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Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität München Munich, September 11-13,1975 Editors W. Bandlow · R.J. Schweyen · D.Y. Thomas K. Wolf · F. Kaudewitz W DE G Walter de Gruyter · Berlin · New York 1976

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Page 1: Genetics, Biogenesis and Bioenergetics of Mitochondria · Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität

Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität München Munich, September 11-13,1975

Editors W. Bandlow · R.J. Schweyen · D.Y. Thomas K. Wolf · F. Kaudewitz

W DE G

Walter de Gruyter · Berlin · New York 1976

Page 2: Genetics, Biogenesis and Bioenergetics of Mitochondria · Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität

Contents

Contributors XI

Session 1. Genetics and Cytology of Mitochondria and Cytoplastic Factors Chairman: P. P. Slonimski

Transmission, Recombination and Segregation of Mitochondrial Genes in Saccharomyces cerevisiae B. Dujon 1

On the Formation of rho~-Petites in Yeast: V. Mapping by Recombina­tion and rho'-Deletion Analysis of Mitochondrial Genes in Saccharomyces cerevisiae R. J. Schweyen, B. Backhaus, S. Mathews, F . Kaudewitz 7

Extrachromosomal Inheritance of Drug Resistance and Respiratory Deficiency in the Fission Yeast Schizosaccharomyces pombe K. Wolf, G. Seitz, B. Lang, G. Burger, F. Kaudewitz 23

Temperature-sensitive Nuclear ,,Petite" Mutants of Saccharomyces cerevisiae G. Burkl, W. Demmer, H. Holzner, E . Schweizer 39

Recombination of Yeast Mitochondrial DNA Segments Conferring Resistance either to Paromomycin or to Chloramphenicol G. Michaelis, Ε. Pratje, Β. Dujon, L. Weill 49

Mapping of Transfer and Ribosomal RNA Genes of Yeast Mitochondria H. Fukuhara, G. Faye, M. Bolotin-Fukuhara, H. J. Hsu, N. Martin, M. Rabinowitz 57

Molecular Genetics of Yeast Mitochondria G. Bernardi 69

Genes on Saccharomyces DNA P. Borst, J. P. M. Sanders, C. Heyting 85

The Effect of Glucose on the Number of Mitochondrial Genomes Participating in Mitochondrial Crosses E . Böker, F. Kaudewitz, Κ. V. Richmond, R. J. Schweyen, D.Y.Thomas 99

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VIII

Packaging and Recombination of Mitochondrial DNA in Vegetatively Growing Yeast Cells D. H.Williamson 117

Mitochondrial Involvement in the Control of Cell Function in Saccharomyces cerevisiae J. H. Evans, V. Egilsson, D. Wilkie 125

Ultrastructural Characterization of Mitochondria from a Yeast Mutant Sensitive to „Petite" Induction (uvsp 72) B. J. Stevens, E . Moustacchi 137

The Killer Character in Yeast: Preliminary Studies of Virus-like Particle Replication E . A. Bevan, A. J. Herring 153

Session 2. Biogenesis of Mitochondria Chairman: P. Borst The Biogenesis of Mitochondria - a Review G. Schatz 163

On the Formation of rho"-Petites in Yeast: VI. Expression of a Mitochondrial Conditional Mutation Controlling Petite Formation in Saccharomyces cerevisiae W. Bandlow 179

Disaggregation of Mitochondrial Translation Products to Small Polypeptides by Performic Acid Oxidation D. E . Leister, P. J . Rogers, H. Küntzel 203

The Synthesis of Yeast Mitochondrial Proteins in Cell-free Systems A. H. Scragg, M. J . Eggitt, D. Y. Thomas 215

Translation of Mitochondrial Messenger RNA's in vitro as an Approach to the Identification of Gene Products of Yeast Mitochondrial DNA A. F . M. Moorman, L . A. Grivell, M. B. Katan, G. J . B. van Ommen, C. M. Meiland 241

Translation Products in Isolated Mitochondria from Neurospora crassa: Partial Assembly of Cytochrome Oxidase Components A. v. Rücker, W. Neupert, S. Werner 259

The Use of Isolated Mitochondria in the Study of Mitochondrial Biogenesis G. S. P. Groot, N. van Harten-Loosbroek 269

