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Induction of petite “mutants” in an ethidium-resistant strain of Saccharomyces cerevisiae by photoaffinity labeling
91
Mutation Research,
80 ( 1 9 8 1 ) 9 1 - - 9 7 © Elsevier/North-Holland Biomedical Press
INDUCTION OF PETITE "MUTANTS" IN AN ETHIDIUM-RESISTANT STRAIN OF $accbaromyces cerevisiae BY PHOTOAFFINITY LABELING
DISTINCTION BETWEEN EARLY AND LATE STEPS *
MASAHITO FUKUNAGA
** a n d K. L E M O N E Y I E L D I N G * * *
Laboratory of Molecular Biology, University of Alabama in Birmingham, University Station, Birmingham, AL 35294 (U.S.A.) (Received 3 December 1979) ( R e v i s i o n r e c e i v e d 11 A u g u s t 1 9 8 0 ) ( A c c e p t e d 12 A u g u s t 1 9 8 0 )
Summary A strain of Saccharomyces cerevisiae (MH41-7B/011) was resistant to petite induction by ethidium bromide at 30 °, but was sensitive to induction by photolabeling with ethidium monoazide. These results suggested a defect in the mutant in metabolic activation of ethidium to account for its resistance. Synchronized cultures of both the mutant and the normal parent strains showed a substantial reduction in petite response to photolabeling in station~y phase cells which could not be accounted for by changes in cell penetration of the drug. The use of photolabeling with normal and mutant cells suggested that petite induction can be divided into early and late steps.
The mechanisms involved in drug-induced mitochondrial petite "mutations" are complex and are strongly influenced by such factors as drug processing and the metabolic and growth status of cells. For example, ethidium bromide is highly efficient in producing mitochondrial petites in yeast cells [6,7,12,15,16, 20]. This drug is activated metabolically to produce covalent adducts [1,16-18], inhibits mitochondrial DNA replication [6], stimulates mitochondrial DNA degradation [5,7]; and its effects can be prevented or reversed by glucose repression [12], visible light exposure [11], elevated temperature [19] or * Supported by NIH Grant No. CA17629 and Cancer Center Core SulSPOrt Grant CA13148. ** Address: D e p a r t m e n t of Microbiology, University of Occupational and Environmental Health Japan, P.O. Orio, Yahata-nishi-ku, Kitakyushu-shi, Fukuoka-ken 807 (Japan). * * * Correspondence addressee.
92 incubation under certain conditions with a drug excess [21] or with the noneffective analog propidium [4]. The induction process can thus be divided into early (noncovalent) binding, metabolic drug activation, covalent attachment, and the late events which include mitochondrial DNA degradation and/or other irreversible events. The precise definition of each relevant step and the relationship to other cellular events is difficult because of the continual presence of the drug and the complex kinetics involved. Covalent drug fixation can be accomplished under specific conditions through use of a photosensitive analog of ethidium which mimics the interactions and biological effects of ethidium [9]. Photolysis of this photoaffinity probe, 8-azido-3-amino-5-ethyl-6-phenyl phenanthridine, with yeast cells results in covalent attachment to cellular macromolecules, and produces petite mutants at high efficiency. Furthermore, metabolic suppression of ethidium mutagenesis by high glucose concentrations can be bypassed by this photolytic drug activation [ 10]. The drug which is photolyzed free in solution loses its p o t e n c y [9]. The present work makes use of these properties to study ethidium effects in a strain of Saccharomyces resistant to the parent drug, and to examine the effects of cellular metabolic state on the consequences of covalent drug fixation in contrast to the early steps leading to petite induction. Materials and methods A haploid strain of Saccharomyces cerevisiae MH 41-7B (a, ade2, hiss) (wildtype) and the derived nuclear m u t a n t strain MH 41-7B/011, resistant to petite induction by ethidium bromide, were kindly provided by Dr. Hiroshi Fukuhara [2]. The m u t a n t strain is resistant to ethidium at 30 ° and normally sensitive at 20 ° " Yeast cells were grown in a YPD medium containing 1% yeast extract, (DIFCO), 2% b a c t o p e p t o n e (DIFCO) and 1% dextrose as a carbon source, and 50 #g/ml of adenine (Eastman). Determination of petites, cell survival, cell density, and the binding experiments using radiolabeled ethidium azide ([14C]ethidium azide, 17.7 mCi/mmole, synthesized in this laboratory [ 8 ] ) w e r e performed as described previously [3,4]. Cultivations, incubations and drug treatment were performed in the dark, more than 500 surviving cells were tested for each sample, and all experiments were duplicated. All other experimental conditions are described in the figure legends. Ethidium bromide and propidium iodide were purchased from Calbiochem, and all other chemicals were reagent grade. Ethidium monoazide (8-azido-3amino-5-ethyl-6-phenylphenanthridinium chloride) was synthesized by David Graves of this laboratory [8]. Results
The effects of ethidium on petite induction at 30 ° in cells of the wild-type and mutant strains derived from different phases of growth are shown in Fig. 1. There was no significant induction of petites by ethidium observed with any of the cells of the m u t a n t strain over the incubation period of 5 h.
93 Early Exponential Phase
Late Exponential Phase
Starved
Stationary Phase
I00
t0
J
75
25
2
4
2
4
2
4
Incubation Time (h) Fig. 1. P e t i t e i n d u c t i o n b y e t h i d i u m b r o m i d e in n o r m a l and m u t a n t cells derived f r o m d i f f e r e n t c u l t u r e stages. Y e a s t cells w e r e c u l t i v a t e d at 3 0 ° in Y P D m e d i u m c o n t a i n i n g a d e n i n e 5 0 /ag]ml f o r 1 5 h ( l a t e e x p o n e n t i a l - p h a s e cells) and 4 8 h ( s t a t i o n a r y - p h a s e cells), h a r v e s t e d and w a s h e d in p h o s p h a t e b u f f e r ( 0 . 0 6 7 M, p H 7 . 0 ) 3 t i m e s . S t a t i o n a r y - p h a s e cells w e r e starved for 1 5 h in t h e s a m e b u f f e r a t 3 0 ° C . Starved s t a t i o n a r y cells w e r e s u s p e n d e d in Y P D m e d i u m and i n c u b a t e d for 2 . 5 h, w a s h e d in p h o s p h a t e b u f f e r and used as e a r l y e x p o n e n t i a l - p h a s e cells. Cells o f e a c h phase w e r e s u s p e n d e d in b u f f e r a t a cell d e n s i t y o f 1 0 6 / m l and i n c u b a t e d w i t h 2 5 ~M e t h i d i u m b r o m i d e at 3 0 ° C . A t e a c h interval i n d i c a t e d cells w e r e p l a t e d f o r d e t e r m i n a t i o n o f p e t i t e m u t a t i o n i n d u c t i o n [ 4 ] . N o r m a l cells, S. cerevisiae M H 4 1 - 7 B (o); m u t a n t cells, M H 4 1 - 7 B / 0 1 1 (A).
