Bioehimica et Biophysica Acta, 454 (1976) 375--381 © Elsevier/North-HollandBiomedical Press
B B A Report BBA 91438 THE ABSENCE OF DNA PHOTOREACTIVATION ENZYME IN YEAST MITOCHONDRIA*
o. PATRICK DAILY**, F R A N K F. CUTTITTA JR. and A N T H O N Y M. MACQUILLAN*** Department of Microbiology, University of Maryland, College Park, Md. 20742 (U.S.A.) (Received September 16th, 1976)
Summary Mitochondria isolated from haploid yeast cells by spheroplast lysis were purified by flotation on renografin gradients. Electron micrographs and respiratory control ratios revealed that the purified mitochondria were stillintact and functional. Assays for photoreactivation enzyme using as substrate [3HIthymine-labeled Escherichia coli D N A were performed on crude and purified mitochondrial preparations. While the crude preparation contained high amounts of photoreactivation enzyme, it appeared to be associated with contaminating nuclei. The purified mitochondria lacked any photoreactivation enzyme activity.W e suggest that yeast mitochondria do not normally contain photoreactivation enzyme.
The ultraviolet irradiation-induced damage leading to petite (rho-) mutations in Saccharomyces cerevisiae can be reversed by exposing cellsto photoreactivating light [1--4]. The mechanism of petite induction initiated by the irradiation and the modes and mechanisms of its repair are unclear. Several authors [1,5--8] have implicated mitochondrial D N A ( m t D N A ) as the target for ultravioletlight induction of petites and have postulated that photorepair of petite mutational damage was due to a splittingof pyrimidine dimers in m t D N A by a photoreactivation enzyme. Only very recently has direct evidence been presented that suggests that this m a y be the case. Waters and Moustacchi [6] have shown, by CsCl ultracentrifugation, that dimers disappear from rntDNA of ultravioletlight-irradiatedyeast cells after a period of photoreactivation. These findings have been confirmed by Prakash [8]. In an *This w o r k was c a r r i e d o u t b y O.P.D. in i)artial f u l f i l l m e n t o f t h e University o5 M a r y l a n d for the Ph.D. degree. Portions o f this paper w e r e p r e s e n t e d at the 7 6 t h A n n u a l Meeting o f the A m e r i c a n S o c i e t y for M i c r o b i o l o g y , Atlantic City, N.J., 2--7 May, 1 9 7 6 . **Present address: D e p a r t m e n t o f M i c r o b i o l o g y , Naval Medical R e s e a r c h I n s t i t u t e , N a t i o n a l Naval Medical Center, Bethesda, Md. 2 0 0 1 4 , U.S.A. * * * T o w h o m inquiries a n d r e p r i n t r e q u e s t s s h o u l d b e addressed.
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Fig. 1. E l e c t r o n m i c r o g r a p h s o f (a) c r u d e m i t o c h o n d r i a l p r e p a r a t i o n a n d (b) r e n o g r a f i n - p u r i f i e d m i t o c h o n d r i a . M i t o c h o n d r i a l p r e p a r a t i o n s in 0 . 0 5 M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 6 . 8 ) c o n t a i n i n g 20% s u c r o s e a n d 1 • 10 -s M E D T A w e r e t r e a t e d w i t h 2.5% g l u t a r a i d e h y d e 0 f i x e d in 1.0% o s m i u m t e t r o x i d e , e m b e d d e d in e p o n 8 1 2 ( D u P o n t ) , s e c t i o n e d , a n d s t a i n e d w i t h u r a n y l a c e t a t e a n d lead c i t r a t e . Magnifi c a t i o n 1 7 0 0 X.
effort to elucidate further the mechanism involved in the photorepair of ultraviolet-induced petite mutational damage we elected to determine whether or n o t yeast mitochondria possess photoreactivation enzyme. In this report we present evidence which suggests that yeast mitochondria do n o t normally contain detectable photoreactivation enzyme.