Comparative Immunological and Electrophoretic Studies of the Cytochrome bcx Complex of Different Organisms Μ. B. Katan 279

Page 4: Genetics, Biogenesis and Bioenergetics of Mitochondria · Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität

I X

Subunit Structure and Biogenesis of Cytochrome b from Neurospora crassa H. Weiss, B. Ziganke, H. J . Kolb 289

Compared Properties of the Oligomycin-sensitive ATPase in Fungi with short and long Mitochondrial DNA A. Goffeau, Y . Landry 303

Session 3. Components and Mechanisms of Electron Transfer and Oxidative Phosphorylation Chairmen: M. Klingenberg and D. F . Wilson

Composition and Function of Complex III from Beef Heart Mitochondria P. Gellerfors, M. Lunden, B. D. Nelson 309

Factors Controlling Electron Flow in Liposomes Containing Complex III from Beef Heart B. D. Nelson, J . Mandel-Hartvig, F . Guerrieri, P. Gellerfors 315

On the Role of Conformational Changes in Cytochrome c Oxidase in the Mechanism and Control of Oxidative Phosphorylation Μ. K. F . Wikström 325

The Significance of the Radicals of Ubiquinone in Electron Transport as Judged from their Chemical Properties A. Kröger 343

The Inhibition of Electron Transfer from Cytochrome b to Ubiquinone by Antimycin G. v. Jagow 353

Effects of Antimycin on the ^-Cytochromes in Yeast. Analysis of Antimycin-induced Effects on Cytochrome b Reduction in the Wild Type of the Fission Yeast Schizosaccharomyces pombe R. W. Manhard, W. Bandlow 365

Characterization of Respiratory-deficient and Antimycin-resistant Mutants of Schizosaccharomyces pombe with Extrachromosomal Inheritance B. Lang, G. Burger, K. Wolf, W. Bandlow, F . Kaudewitz 379

Models for Antimycin Binding in Wild Type of Candida utilis and Antimycin-resistant Mutants C. J . P. Grimmelikhuijzen, E . C. Slater 389

Page 5: Genetics, Biogenesis and Bioenergetics of Mitochondria · Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität

χ

The Inhibitory Effect of HQNO Compared With Extrareduction and Binding in the Wild Type and in an Antimycin-resistant Mutant, ANT8, in Schizosaccharomyces pombe G. Burger, B. Lang, K. Wolf, W. Bandlow, F . Kaudewitz 399

The Binding of 2-n-Heptyl-4-hydroxyquinoline-N-oxide to Submito­chondrial Particles and Inhibition of Electron Transport J. A. Berden, G. van Ark 409

Page 6: Genetics, Biogenesis and Bioenergetics of Mitochondria · Genetics, Biogenesis and Bioenergetics of Mitochondria Proceedings of a Symposium held at the Genetisches Institut der Universität

Translation Products in Isolated Mitochondria from Neurospora Crassa: Partial Assembly of Cytochrome Oxidase Components

A. v. Rücker, W. Neupert, S. Werner

I n t r o d u c t i o n

The f o r m a t i o n of a m i t o c h o n d r i a l membrane w h i c h i s f u l l y competent i n e l e c t r o n t r a n s f e r and o x i d a t i v e p h o s p h o r y l a t i o n r e q u i r e s t h e c o o p e r a t i o n o f c y t o p l a s m i c and m i t o c h o n d r i a l p r o t e i n s y n t h e s i s . P a r t i c u l a r l y , t h i s h o l d s f o r t h e membrane c o n s t i t u e n t cytochrome o x i d a s e . T h i s enzyme i s composed of s u b u n i t s w h i c h c a n be d i v i d e d i n t o two groups : one group i s s y n t h e s i z e d on m i t o c h o n d r i a l r i b o s o m e s ( s u b u n i t s 1, 2 and 3) and t h e second group on c y t o p l a s m i c r i b o s o m e s ( s u b u n i t s 4, 5, 6 and 7 ) ( 1 , 2) . T h i s f i n d i n g i m p l i e s t h a t many complex p r o c e s s e s must be i n v o l v e d i n t h e f o r m a t i o n o f t h e enzyme. A c c o r d i n g l y , we a r e f a c e d w i t h a l a r g e number of open q u e s t i o n s , c o n c e r n i n g e. g. t h e r e g u l a t i o n of t h e s y n t h e s i s of t h e two groups of s u b u n i t s , t h e t r a n s f e r o f t h e s u b u n i t p r e c u r s o r s t o t h e s i t e s o f assembly, t h e sequence o f t h e a s s e m b l y r e a c t i o n , e t c .