The effects of ethidium on strain MH 41-7B/011 for longer periods and at both permissive and non-permissive temperatures, are illustrated in Fig. 2. The drug was ineffective on growth or petite induction at 30 ° with only 10% petites after 24 h of incubation. At 20 °, petite induction was 100% after 24 h. The response of the mutant strain to propidium was also examined in these experiments in light of our previous observations that this analog could stimulate the ethidium effect in growing cells [4,5]. At 20 °, the combined effects of propidium and ethidium were the same as reported for wild-type cells. At 30 ° , Petite Induction by IO,u.M Ethidium ioo
8O
a_ 4 0 2O 0
r~
2 4 6 8 Incubation Time (h)
,
24
Fig. 2. P e t i t e i n d u c t i o n in m u t a n t cells b y e t h i d i u m b r o m i d e . Late e x p o n e n t i a l - p h a s e cells in Y P D m e d i u m + 5 0 ~ g / m l o f a d e n i n e at 3 0 ° C w e r e h a r v e s t e d , w a s h e d and r e s u s p e n d c d in 1 0 m l o f t h e s a m e m e d i u m w i t h or w i t h o u t drugs at a c e l l d e n s i t y o f 1 0 6 / m l . Cell s u s p e n s i o n s w e r e i n c u b a t e d at 2 0 ° C Cftlled s y m b o l s ) o r 3 0 ~ C ( o p e n s y m b o l s ) in a s h a k i n g w a t e r b a t h . A f t e r e a c h interval, s a m p l e s o f ceils w e r e c o u n t e d in a h a e m o c y t o m e t e r and p l a t e d o n Y P D solid m e d i a a f t e r suitable d i l u t i o n s . Plates w e r e i n c u b a t e d for 3 d a y s at e a c h t e m p e r a t u r e , and p e t i t e m u t a t i o n s w e r e d e t e r m i n e d as desCribed p r e v i o u s l y [ 4 ] . C o n t r o l , n o drugs ( o , o ) ; e t h i d i u m b r o m i d e 1 0 / ~ M ( A, A); p r o p i d i u m i o d i d e 1 0 0 ~ M ( ~ , v ) ; e t h i d i u m b r o m i d e 1 0 / ~ M + p r o p i d i u m i o d i d e 1 0 0 / ~ M Co. m).
94
Early Exponential Phase
Late Exponential Phase
Starved Stationary Phase
'°° I 5O 25 0
5
I©
5
I0
5
10
Ethidium Azide Concentration (/zM) Fig. 3. P e t i t e i n d u c t i o n b y e t h i d i u m a z i d e in n o r m a l a n d m u t a n t cells d e r i v e d f r o m d i f f e r e n t c u l t u r e stages. Cells w e r e p r e p a r e d as d e s c r i b e d in t h e l e g e n d f o r Fig. 1 a n d s u s p e n d e d in b u f f e r at 1 0 5 c e l l s / m l w i t h e t h i d i u m a z i d e at t h e c o n c e n t r a t i o n s s h o w n . Cell s u s p e n s i o n s w e r e i n c u b a t e d f o r 1 h in t h e d a r k a n d e x p o s e d t o l i g h t f o r 1 h as d e s c r i b e d e l s e w h e r e [ 4 ] . All o t h e r e x p e r i m e n t a l c o n d i t i o n s a n d s y m b o l s w e r e t h e s a m e as in Fig. 1.
the propidium enhancement was still apparent, suggesting that the m u t a n t cells are permeable to the drug and that the propidium-sensitive process is operative. These results could be consistent with a defect in the m u t a n t strain in ethidium activation and an enhancement by propidium of a late step in mutagenesis. To test this hypothesis, ethidium azide was used to provide photolytic drug activation and attachment in the drug-resistant mutant. These results are presented in Fig. 3 for both the parent (wild-type) strain and the mutant. In cells derived from early exponential cultures, photolytic drug treatment produced 100% petites in both the normal and mutant strains, although the mutant was slightly less responsive as shown by the dose--response curve. In late log cells, the dose--response curves were suppressed for both strains, and on cells derived from stationary phase cultures, the responses were almost completely suppressed. Cell survival was more than 60% in every case. These experiments suggested two important conclusions. First, the ethidium-resistant strain appears to be blocked principally in its metabolic activation of ethidium; and second, when the ethidium attachment is limited over a short period, even in normal cells, there is a significant decrease in responsiveness of cells which are stationary at the time of exposure. The lack of dependence of ethidium bromide effect on the culture phase (Fig. 1) may be explained by the fact that the bulk of the drug persists in the cells and can be activated and b o u n d after growth is initiated. In contrast, the ethidium azide is converted either to the b o u n d form or to an inactive form [9] by the photolytic process. The question of variations in penetration and binding by the photoaffinity probe in cells derived from early exponential and stationary cultures was examined by following the subcellular distribution of the radiolabeled c o m p o u n d after photolysis. These results, shown in Table I, showed good cell penetration since there were no gross differences in binding between parent and m u t a n t strains, nor between early exponential and stationary cells. Since binding experiments (Table I) were done at a drug concentration of 50 #M, the Expts. in Fig. 3 were extended by examining t h e effects of 50 pM ethidium with stationary phase cells. In 2 sets of Expts. using cell densities of 10S/ml and 106/ml,