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Mitochondria from ~ cerevisiae haploid cells (strain a31-5) were isolated from lysed spheroplasts prepared in 2 M sorbitol buffer by the procedure of DueU et al. [9]. Respiratory control ratios (QO2 with ADP/QO2 without ADP) of 12.9 and 2.8 were determined for ~-ketoglutarate and succinate oxidation respectively, suggesting minimal mitochondrial damage [9,10]. However, electron micrographs of these mitochondria revealed significant contamination with nuclei, Fig. la. A further purification of the mitochondria was achieved by renografin
378 density-gradient flotation [11,12]. Mitochondria were resuspended in renografin at 1.25 d (g/cm 3) and overlayered with 6 volumes of 1.15 d renografin. Centrifugation was carried out in a Beckman Type 21 rotor for 2 h at 21 000 rev./min. The intact mitochondria were isolated from the top of the gradient. Electron micrographs, Fig. lb, reveal that this material consisted predominantly of intact mitochondria relatively free of contaminating nuclei. Respiratory control ratios of 2.35 for a-ketoglutarate and 1.3 for succinate, although lower than the values of the crude preparation, indicate that the purified mitochondria were physiologically intact. In addition, double membrane-bound structures in Fig. l b suggest that mitochondrial integrity survived the renografin treatment. Assays for photoreactivation e n z y m e were based on the photorepair of DNA containing t h y m i n e dimers as described by Cook and Worthy [13]. Purified [3H]thymine-labeled Escherichia coli DNA (1.4 × l 0 s dpm/pg) was irradiated with ultraviolet light in reduced 02 in the presence of 0.01 M acetophenone. This procedure converted approx. 25% of the labeled t h y m i n e into the t h y m i n e dimer form. After removal of the acetophenone by dialysis, this dimerized DNA in 0.15 M NaC1 was used as the substrate for photoreactiration e n z y m e in yeast cell extracts and in lysed, homogenized mitochondrial preparations. Conversion of t h y m i n e dimers to monomers by photoreactivation e n z y m e was monitored by one-dimensional paper chromatography. The crude mitochondrial preparation and renografin-purified mitochondria were osmotically lysed, homogenized, and tested for photoreactivation enzyme activity. As shown in Fig. 2, photoreactivation enzyme activity was found in the crude mitochondrial preparation but was not detected in the purified mitochondria. Possible denaturation of a potential mitochondrial photoreactivation enzyme by the renografin t r e a t m e n t was ruled unlikely since renografin exposure failed to alter the photoreactivation enzyme activity of a yeast cell extract. Specific activities (see legend to Fig. 3) of the extract before and after a 1-h exposure to renografin, followed by dialysis, were 0.173 and 0.175 respectively. To insure that a potential mitochondrial photoreactivation enzyme had access to the labeled substrate the mitochondria were solubilized with Triton X-100 [11 ], rather than lysed by osmotic and mechanical means. Mitochondria solubilized in this way failed to exhibit any photoreactivation enzyme activity. Triton X-100 only marginally altered the specific photoreactivation e n z y m e activity of a yeast extract from 0.120 to 0.109, which would seem to exclude the possible inactivation of a potential mitochondrial photoreactivation e n z y m e by the detergent. It was conceivable that a mitochondrial photoreactivation enzyme might exhibit a markedly different ionic strength (g) requirement. We, therefore, compared the photoreactivation e n z y m e activity of purified mitochondria with that of yeast extracts over a wide range of ionic strengths (p = 0.036-0.385). Fig. 3 shows the results of these experiments. The yeast extract showed optimal photoreactivation e n z y m e activity at p = 0.15--0.16 [ 13 ], while purified mitochondria failed to show photoreactivation enzyme activity over the entire range of ionic strengths tested. Our results suggest that mitochondria isolated from S. cerevisiae do not