Two t y p e s o f app r o a c h e s t o answer t h e s e q u e s t i o n s appear t o be p r o m i s i n g : a) i n v i v o s t u d i e s , employing r a d i o a c t i v e p u l s e - c h a s e l a b e l i n g t e c h n i q u e s t o g e t h e r w i t h s p e c i f i c i n ­h i b i t o r s o f m i t o c h o n d r i a l and c y t o p l a s m i c t r a n s l a t i o n ( 3 ) ;

i n v i t r o s t u d i e s w i t h c e l l f r a c t i o n s and r e c o n s t i t u t e d s y s t e m s .

We have d e s c r i b e d a s y s t e m i n w h i c h i s o l a t e d m i t o c h o n d r i a a r e a b l e t o form s u b u n i t s 1, 2 and 3 of cytochrome o x i d a s e

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( 4 ) . Here we p r e s e n t e x p e r i m e n t s w h i c h show t h a t t h e c y t o ­p l a s m i c f r a c t i o n o f N e u r o s p o r a c o n t a i n s components w h i c h have a s t i m u l a t o r y e f f e c t on p o l y p e p t i d e s y n t h e s i s i n i s o ­l a t e d m i t o c h o n d r i a and on t h e f o r m a t i o n of s u b u n i t s of c y t o ­chrome o x i d a s e . F u r t h e r m o r e , t h e c y t o p l a s m i c f r a c t i o n i n h i b i t s p r o t e o l y s i s o f i n v i t r o formed p o l y p e p t i d e s . F i n a l l y , i t i s shown t h a t p a r t i a l assembly o f s u b u n i t p r e ­c u r s o r s of cytochrome o x i d a s e o c c u r s i n su c h a r e c o n s t i t u t e d s ystem.

R e s u l t s

1. Amino a c i d i n c o r p o r a t i o n i n t o t h e p r o t e i n of i s o l a t e d m i t o c h o n d r i a

F i g . 1 shows t h e r a t e o f i n c o r p o r a t i o n of l e u c i n e i n t o t h e p r o t e i n o f i s o l a t e d m i t o c h o n d r i a . I n c o r p o r a t i o n i n an i n c u ­b a t i o n medium ( s e e r e f . 4) c o n t a i n i n g 3 - H - l e u c i n e , p r o c e e d s f o r about 30 min. When a c h a s e o f u n l a b e l e d l e u c i n e i s g i v e n a f t e r 30 min i t can be o b s e r v e d t h a t l e u c i n e w h i c h was a l ­r e a d y i n c o r p o r a t e d i s l o s t from T C A - p r e c i p i t a b l e p r o t e i n . T h i s i n d i c a t e s a breakdown o f newly formed c h a i n s . T hus, i n i s o l a t e d m i t o c h o n d r i a f o r m a t i o n and breakdown of p o l y p e p t i d e c h a i n s t a k e p l a c e s i m u l t a n e o u s l y .

When i s o l a t e d m i t o c h o n d r i a a r e r e s u s p e n d e d i n an i n c u b a t i o n medium w h i c h c o n t a i n s p o s t - r i b o s o m a l c y t o p l a s m i c s u p e r n a t a n t , i n c o r p o r a t i o n o f l e u c i n e i s s t r o n g l y s t i m u l a t e d . A f t e r a c h a s e o f l e u c i n e , t h e amount o f i n c o r p o r a t e d l e u c i n e does not d e c r e a s e . T h u s , t h e c y t o p l a s m i c s u p e r n a t a n t has two e f f e c t s : i t s t i m u l a t e s p r o t e i n s y n t h e s i s and i n h i b i t s b r e a k ­down. T h e s e two e f f e c t s may be ba s e d on d i f f e r e n t mechanisms s i n c e i t was found t h a t a p r o t e i n a s e i n h i b i t o r f r a c t i o n i s o l a t e d from N e u r o s p o r a b l o c k s breakdown but does not s t i m u l a t e t h e i n i t i a l r a t e of l e u c i n e i n c o r p o r a t i o n (unpub­l i s h e d r e s u l t s ) .