95 TABLE 1 ETHIDIUM AZIDE BINDING TO YEAST SUBCELLULAR FRACTIONS E a r l y - e x p o n e n t i a l cells a n d s t a r v e d - s t a t i o n a r y cells w e r e p r e p a r e d as d e s c r i b e d in t h e l e g e n d o f Fig. 1. Cells w e r e s u s p e n d e d in 10 m l o f b u f f e r w i t h 5 0 /aM e t h i d i u m a z i d e ( 6 - [ 1 4 C ] , 17.7 m C i / m o l e ) a t a cell p o p u l a t i o n of 108 cells/ml. All s u s p e n s i o n s w e r e i n c u b a t e d in t h e d a r k at 3 0 ° C f o r 1 h a n d l i g h t - e x p o s e d f o r 1 h as d e s c r i b e d e l s e w h e r e [ 3 ] . A f t e r p h o t o l y t i c t r e a t m e n t , cells w e r e w a s h e d 4 t i m e s in b u f f e r a n d s u s p e n d e d in 0.1 M Tris, 1 0 m M E D T A , p H 8.0 a n d m i x e d w i t h glass b e a d s (0.45---0.50 r a m ) . Cells w e r e d i s r u p t e d a c c o r d i n g to t h e " h a n d - s h a k e " m e t h o d d e s c r i b e d b y L a n g et al. [ 1 3 ] , a n d t h e glass b e a d s a n d cell d e b r i s r e m o v e d b y c e n t r i f u g a t i o n f o r 10 rain a t 2 0 0 0 g. S u p e r n a t a n t was r e m o v e d a n d t h e m i t o c h o n d r i a l f r a c t i o n c o l l e c t e d b y c e n t r i f u g a t i o n ( 1 0 0 0 0 g × 1 0 rain) a n d w a s h e d . T h e 10 0 0 0 X g supern a t a n t w a s u s e d as t h e soluble f r a c t i o n . A s m a l l a l i q u o t o f e a c h f r a c t i o n was u s e d f o r p r o t e i n e s t i m a t i o n b y t h e m e t h o d of L o w r y et al. [ 1 4 ] a n d t h e a c i d - i n s o l u b l e f r a c t i o n s ( t r i c h l o r o a c e t i c a c i d ) w e r e c o l l e c t e d o n filter p a p e r ( W h a t m a n G F / c ) a n d c o u n t e d as d e s c r i b e d p r e v i o u s l y [ 3 ] . Cell p r e p a r a t i o n
Cell t y p e
G r o w t h phase
R e s u l t s of labeling w i t h [ 14C]ethidium monoazide Expt. I
I n t a c t cells (results, c p m / 1 0 7 cells)
Mitochondria fraction (results, c p m / m g p r o t e i n )
Soluble fraction (results, c p m / m g p r o t e i n )
E x p t . II
(Avg.)
Wild-type
S t a t i o n a r y (S) E x p o n e n t i a l (E)
6128 5015
7916 7237
7022 6126
Mutant
S E
7327 6452
5327 4769
6327 5611
Wild-type
S E
18 1 8 4 25 2 9 6
15 5 4 0 21 8 1 8
16 8 6 2 23 557
Mutant
S E
18 4 1 6 21 8 6 9
11 5 0 0 20 1 9 6
14 958 21 0 3 3
Wild-type
S E
4428 6271
3034 4308
3731 5290
Mutant
S E
2633 4054
2662 3613
2648 3834
petite induction was only 31% and 35%, resp. It could n o t be concluded whether a small decrease in binding to mitochondrial fractions could a c c o u n t for the loss of petite induction, b u t it did seem clear that the lack of response could n o t be explained simply by lack of cell permeability. Discussion Ethidium effects in mitochondria involve several steps which are paraphrased in Fig. 4. An initial reversible step is followed by covalent fixation either through metabolic or photolytic activation. Both the reversible and irreversible complexes are thought to involve mitochondrial D N A and/or mitochondrial membranes. The late steps then involve D N A degradation and preN O N - C O V A L E N T D R U G C O M P L E X --~ C O V A L E N T A D D U C T (REVERSIBLE) (IRREVERSIBLE)
~ ~ D I
R
~ / ~
UPTION OF mDNA ~ REPLICATIO N Fig. 4. M o d e l f o r p e t i t e i n d u c t i o n .
mDNAase ) DEGRADATION OF PRE-EXISTING mDNA
(LATE) CELL GROWTH
1
~PETITES
96 sumed disruption of DNA replication. The importance of activation and covalent attachment has been emphasized by studies with the photoaffinity probe showing its light-dependent petite-inducing potency and bypass of the glucose repression of ethidium mutagenesis [9,10]. The present experiments have shown that the ethidium-resistant mutant MH 41-7B/011 is sensitive at non-permissive temperature to the ethidium moiety when drug attachment is accomplished by photoaffinity labeling with ethidium monoazide. This finding indicates that the mutant is most likely blocked in an early step concerned with drug processing and attachment, and this block can also be bypassed by the photolytic mechanism. The mutant, therefore, provides an opportunity to study, separately, the early and late events in petite induction. The observation here that stationary phase cells are non-responsive to the covalent attachment of the ethidium moiety offers an excellent opportunity to study the late events in ethidium mutagenesis. The lack of effect on cells labeled in stationary phase suggests that the nature of the adduct formed may be different due to a difference in the DNA-membrane structural state, or that the handling of the adduct formed may be different. Additional experiments should distinguish between these possibilities. References 1 Bastos, R.N., and H.R. Mahler, Modulation of petite induction by low concentrations of ethidinm b r o m i d e , B i o c h e m . B i o p h y s . Res. C o m m u n . , 6 9 ( 1 9 7 6 ) 5 2 8 - - 5 3 7 . 