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TIME (min) Fig. 2. P h o t o r e a c t i v a t i o n e n z y m e a c t i v i t y o f c r u d e a n d p u r i f i e d y e a s t m i t o c h o n c L r i a . Y e a s t m i t o e h o n d r i a l prepaxations were washed in 0.05 M potassium phosphate buffer (pH 6.8) eontslninS 20% sucrose and 1 • 1 0 -3 M E D T A , r e s u s p e n d e d t o 2 9 0 / ~ g p r o t e i n l m l , l y s e d in 0 . 0 6 7 M p o t a s ~ u m p h o s p h a t e b u f f e r (pH 7.0) and homogenized with a Potter-Elvejhem tissue ~dnder. This material was mixed with an equal v o l u m e of [ S H ] t h y m i n e - l a b e l e d E. coil D N A , p r e v i o u s l y i r r a d i a t e d w i t h ultraviolet light in the p r e s e n c e o f a c e t o p h e n o n e so t h a t 2 5 % o f t h e t h y m i n e w a s i n t h e d i m e r f o r m . P h o t o r e a c t i v a t i o n a t 3 0 ° C w a s c a r r i e d o u t b y i l l u m i n a t i n g t h e r e a c t i o n m i x t u r e s w i t h f o u r b l a c k l i g h t s ( G e n e r a l E l e c t r i c F 1 5 T S - B L B ) a t a disfence of 10 cm. Samples were removed from the reaction mixtu~s, precipitated with trichloroacetic a c i d , h y d r o l y z e d , c h r o m a t o g r a p h e d a n d c o u n t e d as d e s c r i b e d b y C o o k a n d W o r t h y [ 1 3 ] , T h e r e s u l t s w e r e p l o t t e d as S / S o vs. t i m e , w h e r e S 0 is t h e i n i t i a l s u b s t r a t e c o n c e n t r a t i o n ( t h y m i n e d i m • r ) a t t i m e 0, a n d S is t h e s u b s t r a t e c o n c e n t r a t i o n a t a g i v e n t i m e . o o, purified mitochondrla; • •, crude mitochondria.
possess measurable amounts of photoreactivation enzyme. The activity in the crude mitochondrial fraction is due to contaminating nuclei. Renografin purification reduces nuclear contamination with a concomitant loss of photoreactivation enzyme activity. The lack of this activity in the purified mitochondria would seem n o t to be due to renografin denaturation of a possible mitochondrial photoreactivation enzyme, to a different ionic strength requirement, nor to a lack of enzyme-substrate contact with incompletely lysed mitochondria. The fact that these mitochondria are still intact as judged by electron microscopy and physiological parameters mitigates against leakage of a potential photoreactivation enzyme from the mitochondria. A lack of
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IONIC S T R E N G T H (~1) Fig. 3. E f f e c t o f i o n i c s t r e n g t h o n p h o t o r e a c t i v a t i o n e n z y m e specific a c t i v i t y o f y e a s t e x t r a c t s a n d p u r i f i e d m i t o c h o n d r i a . P u r i f i e d m i t o c h o n d r i a a n d y e a s t e x t r a c t s w e r e d i l u t e d t o 3 0 0 ?tg p r o t e i n / m l in 0 . 0 6 7 p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7 . 0 ) a n d a s s a y e d f o r p h o t o r e a c t i v a t i o n e n z y m e , as d e s c r i b e d in Fig. 2, a t t h e i n d i c a t e d ionic s t r e n g h t s (/~). The p h o s p h a t e c o n c e n t r a t i o n w a s h e l d c o n s t a n t w h i l e t h e c o n c e n t r a t i o n o f NaCI was a d j u s t e d t o a l t e r t h e ionic strength. Specific activity o f p h o t o r e a e t i v a t i o n e n z y m e is d e f i n e d as t h e s l o p e , d e t e r m i n e d b y regression analysis, o f e a c h p l o t o f S / S o v s . t i m e (e.g. Fig. 2) d i v i d e d b y t h e protein c o n c e n t r a t i o n in m g . o o, p u r i f i e d m i t o c h o n d r i a ; • o0 y e a s t extracts.