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Ο 30 60 90

Time of incubation (min)

F i g . 1, I n c o r p o r a t i o n o f l e u ­c i n e i n t o t h e p r o t e i n of i s o l a t e d m i t o c h o n d r i a from N e u r o s p o r a c r a s s a

I s o l a t e d m i t o c h o n d r i a were i n c u b a t e d w i t h 3 - H - l e u c i n e i n a medium o p t i m i z e d f o r amino a c i d i n c o r p o r a t i o n (4) w i t h ­out and w i t h c y t o p l a s m i c s u p e r n a t a n t . -o o- m i t o c h o n d r i a i n i n c u ­

b a t i o n medium -· ·- m i t o c h o n d r i a i n medium

c o n t a i n i n g c y t o p l a s m i c s u p e r n a t a n t

C y t o p l a s m i c s u p e r n a t a n t was p r e p a r e d by g r i n d i n g c e l l s w i t h sand i n t h e p r e s e n c e o f i n c u b a t i o n medium (3 ml/g hyphae wet w e i g h t ) . The r e ­s u l t i n g s u s p e n s i o n was c e n t r i -fuged t o remove c e l l s w a l l s , m i t o c h o n d r i a and c y t o p l a s m i c r i b o s o m e s . At t h e t i m e i n d i c a t e d by a r r o w s , a c h a s e o f u n l a b e l e d l e u c i n e (5 mM) was g i v e n t o s t o p i n c o r p o r a t i o n of 3-H-l e u c i n e

2. P r o d u c t s of t r a n s l a t i o n i n i s o l a t 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 were i s o l a t e d from c e l l s w h i c h were p r e i n c u b a t e d w i t h 1 4 - C - l e u c i n e t o o b t a i n homogeneous l a b e l i n g of t o t a l m i t o c h o n d r i a l p r o t e i n . Sarnies of m i t o c h o n d r i a were i n c u b a t e d f o r 30 min w i t h 3 - H - l e u c i n e i n t h e a b s e n c e and p r e s e n c e of c y t o p l a s m i c s u p e r n a t a n t . Then a c h a s e of u n l a b e l e d l e u c i n e was g i v e n f o r 60 min. The p o l y p e p t i d e s s y n t h e s i z e d under t h e s e c o n d i t i o n s were a n a l y s e d by SDS-gel e l e c t r o p h o r e s i s . F i g . 2 shows t h e d i s t r i b u t i o n of 3-H- and 1 4 - C - l a b e l on t h e g e l s . The homogeneous 1 4 - C - l a b e l d i s p l a y s t h e same pattern i n t h e two s a m p l e s , w i t h o u t (A) and w i t h (B) added c y t o -

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p l a s m i c s u p e r n a t a n t . T h i s p a t t e r n i s v e r y s i m i l a r t o t h e ab-s o r b a n c e p a t t e r n a f t e r s t a i n i n g g e l s w i t h Coomassie B r i l l i a n t B l u e o b t a i n e d when i s o l a t e d m i t o c h o n d r i a were s u b j e c t e d t o g e l e l e c t r o p h o r e s i s . I t i s c o n c l u d e d t h a t d u r i n g t h e i n c u b a ­t i o n no s i g n i f i c a n t breakdown o f m i t o c h o n d r i a l p r o t e i n s has t a k e n p l a c e .

600-

10~3 apparent M r

80 4) 20 10

10 -20 30 40 50

Number of gel slice 50

-100

75

50

-25

200

150

100

50

F i g . 2. G e l e l e c t r o p h o r e t i c a n a l y s i s of p o l y p e p t i d e s s y n ­t h e s i z e d i n i s o l a t 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 were i s o l a t e d from c e l l s l a b e l e d w i t h 14-C-l e u c i n e f o r 2 h r s . A f t e r 30 min i n c u b a t i o n w i t h 3-H-l e u c i n e and a 60 min c h a s e o f u n l a b e l e d l e u c i n e , m itochon­d r i a were d i s s o l v e d i n SDS c o n t a i n i n g b u f f e r , d i a l y s e d , and s u b j e c t e d t o g e l e l e c t r o ­p h o r e s i s (4) .