2 F u k u h a r a , H., E. M o u s t a c c h i a n d M. W e s o l o w s k i , P r e f e r e n t i a l d e l e t i o n s o f a s p e c i f i c r e g i o n o f m i t o c h o n d r i a l D N A in Saccharomyces cerevisiae b y e t h i d i u m b r o m i d e a n d 3 - c a r b e t h o x y - p s o r a l e n , Direct i o n a l r e t e n t i o n s o f D N A s e q u e n c e , Mol. G e n . G e n e t . , 1 6 2 ( 1 9 7 8 ) 1 9 1 - - 2 0 1 . 3 F u k u n a g a , M., a n d K . L . Y i e l d i n g , I n t r a c e l l u l a r b i n d i n g o f e t h i d i u m s t u d i e d b y p h o t o a f f i n i t y l a b e l i n g i n vivo, B i o c h i m . B i o p h y s . A c t a , 5 8 5 ( 1 9 7 9 ) 2 9 3 - - 2 9 9 . 4 F u k u n a g a , M., a n d K . L . Y i e l d i n g , P r o p i d i u r n : I n d u c t i o n o f p e t i t e s a n d r e c o v e r y f r o m e t h i d i u m m u t a genesis in Saccharomyces cerevisiae, B i o c h e m . B i o p h y s . Res. C o m m u n . , 8 4 ( 1 9 7 8 ) 5 0 1 - - 5 0 7 . 5 F u k u n a g a , M., a n d K . L . Y i e l d i n g , C o - m u t a g e n i c e f f e c t s o f p r o p i d i u m o n p e t i t e i n d u c t i o n b y e t h i d i u m i n Saccharomyces cerevisiae, M u t a t i o n Res., 6 9 ( 1 9 8 0 ) 4 3 - - 5 0 . 6 G o l d r i n g , E . S . , L . I . G r o s s m a n , D. K m p n i c k , D . R . C r y e r a n d J . M a r m u r , T h e p e t i t e m u t a t i o n i n y e a s t , L o s s o f m i t o c h o n d r i a l d e o x y r i b o n u c l e i c a c i d d u r i n g i n d u c t i o n o f p e t i t e s w i t h e t h i d i u m b r o m i d e , J. Mol. Biol., 5 2 ( 1 9 7 0 ) 3 2 3 - - 3 3 5 . 7 G o l d r i n g , E.S., L.I. G r o s s m a n a n d J. M a r r n u r , P e t i t e m u t a t i o n s in y e a s t , II. I s o l a t i o n o f m u t a n t s c o n t a l n i n g m i t o c h o n d r i a l d e o x y r i b o n u c l e i c a c i d o f r e d u c e d size, J. B a c t e r i o l . , 1 0 7 ( 1 9 7 1 ) 3 7 7 - - 3 8 1 . 8 Graves, D . E . , L.W. Y i e l d i n g , C.L. W a t k i n s a n d K . L . 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I r o n s , R e v e r s a l o r p r o t e c t i o n b y l i g h t o f t h e e t h i d i n m b r o m i d e i n d u c e d p e t i t e m u t a t i o n in y e a s t , Mol. G e n . G e n e t . , 1 6 9 ( 1 9 7 9 ) 6 3 - - 6 6 . 1 2 H o L l e n b e r g , C.P., a n d P. B u r s t , C o n d i t i o n s t h a t p r e v e n t p - i n d u c t i o n b y e t h i d i u m b r o m i d e , B i o c h e m . B i o p h y s . l%es. C o m m u n . , 4 5 ( 1 9 7 1 ) 1 2 5 0 - - 1 2 5 4 . 13 L a n g , B., G. B u r g e r , I. D o x i a d i s , D . Y . T h o m a s , W. B a n d l o w a n d F. K a n d e w i t z , A s i m p l e m e t h o d f o r the large-scale preparation of mitochondria from microorganisms. Anal. Biochem., 77 (1977) 110-121. 1 4 L o w r y , O . H . , N.J. R o s e n b r o u g h , A . L . F a r r a n d R . J . R a n d a l l , P r o t e i n m e a s u r e m e n t w i t h t h e f o l i n p h e n o l r e a g e n t , J. Biol. C h e m . , 1 9 3 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 .
97 1 5 M a h l e r , H . R . , a n d P.S. P e r l m a n , E f f e c t s o f m u t a g e n i c t r e a t m e n t b y e t h i d i u m b r o m i d e o n c e l l u l a r a n d mitochondrial phenotype, Arch. Biochem. Biophys., 148 (1972) 115--129. 1 6 M a h l e r , H . R . , a n d P.S. P e r l m a n , M i t o c h o n d r i a l m e m b r a n e s a n d m u t a g e n e s i s b y e t h i d i u m b r o m i d e , J. Supramol. Struct., 1 (1972) 105--124. 17 M a h l e r , H . R . , a n d R . N . B a s t o s , C o u p l i n g b e t w e e n m i t o c h o n d r i a l m u t a t i o n a n d e n e r g y t r a n s d u c t i o n , P r o c . Natl. A c a d . Sct. ( U . S . A . ) , 7 1 ( 1 9 7 4 ) 2 2 4 1 - - 2 2 4 5 . 18 Mahler, H.R., and R.N. Bastos, A novel reaction of mitochondrial DNA with ethidium bromide, FEBS Lett., 39 (1974) 27--34. 19 P e r l m a n , P.S., a n d H . R . M a h l e r , A p r e s e n t a t i o n a l s t a t e i n d u c e d in y e a s t b y e t h i d i u m b r o m i d e , Bioc h e m . B i o p h y s . Res. C o m m u n . , 4 4 ( 1 9 7 1 ) 2 6 1 - - 2 6 7 . 2 0 S l o n i m s k i , P.P., G. P e r r o d i n a n d J . H . C r o f t , E t h i d i u m b r o m i d e i n d u c e d m u t a t i o n o f y e a s t m i t o c h o n d r i a , C o m p l e t e t r a n s f o r m a t i o n o f cells i n t o r e s p i r a t o r y d e f i c i e n t n o n - c h r o m o s o m a l " p e t i t e s " , Bioc h e m . B i o p h y s . Res. C o m m u n . , 3 0 ( 1 9 6 8 ) 2 3 2 - - 2 3 9 . 21 Wheelis, L., M . K . T r e m b a t h a n d R.S. C r i d d l e , P e t i t e i n d u c t i o n a n d r e c o v e r y in t h e p r e s e n c e o f h i g h levels o f e t h i d i u m b r o m i d e , B i o e h e m . B i o p h y s . Res. C o m m u n . , 6 5 ( 1 9 7 5 ) 8 3 8 - - 8 4 5 .