respiratory control in mitochondria which we purified by sucrose density gradient centrifugation precluded meaningful photoreactivation enzyme assays of these preparations. For a non-mitochondrial photoreactivation enzyme to reach mtDNA it would presumably have to be transported, by some mechanism, into the mitochondria. Transfer of proteins across membranes probably occurs in most cells [ 14], and transport specifically into mitochondria has been suggested by a number of authors [15--19]. Diamond et al. [20] have proposed that cytoplasmically synthesized photoreactivation enzyme in Euglena can penetrate chloroplast, mitochondrial and nuclear membranes. The recently reported evidence for dimer disappearance in mtDNA after photoreactivation [6,8], coupled with our data, leads us to conclude that yeast mitochondria do not normally contain photoreactivation enzyme; rather photorepair is accomplished by a non-mitochondrial enzyme that presumably gains access to the damaged mtDNA. This possibility might be tested by examining mitochondria from ultraviolet irradiated cells for photoreactivation enzyme activity. However, the large numbers of cells needed to obtain sufficient mitochondria, free of nuclei, for the assay, and the cumbersome irradiation procedure present technical obstacles which would be difficult to overcome. Our results also imply that the photoreactivation enzyme responsible for
381
photorepair of yeast mtDNA is probably not encoded within the mitochondrion, but rather by nuclear DNA. This work was supported by grants to A.M.M. from Research Corporation and the United States Public Health Service (5 RO1 GM 18490). References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Wflkie, D. ( 1 9 6 3 ) J. MoL Biol. 7 , 5 2 7 - - 5 3 3 . Pittman, D., Ranganathan, B. and Wilson, F. (1959) Exp. Cell Ites. 17, 368--377. Ss,rachek, A. (1958) Cytologia 23, 143--158. Pittman, D.D. and Pedigo, P.R. (1959) Exp. Cell Res. 17, 359--367. Allen, N.E. and MacQufllan, A.M. (1969) J. Bacterlol. 97, 1142--1148. Waters, It. and Moustacchi, E. (1974) Biochim. Biophys. Acta 366, 241--250. Maroudas, N.G. and Wilkie, D. (1968) Binchim. Biophys. Acta 166, 661--688. 1~,akash, L. (1975) J. Mol. Biol. 96, 761--795. Duell, E.A., Inoue, S. and Utter, M.F. (1964) J. Bacter[ol. S6, 1762--1773. Lloyd, D. (1974) in The Mitochondtla of Microorganisms, pp. 69--90, Academic Press, New York, Zeman, L. and Lusena, C.V. (1974) FEBS Lett. 40, 84--67. Schatz, G., Haslhrunner, E. and Tuppy, H. (1964) Biochem. Biophys. Res. Commun. 15, 127--132. Cook, J.S. and Worthy, T.E. (1972) Biochemistry 11,368---393. Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67, 635--851. Bingham, It.W. and Campbell, P.N. (1972) Biochem. J. 126, 211--215. Kadenbach, B. (1970) Eur. J. Biochem. 12, 392--398. Stratman, F.W., Zahlten, I t . N , Hochberg, A, and Lardy, H.A. (1972) Biochemistry 11, 3154--3162. Kellems, R.E. and Butow, It.A. (1974) J. Biol. Chem, 249, 3304--3310. Mahler, H.R. (1973) CItC Crit. Rev. Biochem. 1, 361--460. Diamond, J,, Schiff, J.A. and Kelner, A. (1975) Arch. Biochem. Biophys. 167, 603--614.
B B A Report BBA 91438 THE ABSENCE OF DNA PHOTOREACTIVATION ENZYME IN YEAST MITOCHONDRIA*
o. PATRICK DAILY**, F R A N K F. CUTTITTA JR. and A N T H O N Y M. MACQUILLAN*** Department of Microbiology, University of Maryland, College Park, Md. 20742 (U.S.A.) (Received September 16th, 1976)
Summary Mitochondria isolated from haploid yeast cells by spheroplast lysis were purified by flotation on renografin gradients. Electron micrographs and respiratory control ratios revealed that the purified mitochondria were stillintact and functional. Assays for photoreactivation enzyme using as substrate [3HIthymine-labeled Escherichia coli D N A were performed on crude and purified mitochondrial preparations. While the crude preparation contained high amounts of photoreactivation enzyme, it appeared to be associated with contaminating nuclei. The purified mitochondria lacked any photoreactivation enzyme activity.W e suggest that yeast mitochondria do not normally contain photoreactivation enzyme.
