of form II is always associated with the disappearance of form I. The failure to detect an inactive form II precursor, different from form I, also argues in favor of this conclusion. However, we cannot completely exclude the possibility of a form II origin independent of form I. Extensive purification of both DNAases form I and form II, and gel electrophoresis analysis of the polypeptides would allow one to analyse clearly the conversion process. However, as already noted by Rosamond [5], a major problem in this work is the great instability of the enzyme(s) during the purification. Our results allow us to define more accurately the conditions required to protect, and then purify, either one of these enzyme forms. Conversion of form I to form II was found to depend on such factors as the storage of the mitochondrial extract or the physiological state of the yeast cells. An important question was as to whether this conversion occurs only in vitro, during the enzyme purification, or whether it may also occur in vivo. Conversion of form I to form II, observed in cells harvested at early stationary phase or later, coincides with yeast proteinase increment [9,10], and is blocked by the early addition of proteinase inhibitors to the mitochondrial extract. This shows that conversion of form I to form II, described in this work, involves the yeast endogenous proteinases, but occurs in vitro. The fact that this conversion can be unspecifically reproduced in vitro with a-chymotrypsin does not exclude the existence, in vivo, of a much more specific process, implying the involvement of a membrane mitochondrial proteinase. Indirect support for an in vivo transformation of form I to form II is provided by the work of Foury [12], who isolated a mutant which carries a temperature-sensitive mitochondrial membrane deoxyribonuclease. This enzyme degrades mtDNA, at acid pH, in the presence of Triton X-100, and its activity is increased in the presence of ethidium bromide. These prop-

260

erties, which are very close to those of the ethidium bromide-activated DNAase [2], suggest that form II might be present in vivo, and, therefore, proteolysis, of form I to form II might also occur in vivo. Other examples of proteolytic transformation of nucleases have been described. Kwong and Fraser [13] have shown that, in Neurospora crassa, an endonuclease, initially synthesized as an inactive precursor, or tightly complexed in the cell with an inhibitor, is progressively transformed by proteolysis into an active endoexonuclease, and then a nuclease specific for ss-DNA. Mutants presenting a defect in this conversion have been isolated [14]. Recently, Fraser and Cohen [15] showed that most of the inactive precursor and part of the active enzyme are located in the inner mitochondrial membrane. Finally, another puzzling problem is the apparent specificity of the ethidium bromide effect, which in vitro, stimulates only the enzyme form II. In a phenanthridinium series, this effect was correlated with the mutagenic efficiency of the drugs [3]. According to our previous results [16], this activation would result from the formation of a ternary complex between the DNA, the drug, and the detergent required for the enzyme solubilization. When this complex is formed, the DNA molecule is surrounded with Triton X-100 molecules, which constitute an hydrophobic environment, and make the substrate more prone to interaction with the enzyme. One possibility would then be that the structural change, resulting from the proteolysis of form I, would make form II more hydrophobic, and therefore more dependent on this effect. The 4.5 S enzyme, previously designated ethidium bromide DNAase, then appears in vitro as the product of the proteolytic conversion of a precursor form.

Acknowledgements The technical assistance of B. Lecomte is gratefully acknowledged. We are indebted to F. Foury for stimulating discussion. Financial support was provided by the Centre National de la Recherche Scientiflque (L.A. 147), the Institut National de la Sant6 et de la Recherche M6dicale (U 140, and Contrat libre), and the Commissariat ~t l'Energie Atomique.

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