Mutation Research,
80 ( 1 9 8 1 ) 9 1 - - 9 7 © Elsevier/North-Holland Biomedical Press
INDUCTION OF PETITE "MUTANTS" IN AN ETHIDIUM-RESISTANT STRAIN OF $accbaromyces cerevisiae BY PHOTOAFFINITY LABELING
DISTINCTION BETWEEN EARLY AND LATE STEPS *
MASAHITO FUKUNAGA
** a n d K. L E M O N E Y I E L D I N G * * *
Laboratory of Molecular Biology, University of Alabama in Birmingham, University Station, Birmingham, AL 35294 (U.S.A.) (Received 3 December 1979) ( R e v i s i o n r e c e i v e d 11 A u g u s t 1 9 8 0 ) ( A c c e p t e d 12 A u g u s t 1 9 8 0 )
Summary A strain of Saccharomyces cerevisiae (MH41-7B/011) was resistant to petite induction by ethidium bromide at 30 °, but was sensitive to induction by photolabeling with ethidium monoazide. These results suggested a defect in the mutant in metabolic activation of ethidium to account for its resistance. Synchronized cultures of both the mutant and the normal parent strains showed a substantial reduction in petite response to photolabeling in station~y phase cells which could not be accounted for by changes in cell penetration of the drug. The use of photolabeling with normal and mutant cells suggested that petite induction can be divided into early and late steps.
The mechanisms involved in drug-induced mitochondrial petite "mutations" are complex and are strongly influenced by such factors as drug processing and the metabolic and growth status of cells. For example, ethidium bromide is highly efficient in producing mitochondrial petites in yeast cells [6,7,12,15,16, 20]. This drug is activated metabolically to produce covalent adducts [1,16-18], inhibits mitochondrial DNA replication [6], stimulates mitochondrial DNA degradation [5,7]; and its effects can be prevented or reversed by glucose repression [12], visible light exposure [11], elevated temperature [19] or * Supported by NIH Grant No. CA17629 and Cancer Center Core SulSPOrt Grant CA13148. ** Address: D e p a r t m e n t of Microbiology, University of Occupational and Environmental Health Japan, P.O. Orio, Yahata-nishi-ku, Kitakyushu-shi, Fukuoka-ken 807 (Japan). * * * Correspondence addressee.
92 incubation under certain conditions with a drug excess [21] or with the noneffective analog propidium [4]. The induction process can thus be divided into early (noncovalent) binding, metabolic drug activation, covalent attachment, and the late events which include mitochondrial DNA degradation and/or other irreversible events. The precise definition of each relevant step and the relationship to other cellular events is difficult because of the continual presence of the drug and the complex kinetics involved. Covalent drug fixation can be accomplished under specific conditions through use of a photosensitive analog of ethidium which mimics the interactions and biological effects of ethidium [9]. Photolysis of this photoaffinity probe, 8-azido-3-amino-5-ethyl-6-phenyl phenanthridine, with yeast cells results in covalent attachment to cellular macromolecules, and produces petite mutants at high efficiency. Furthermore, metabolic suppression of ethidium mutagenesis by high glucose concentrations can be bypassed by this photolytic drug activation [ 10]. The drug which is photolyzed free in solution loses its p o t e n c y [9]. The present work makes use of these properties to study ethidium effects in a strain of Saccharomyces resistant to the parent drug, and to examine the effects of cellular metabolic state on the consequences of covalent drug fixation in contrast to the early steps leading to petite induction. Materials and methods A haploid strain of Saccharomyces cerevisiae MH 41-7B (a, ade2, hiss) (wildtype) and the derived nuclear m u t a n t strain MH 41-7B/011, resistant to petite induction by ethidium bromide, were kindly provided by Dr. Hiroshi Fukuhara [2]. The m u t a n t strain is resistant to ethidium at 30 ° and normally sensitive at 20 ° " Yeast cells were grown in a YPD medium containing 1% yeast extract, (DIFCO), 2% b a c t o p e p t o n e (DIFCO) and 1% dextrose as a carbon source, and 50 #g/ml of adenine (Eastman). Determination of petites, cell survival, cell density, and the binding experiments using radiolabeled ethidium azide ([14C]ethidium azide, 17.7 mCi/mmole, synthesized in this laboratory [ 8 ] ) w e r e performed as described previously [3,4]. Cultivations, incubations and drug treatment were performed in the dark, more than 500 surviving cells were tested for each sample, and all experiments were duplicated. All other experimental conditions are described in the figure legends. Ethidium bromide and propidium iodide were purchased from Calbiochem, and all other chemicals were reagent grade. Ethidium monoazide (8-azido-3amino-5-ethyl-6-phenylphenanthridinium chloride) was synthesized by David Graves of this laboratory [8]. Results
The effects of ethidium on petite induction at 30 ° in cells of the wild-type and mutant strains derived from different phases of growth are shown in Fig. 1. There was no significant induction of petites by ethidium observed with any of the cells of the m u t a n t strain over the incubation period of 5 h.
93 Early Exponential Phase
Late Exponential Phase
Starved
Stationary Phase
I00
t0
J
75
25
2
4
2
4
2
4
Incubation Time (h) Fig. 1. P e t i t e i n d u c t i o n b y e t h i d i u m b r o m i d e in n o r m a l and m u t a n t cells derived f r o m d i f f e r e n t c u l t u r e stages. Y e a s t cells w e r e c u l t i v a t e d at 3 0 ° in Y P D m e d i u m c o n t a i n i n g a d e n i n e 5 0 /ag]ml f o r 1 5 h ( l a t e e x p o n e n t i a l - p h a s e cells) and 4 8 h ( s t a t i o n a r y - p h a s e cells), h a r v e s t e d and w a s h e d in p h o s p h a t e b u f f e r ( 0 . 0 6 7 M, p H 7 . 0 ) 3 t i m e s . S t a t i o n a r y - p h a s e cells w e r e starved for 1 5 h in t h e s a m e b u f f e r a t 3 0 ° C . Starved s t a t i o n a r y cells w e r e s u s p e n d e d in Y P D m e d i u m and i n c u b a t e d for 2 . 5 h, w a s h e d in p h o s p h a t e b u f f e r and used as e a r l y e x p o n e n t i a l - p h a s e cells. Cells o f e a c h phase w e r e s u s p e n d e d in b u f f e r a t a cell d e n s i t y o f 1 0 6 / m l and i n c u b a t e d w i t h 2 5 ~M e t h i d i u m b r o m i d e at 3 0 ° C . A t e a c h interval i n d i c a t e d cells w e r e p l a t e d f o r d e t e r m i n a t i o n o f p e t i t e m u t a t i o n i n d u c t i o n [ 4 ] . N o r m a l cells, S. cerevisiae M H 4 1 - 7 B (o); m u t a n t cells, M H 4 1 - 7 B / 0 1 1 (A).