The ultraviolet irradiation-induced damage leading to petite (rho-) mutations in Saccharomyces cerevisiae can be reversed by exposing cellsto photoreactivating light [1--4]. The mechanism of petite induction initiated by the irradiation and the modes and mechanisms of its repair are unclear. Several authors [1,5--8] have implicated mitochondrial D N A ( m t D N A ) as the target for ultravioletlight induction of petites and have postulated that photorepair of petite mutational damage was due to a splittingof pyrimidine dimers in m t D N A by a photoreactivation enzyme. Only very recently has direct evidence been presented that suggests that this m a y be the case. Waters and Moustacchi [6] have shown, by CsCl ultracentrifugation, that dimers disappear from rntDNA of ultravioletlight-irradiatedyeast cells after a period of photoreactivation. These findings have been confirmed by Prakash [8]. In an *This w o r k was c a r r i e d o u t b y O.P.D. in i)artial f u l f i l l m e n t o f t h e University o5 M a r y l a n d for the Ph.D. degree. Portions o f this paper w e r e p r e s e n t e d at the 7 6 t h A n n u a l Meeting o f the A m e r i c a n S o c i e t y for M i c r o b i o l o g y , Atlantic City, N.J., 2--7 May, 1 9 7 6 . **Present address: D e p a r t m e n t o f M i c r o b i o l o g y , Naval Medical R e s e a r c h I n s t i t u t e , N a t i o n a l Naval Medical Center, Bethesda, Md. 2 0 0 1 4 , U.S.A. * * * T o w h o m inquiries a n d r e p r i n t r e q u e s t s s h o u l d b e addressed.
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Fig. 1. E l e c t r o n m i c r o g r a p h s o f (a) c r u d e m i t o c h o n d r i a l p r e p a r a t i o n a n d (b) r e n o g r a f i n - p u r i f i e d m i t o c h o n d r i a . M i t o c h o n d r i a l p r e p a r a t i o n s in 0 . 0 5 M p o t a s s i u m p h o s p h a t e b u f f e r ( p H 6 . 8 ) c o n t a i n i n g 20% s u c r o s e a n d 1 • 10 -s M E D T A w e r e t r e a t e d w i t h 2.5% g l u t a r a i d e h y d e 0 f i x e d in 1.0% o s m i u m t e t r o x i d e , e m b e d d e d in e p o n 8 1 2 ( D u P o n t ) , s e c t i o n e d , a n d s t a i n e d w i t h u r a n y l a c e t a t e a n d lead c i t r a t e . Magnifi c a t i o n 1 7 0 0 X.
effort to elucidate further the mechanism involved in the photorepair of ultraviolet-induced petite mutational damage we elected to determine whether or n o t yeast mitochondria possess photoreactivation enzyme. In this report we present evidence which suggests that yeast mitochondria do n o t normally contain detectable photoreactivation enzyme.
377
Mitochondria from ~ cerevisiae haploid cells (strain a31-5) were isolated from lysed spheroplasts prepared in 2 M sorbitol buffer by the procedure of DueU et al. [9]. Respiratory control ratios (QO2 with ADP/QO2 without ADP) of 12.9 and 2.8 were determined for ~-ketoglutarate and succinate oxidation respectively, suggesting minimal mitochondrial damage [9,10]. However, electron micrographs of these mitochondria revealed significant contamination with nuclei, Fig. la. A further purification of the mitochondria was achieved by renografin
378 density-gradient flotation [11,12]. Mitochondria were resuspended in renografin at 1.25 d (g/cm 3) and overlayered with 6 volumes of 1.15 d renografin. Centrifugation was carried out in a Beckman Type 21 rotor for 2 h at 21 000 rev./min. The intact mitochondria were isolated from the top of the gradient. Electron micrographs, Fig. lb, reveal that this material consisted predominantly of intact mitochondria relatively free of contaminating nuclei. Respiratory control ratios of 2.35 for a-ketoglutarate and 1.3 for succinate, although lower than the values of the crude preparation, indicate that the purified mitochondria were physiologically intact. In addition, double membrane-bound structures in Fig. l b suggest that mitochondrial integrity survived the renografin treatment. Assays for photoreactivation e n z y m e were based on the photorepair of DNA containing t h y m i n e dimers as described by Cook and Worthy [13]. Purified [3H]thymine-labeled Escherichia coli DNA (1.4 × l 0 s dpm/pg) was irradiated with ultraviolet light in reduced 02 in the presence of 0.01 M acetophenone. This procedure converted approx. 25% of the labeled t h y m i n e into the t h y m i n e dimer form. After removal of the acetophenone by dialysis, this dimerized DNA in 0.15 M NaC1 was used as the substrate for photoreactiration e n z y m e in yeast cell extracts and in lysed, homogenized mitochondrial preparations. Conversion of t h y m i n e dimers to monomers by photoreactivation e n z y m e was monitored by one-dimensional paper chromatography. The crude mitochondrial preparation and renografin-purified mitochondria were osmotically lysed, homogenized, and tested for photoreactivation enzyme activity. As shown in Fig. 2, photoreactivation enzyme activity was found in the crude mitochondrial preparation but was not detected in the purified mitochondria. Possible denaturation of a potential mitochondrial photoreactivation enzyme by the renografin t r e a t m e n t was ruled unlikely since renografin exposure failed to alter the photoreactivation enzyme activity of a yeast cell extract. Specific activities (see legend to Fig. 3) of the extract before and after a 1-h exposure to renografin, followed by dialysis, were 0.173 and 0.175 respectively. To insure that a potential mitochondrial photoreactivation enzyme had access to the labeled substrate the mitochondria were solubilized with Triton X-100 [11 ], rather than lysed by osmotic and mechanical means. Mitochondria solubilized in this way failed to exhibit any photoreactivation enzyme activity. Triton X-100 only marginally altered the specific photoreactivation e n z y m e activity of a yeast extract from 0.120 to 0.109, which would seem to exclude the possible inactivation of a potential mitochondrial photoreactivation e n z y m e by the detergent. It was conceivable that a mitochondrial photoreactivation enzyme might exhibit a markedly different ionic strength (g) requirement. We, therefore, compared the photoreactivation e n z y m e activity of purified mitochondria with that of yeast extracts over a wide range of ionic strengths (p = 0.036-0.385). Fig. 3 shows the results of these experiments. The yeast extract showed optimal photoreactivation e n z y m e activity at p = 0.15--0.16 [ 13 ], while purified mitochondria failed to show photoreactivation enzyme activity over the entire range of ionic strengths tested. Our results suggest that mitochondria isolated from S. cerevisiae do not
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TIME (min) Fig. 2. P h o t o r e a c t i v a t i o n e n z y m e a c t i v i t y o f c r u d e a n d p u r i f i e d y e a s t m i t o c h o n c L r i a . Y e a s t m i t o e h o n d r i a l prepaxations were washed in 0.05 M potassium phosphate buffer (pH 6.8) eontslninS 20% sucrose and 1 • 1 0 -3 M E D T A , r e s u s p e n d e d t o 2 9 0 / ~ g p r o t e i n l m l , l y s e d in 0 . 0 6 7 M p o t a s ~ u m p h o s p h a t e b u f f e r (pH 7.0) and homogenized with a Potter-Elvejhem tissue ~dnder. This material was mixed with an equal v o l u m e of [ S H ] t h y m i n e - l a b e l e d E. coil D N A , p r e v i o u s l y i r r a d i a t e d w i t h ultraviolet light in the p r e s e n c e o f a c e t o p h e n o n e so t h a t 2 5 % o f t h e t h y m i n e w a s i n t h e d i m e r f o r m . P h o t o r e a c t i v a t i o n a t 3 0 ° C w a s c a r r i e d o u t b y i l l u m i n a t i n g t h e r e a c t i o n m i x t u r e s w i t h f o u r b l a c k l i g h t s ( G e n e r a l E l e c t r i c F 1 5 T S - B L B ) a t a disfence of 10 cm. Samples were removed from the reaction mixtu~s, precipitated with trichloroacetic a c i d , h y d r o l y z e d , c h r o m a t o g r a p h e d a n d c o u n t e d as d e s c r i b e d b y C o o k a n d W o r t h y [ 1 3 ] , T h e r e s u l t s w e r e p l o t t e d as S / S o vs. t i m e , w h e r e S 0 is t h e i n i t i a l s u b s t r a t e c o n c e n t r a t i o n ( t h y m i n e d i m • r ) a t t i m e 0, a n d S is t h e s u b s t r a t e c o n c e n t r a t i o n a t a g i v e n t i m e . o o, purified mitochondrla; • •, crude mitochondria.