The effects of ethidium on strain MH 41-7B/011 for longer periods and at both permissive and non-permissive temperatures, are illustrated in Fig. 2. The drug was ineffective on growth or petite induction at 30 ° with only 10% petites after 24 h of incubation. At 20 °, petite induction was 100% after 24 h. The response of the mutant strain to propidium was also examined in these experiments in light of our previous observations that this analog could stimulate the ethidium effect in growing cells [4,5]. At 20 °, the combined effects of propidium and ethidium were the same as reported for wild-type cells. At 30 ° , Petite Induction by IO,u.M Ethidium ioo
8O
a_ 4 0 2O 0
r~
2 4 6 8 Incubation Time (h)
,
24
Fig. 2. P e t i t e i n d u c t i o n in m u t a n t cells b y e t h i d i u m b r o m i d e . Late e x p o n e n t i a l - p h a s e cells in Y P D m e d i u m + 5 0 ~ g / m l o f a d e n i n e at 3 0 ° C w e r e h a r v e s t e d , w a s h e d and r e s u s p e n d c d in 1 0 m l o f t h e s a m e m e d i u m w i t h or w i t h o u t drugs at a c e l l d e n s i t y o f 1 0 6 / m l . Cell s u s p e n s i o n s w e r e i n c u b a t e d at 2 0 ° C Cftlled s y m b o l s ) o r 3 0 ~ C ( o p e n s y m b o l s ) in a s h a k i n g w a t e r b a t h . A f t e r e a c h interval, s a m p l e s o f ceils w e r e c o u n t e d in a h a e m o c y t o m e t e r and p l a t e d o n Y P D solid m e d i a a f t e r suitable d i l u t i o n s . Plates w e r e i n c u b a t e d for 3 d a y s at e a c h t e m p e r a t u r e , and p e t i t e m u t a t i o n s w e r e d e t e r m i n e d as desCribed p r e v i o u s l y [ 4 ] . C o n t r o l , n o drugs ( o , o ) ; e t h i d i u m b r o m i d e 1 0 / ~ M ( A, A); p r o p i d i u m i o d i d e 1 0 0 ~ M ( ~ , v ) ; e t h i d i u m b r o m i d e 1 0 / ~ M + p r o p i d i u m i o d i d e 1 0 0 / ~ M Co. m).
94
Early Exponential Phase
Late Exponential Phase
Starved Stationary Phase
'°° I 5O 25 0
5
I©
5
I0
5
10
Ethidium Azide Concentration (/zM) Fig. 3. P e t i t e i n d u c t i o n b y e t h i d i u m a z i d e in n o r m a l a n d m u t a n t cells d e r i v e d f r o m d i f f e r e n t c u l t u r e stages. Cells w e r e p r e p a r e d as d e s c r i b e d in t h e l e g e n d f o r Fig. 1 a n d s u s p e n d e d in b u f f e r at 1 0 5 c e l l s / m l w i t h e t h i d i u m a z i d e at t h e c o n c e n t r a t i o n s s h o w n . Cell s u s p e n s i o n s w e r e i n c u b a t e d f o r 1 h in t h e d a r k a n d e x p o s e d t o l i g h t f o r 1 h as d e s c r i b e d e l s e w h e r e [ 4 ] . All o t h e r e x p e r i m e n t a l c o n d i t i o n s a n d s y m b o l s w e r e t h e s a m e as in Fig. 1.
the propidium enhancement was still apparent, suggesting that the m u t a n t cells are permeable to the drug and that the propidium-sensitive process is operative. These results could be consistent with a defect in the m u t a n t strain in ethidium activation and an enhancement by propidium of a late step in mutagenesis. To test this hypothesis, ethidium azide was used to provide photolytic drug activation and attachment in the drug-resistant mutant. These results are presented in Fig. 3 for both the parent (wild-type) strain and the mutant. In cells derived from early exponential cultures, photolytic drug treatment produced 100% petites in both the normal and mutant strains, although the mutant was slightly less responsive as shown by the dose--response curve. In late log cells, the dose--response curves were suppressed for both strains, and on cells derived from stationary phase cultures, the responses were almost completely suppressed. Cell survival was more than 60% in every case. These experiments suggested two important conclusions. First, the ethidium-resistant strain appears to be blocked principally in its metabolic activation of ethidium; and second, when the ethidium attachment is limited over a short period, even in normal cells, there is a significant decrease in responsiveness of cells which are stationary at the time of exposure. The lack of dependence of ethidium bromide effect on the culture phase (Fig. 1) may be explained by the fact that the bulk of the drug persists in the cells and can be activated and b o u n d after growth is initiated. In contrast, the ethidium azide is converted either to the b o u n d form or to an inactive form [9] by the photolytic process. The question of variations in penetration and binding by the photoaffinity probe in cells derived from early exponential and stationary cultures was examined by following the subcellular distribution of the radiolabeled c o m p o u n d after photolysis. These results, shown in Table I, showed good cell penetration since there were no gross differences in binding between parent and m u t a n t strains, nor between early exponential and stationary cells. Since binding experiments (Table I) were done at a drug concentration of 50 #M, the Expts. in Fig. 3 were extended by examining t h e effects of 50 pM ethidium with stationary phase cells. In 2 sets of Expts. using cell densities of 10S/ml and 106/ml,
95 TABLE 1 ETHIDIUM AZIDE BINDING TO YEAST SUBCELLULAR FRACTIONS E a r l y - e x p o n e n t i a l cells a n d s t a r v e d - s t a t i o n a r y cells w e r e p r e p a r e d as d e s c r i b e d in t h e l e g e n d o f Fig. 1. Cells w e r e s u s p e n d e d in 10 m l o f b u f f e r w i t h 5 0 /aM e t h i d i u m a z i d e ( 6 - [ 1 4 C ] , 17.7 m C i / m o l e ) a t a cell p o p u l a t i o n of 108 cells/ml. All s u s p e n s i o n s w e r e i n c u b a t e d in t h e d a r k at 3 0 ° C f o r 1 h a n d l i g h t - e x p o s e d f o r 1 h as d e s c r i b e d e l s e w h e r e [ 3 ] . A f t e r p h o t o l y t i c t r e a t m e n t , cells w e r e w a s h e d 4 t i m e s in b u f f e r a n d s u s p e n d e d in 0.1 M Tris, 1 0 m M E D T A , p H 8.0 a n d m i x e d w i t h glass b e a d s (0.45---0.50 r a m ) . Cells w e r e d i s r u p t e d a c c o r d i n g to t h e " h a n d - s h a k e " m e t h o d d e s c r i b e d b y L a n g et al. [ 1 3 ] , a n d t h e glass b e a d s a n d cell d e b r i s r e m o v e d b y c e n t r i f u g a t i o n f o r 10 rain a t 2 0 0 0 g. S u p e r n a t a n t was r e m o v e d a n d t h e m i t o c h o n d r i a l f r a c t i o n c o l l e c t e d b y c e n t r i f u g a t i o n ( 1 0 0 0 0 g × 1 0 rain) a n d w a s h e d . T h e 10 0 0 0 X g supern a t a n t w a s u s e d as t h e soluble f r a c t i o n . A s m a l l a l i q u o t o f e a c h f r a c t i o n was u s e d f o r p r o t e i n e s t i m a t i o n b y t h e m e t h o d of L o w r y et al. [ 1 4 ] a n d t h e a c i d - i n s o l u b l e f r a c t i o n s ( t r i c h l o r o a c e t i c a c i d ) w e r e c o l l e c t e d o n filter p a p e r ( W h a t m a n G F / c ) a n d c o u n t e d as d e s c r i b e d p r e v i o u s l y [ 3 ] . Cell p r e p a r a t i o n
Cell t y p e
G r o w t h phase
R e s u l t s of labeling w i t h [ 14C]ethidium monoazide Expt. I
I n t a c t cells (results, c p m / 1 0 7 cells)
Mitochondria fraction (results, c p m / m g p r o t e i n )
Soluble fraction (results, c p m / m g p r o t e i n )
E x p t . II
(Avg.)
Wild-type
S t a t i o n a r y (S) E x p o n e n t i a l (E)
6128 5015
7916 7237
7022 6126
Mutant
S E
7327 6452
5327 4769
6327 5611
Wild-type
S E
18 1 8 4 25 2 9 6
15 5 4 0 21 8 1 8
16 8 6 2 23 557
Mutant
S E
18 4 1 6 21 8 6 9
11 5 0 0 20 1 9 6
14 958 21 0 3 3
Wild-type
S E
4428 6271
3034 4308
3731 5290
Mutant
S E
2633 4054
2662 3613
2648 3834
petite induction was only 31% and 35%, resp. It could n o t be concluded whether a small decrease in binding to mitochondrial fractions could a c c o u n t for the loss of petite induction, b u t it did seem clear that the lack of response could n o t be explained simply by lack of cell permeability. Discussion Ethidium effects in mitochondria involve several steps which are paraphrased in Fig. 4. An initial reversible step is followed by covalent fixation either through metabolic or photolytic activation. Both the reversible and irreversible complexes are thought to involve mitochondrial D N A and/or mitochondrial membranes. The late steps then involve D N A degradation and preN O N - C O V A L E N T D R U G C O M P L E X --~ C O V A L E N T A D D U C T (REVERSIBLE) (IRREVERSIBLE)
~ ~ D I
R
~ / ~
UPTION OF mDNA ~ REPLICATIO N Fig. 4. M o d e l f o r p e t i t e i n d u c t i o n .
mDNAase ) DEGRADATION OF PRE-EXISTING mDNA
(LATE) CELL GROWTH
1
~PETITES
96 sumed disruption of DNA replication. The importance of activation and covalent attachment has been emphasized by studies with the photoaffinity probe showing its light-dependent petite-inducing potency and bypass of the glucose repression of ethidium mutagenesis [9,10]. The present experiments have shown that the ethidium-resistant mutant MH 41-7B/011 is sensitive at non-permissive temperature to the ethidium moiety when drug attachment is accomplished by photoaffinity labeling with ethidium monoazide. This finding indicates that the mutant is most likely blocked in an early step concerned with drug processing and attachment, and this block can also be bypassed by the photolytic mechanism. The mutant, therefore, provides an opportunity to study, separately, the early and late events in petite induction. The observation here that stationary phase cells are non-responsive to the covalent attachment of the ethidium moiety offers an excellent opportunity to study the late events in ethidium mutagenesis. The lack of effect on cells labeled in stationary phase suggests that the nature of the adduct formed may be different due to a difference in the DNA-membrane structural state, or that the handling of the adduct formed may be different. Additional experiments should distinguish between these possibilities. References 1 Bastos, R.N., and H.R. Mahler, Modulation of petite induction by low concentrations of ethidinm b r o m i d e , B i o c h e m . B i o p h y s . Res. C o m m u n . , 6 9 ( 1 9 7 6 ) 5 2 8 - - 5 3 7 . 2 F u k u h a r a , H., E. M o u s t a c c h i a n d M. W e s o l o w s k i , P r e f e r e n t i a l d e l e t i o n s o f a s p e c i f i c r e g i o n o f m i t o c h o n d r i a l D N A in Saccharomyces cerevisiae b y e t h i d i u m b r o m i d e a n d 3 - c a r b e t h o x y - p s o r a l e n , Direct i o n a l r e t e n t i o n s o f D N A s e q u e n c e , Mol. G e n . G e n e t . , 1 6 2 ( 1 9 7 8 ) 1 9 1 - - 2 0 1 . 3 F u k u n a g a , M., a n d K . L . Y i e l d i n g , I n t r a c e l l u l a r b i n d i n g o f e t h i d i u m s t u d i e d b y p h o t o a f f i n i t y l a b e l i n g i n vivo, B i o c h i m . B i o p h y s . A c t a , 5 8 5 ( 1 9 7 9 ) 2 9 3 - - 2 9 9 . 4 F u k u n a g a , M., a n d K . L . Y i e l d i n g , P r o p i d i u r n : I n d u c t i o n o f p e t i t e s a n d r e c o v e r y f r o m e t h i d i u m m u t a genesis in Saccharomyces cerevisiae, B i o c h e m . B i o p h y s . Res. C o m m u n . , 8 4 ( 1 9 7 8 ) 5 0 1 - - 5 0 7 . 5 F u k u n a g a , M., a n d K . L . Y i e l d i n g , C o - m u t a g e n i c e f f e c t s o f p r o p i d i u m o n p e t i t e i n d u c t i o n b y e t h i d i u m i n Saccharomyces cerevisiae, M u t a t i o n Res., 6 9 ( 1 9 8 0 ) 4 3 - - 5 0 . 6 G o l d r i n g , E . S . , L . I . G r o s s m a n , D. K m p n i c k , D . R . C r y e r a n d J . M a r m u r , T h e p e t i t e m u t a t i o n i n y e a s t , L o s s o f m i t o c h o n d r i a l d e o x y r i b o n u c l e i c a c i d d u r i n g i n d u c t i o n o f p e t i t e s w i t h e t h i d i u m b r o m i d e , J. Mol. Biol., 5 2 ( 1 9 7 0 ) 3 2 3 - - 3 3 5 . 