possess measurable amounts of photoreactivation enzyme. The activity in the crude mitochondrial fraction is due to contaminating nuclei. Renografin purification reduces nuclear contamination with a concomitant loss of photoreactivation enzyme activity. The lack of this activity in the purified mitochondria would seem n o t to be due to renografin denaturation of a possible mitochondrial photoreactivation enzyme, to a different ionic strength requirement, nor to a lack of enzyme-substrate contact with incompletely lysed mitochondria. The fact that these mitochondria are still intact as judged by electron microscopy and physiological parameters mitigates against leakage of a potential photoreactivation enzyme from the mitochondria. A lack of
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IONIC S T R E N G T H (~1) Fig. 3. E f f e c t o f i o n i c s t r e n g t h o n p h o t o r e a c t i v a t i o n e n z y m e specific a c t i v i t y o f y e a s t e x t r a c t s a n d p u r i f i e d m i t o c h o n d r i a . P u r i f i e d m i t o c h o n d r i a a n d y e a s t e x t r a c t s w e r e d i l u t e d t o 3 0 0 ?tg p r o t e i n / m l in 0 . 0 6 7 p o t a s s i u m p h o s p h a t e b u f f e r ( p H 7 . 0 ) a n d a s s a y e d f o r p h o t o r e a c t i v a t i o n e n z y m e , as d e s c r i b e d in Fig. 2, a t t h e i n d i c a t e d ionic s t r e n g h t s (/~). The p h o s p h a t e c o n c e n t r a t i o n w a s h e l d c o n s t a n t w h i l e t h e c o n c e n t r a t i o n o f NaCI was a d j u s t e d t o a l t e r t h e ionic strength. Specific activity o f p h o t o r e a e t i v a t i o n e n z y m e is d e f i n e d as t h e s l o p e , d e t e r m i n e d b y regression analysis, o f e a c h p l o t o f S / S o v s . t i m e (e.g. Fig. 2) d i v i d e d b y t h e protein c o n c e n t r a t i o n in m g . o o, p u r i f i e d m i t o c h o n d r i a ; • o0 y e a s t extracts.
respiratory control in mitochondria which we purified by sucrose density gradient centrifugation precluded meaningful photoreactivation enzyme assays of these preparations. For a non-mitochondrial photoreactivation enzyme to reach mtDNA it would presumably have to be transported, by some mechanism, into the mitochondria. Transfer of proteins across membranes probably occurs in most cells [ 14], and transport specifically into mitochondria has been suggested by a number of authors [15--19]. Diamond et al. [20] have proposed that cytoplasmically synthesized photoreactivation enzyme in Euglena can penetrate chloroplast, mitochondrial and nuclear membranes. The recently reported evidence for dimer disappearance in mtDNA after photoreactivation [6,8], coupled with our data, leads us to conclude that yeast mitochondria do not normally contain photoreactivation enzyme; rather photorepair is accomplished by a non-mitochondrial enzyme that presumably gains access to the damaged mtDNA. This possibility might be tested by examining mitochondria from ultraviolet irradiated cells for photoreactivation enzyme activity. However, the large numbers of cells needed to obtain sufficient mitochondria, free of nuclei, for the assay, and the cumbersome irradiation procedure present technical obstacles which would be difficult to overcome. Our results also imply that the photoreactivation enzyme responsible for
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photorepair of yeast mtDNA is probably not encoded within the mitochondrion, but rather by nuclear DNA. This work was supported by grants to A.M.M. from Research Corporation and the United States Public Health Service (5 RO1 GM 18490). References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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