7 G o l d r i n g , E.S., L.I. G r o s s m a n a n d J. M a r r n u r , P e t i t e m u t a t i o n s in y e a s t , II. I s o l a t i o n o f m u t a n t s c o n t a l n i n g m i t o c h o n d r i a l d e o x y r i b o n u c l e i c a c i d o f r e d u c e d size, J. B a c t e r i o l . , 1 0 7 ( 1 9 7 1 ) 3 7 7 - - 3 8 1 . 8 Graves, D . E . , L.W. Y i e l d i n g , C.L. W a t k i n s a n d K . L . Y i e l d i n g , S y n t h e s i s , s e p a r a t i o n a n d c h a r a c t e r i z a tion of the mono- and di-azide analogs of ethidium bromide, Biochim. Biophys. Acta, 479 (1977) 98-104. 9 Hixon, S.C., W.E. White Jr. and K.L. Yielding, Selective covalent binding of an ethidium analog to m i t o c h o n d r i a l D N A w i t h p r o d u c t i o n o f p e t i t e m u t a n t s in y e a s t b y p h o t o a f f i n i t y l a b e l i n g , J. MoI. Biol., 9 2 ( 1 9 7 5 ) 3 1 9 - - 3 2 9 . 1 0 H i x o n , S.C., W.E. W h i t e J r . a n d K . L . Y i e l d i n g , B y p a s s b y p h o t o a f f i n i t y l a b e l i n g o f b l o c k e d m e t a b o l i c a c t i v a t i o n o f e t h i d i u m : c o n f i r m a t i o n o f t h e r o l e f o r c o v a l e n t e t h i d i u m a t t a c h m e n t in m i t o c h o n d r i a l m u t a g e n e s i s , B i o c h e m . B i o p h y s . Res. C o m m u n . , 6 6 ( 1 9 7 5 ) 3 1 - - 3 5 . 11 H i x o n , S . C . , A . D . B u r n h a m a n d I%.L. I r o n s , R e v e r s a l o r p r o t e c t i o n b y l i g h t o f t h e e t h i d i n m b r o m i d e i n d u c e d p e t i t e m u t a t i o n in y e a s t , Mol. G e n . G e n e t . , 1 6 9 ( 1 9 7 9 ) 6 3 - - 6 6 . 1 2 H o L l e n b e r g , C.P., a n d P. B u r s t , C o n d i t i o n s t h a t p r e v e n t p - i n d u c t i o n b y e t h i d i u m b r o m i d e , B i o c h e m . B i o p h y s . l%es. C o m m u n . , 4 5 ( 1 9 7 1 ) 1 2 5 0 - - 1 2 5 4 . 13 L a n g , B., G. B u r g e r , I. D o x i a d i s , D . Y . T h o m a s , W. B a n d l o w a n d F. K a n d e w i t z , A s i m p l e m e t h o d f o r the large-scale preparation of mitochondria from microorganisms. Anal. Biochem., 77 (1977) 110-121. 1 4 L o w r y , O . H . , N.J. R o s e n b r o u g h , A . L . F a r r a n d R . J . R a n d a l l , P r o t e i n m e a s u r e m e n t w i t h t h e f o l i n p h e n o l r e a g e n t , J. Biol. C h e m . , 1 9 3 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 .
97 1 5 M a h l e r , H . R . , a n d P.S. P e r l m a n , E f f e c t s o f m u t a g e n i c t r e a t m e n t b y e t h i d i u m b r o m i d e o n c e l l u l a r a n d mitochondrial phenotype, Arch. Biochem. Biophys., 148 (1972) 115--129. 1 6 M a h l e r , H . R . , a n d P.S. P e r l m a n , M i t o c h o n d r i a l m e m b r a n e s a n d m u t a g e n e s i s b y e t h i d i u m b r o m i d e , J. Supramol. Struct., 1 (1972) 105--124. 17 M a h l e r , H . R . , a n d R . N . B a s t o s , C o u p l i n g b e t w e e n m i t o c h o n d r i a l m u t a t i o n a n d e n e r g y t r a n s d u c t i o n , P r o c . Natl. A c a d . Sct. ( U . S . A . ) , 7 1 ( 1 9 7 4 ) 2 2 4 1 - - 2 2 4 5 . 18 Mahler, H.R., and R.N. Bastos, A novel reaction of mitochondrial DNA with ethidium bromide, FEBS Lett., 39 (1974) 27--34. 19 P e r l m a n , P.S., a n d H . R . M a h l e r , A p r e s e n t a t i o n a l s t a t e i n d u c e d in y e a s t b y e t h i d i u m b r o m i d e , Bioc h e m . B i o p h y s . Res. C o m m u n . , 4 4 ( 1 9 7 1 ) 2 6 1 - - 2 6 7 . 2 0 S l o n i m s k i , P.P., G. P e r r o d i n a n d J . H . C r o f t , E t h i d i u m b r o m i d e i n d u c e d m u t a t i o n o f y e a s t m i t o c h o n d r i a , C o m p l e t e t r a n s f o r m a t i o n o f cells i n t o r e s p i r a t o r y d e f i c i e n t n o n - c h r o m o s o m a l " p e t i t e s " , Bioc h e m . B i o p h y s . Res. C o m m u n . , 3 0 ( 1 9 6 8 ) 2 3 2 - - 2 3 9 . 21 Wheelis, L., M . K . T r e m b a t h a n d R.S. C r i d d l e , P e t i t e i n d u c t i o n a n d r e c o v e r y in t h e p r e s e n c e o f h i g h levels o f e t h i d i u m b r o m i d e , B i o e h e m . B i o p h y s . Res. C o m m u n . , 6 5 ( 1 9 7 5 ) 8 3 8 - - 8 4 5 .