A: M i t o c h o n d r i a i n c u b a t e d w i t h o u t c y t o p l a s m i c s u p e r n a ­t a n t

B: M i t o c h o n d r i a i n c u b a t e d w i t h c y t o p l a s m i c s u p e r n a ­t a n t

When m i t o c h o n d r i a a r e i n c u b a t e d w i t h o u t a d d i n g c y t o p l a s m i c s u p e r n a t a n t , t h e b u l k o f m i t o c h o n d r i a l t r a n s l a t i o n p r o d u c t s d i s p l a y s a p p a r e n t m o l e c u l a r w e i g h t s of l e s s t h a n 20 000

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( F i g . 2A, 3 - H - l a b e l ) . I n c o n t r a s t , t h e p o l y p e p t i d e s formed i n i s o l a t e d m i t o c h o n d r i a i n the p r e s e n c e of c y t o p l a s m i c s u p e r n a t a n t have h i g h e r a p p a r e n t m o l e c u l a r w e i g h t s and- mi­g r a t e a s w e l l d e f i n e d bands. T h e s e p o l y p e p t i d e s , as judged by t h e i r e l e c t r o p h o r e t i c m o b i l i t y , a r e v e r y s i m i l a r t o t h o s e formed i n v i v o , when c e l l s a r e l a b e l e d w i t h r a d i o a c t i v e l e u ­c i n e i n the p r e s e n c e of c y c l o h e x i m i d e ( 5 ) . The d a t a from F i g s . 1 and 2 s u g g e s t t h a t t h e m a j o r i t y o f t h e p o l y p e p t i d e s formed i n t h e a b s e n c e o f c y t o p l a s m i c s u p e r n a ­t a n t i s broken down by a s p e c i f i c p r o t e i n a s e , w h i ch a t t a c k s newly formed m i t o c h o n d r i a l t r a n s l a t i o n p r o d u c t s but not m i t o ­c h o n d r i a l p r o t e i n s i n g e n e r a l . T h i s breakdown i s b l o c k e d by c y t o p l a s m i c s u p e r n a t a n t . A f t e r a d d i t i o n of t h e above mentioned p r o t e i n a s e i n h i b i t o r f r a c t i o n t o t h e i n c u b a t i o n medium a l a b e l i n g p a t t e r n v e r y s i m i l a r t o t h a t i n F i g . 2A i s o b s e r v e d .

3. F o r m a t i o n o f s u b u n i t s of cytochrome o x i d a s e

Cytochrome o x i d a s e was i m m u n o p r e c i p i t a t e d from m i t o c h o n d r i a i n c u b a t e d a s d e s c r i b e d i n F i g . 2 w i t h a s p e c i f i c a n t i b o d y a g a i n s t t h e whole enzyme ( 3 ) . The i m m u n o p r e c i p i t a t e was ana­l y s e d by SDS g e l e l e c t r o p h o r e s i s ( F i g . 3 ) . The 1 4 - C - r a d i o -a c t i v i t y peaks i n F i g s . 3A and Β r e p r e s e n t t h e seve n s u b u n i t s o f cytochrome o x i d a s e . When i s o l a t e d m i t o c h o n d r i a a r e i n c u ­b a t e d i n t h e absence of c y t o p l a s m i c s u p e r n a t a n t , o n l y sub-u n i t 3 i s found t o be l a b e l e d w i t h 3 - H - l e u c i n e . The same i s o b s e r v e d when t h e p r o t e i n a s e i n h i b i t o r f r a c t i o n ( s e e above) i s added t o t h e i n c u b a t i o n medium. However, when t h e i n c u ­b a t i o n medium c o n t a i n s c y t o p l a s m i c s u p e r n a t a n t , 3 - H - l a b e l i s a s s o c i a t e d w i t h s u b u n i t s 1, 2 and 3.

T h e s e f i n d i n g s s u g g e s t t h a t t h e c y t o p l a s m i c s u p e r n a t a n t c o n t a i n s a f a c t o r w h i c h i s n e c e s s a r y f o r the i n t e g r a t i o n of s u b u n i t s 1 and 2, and o f h i g h e r amounts of s u b u n i t 3 i n t o a complex which can be i m m u n o p r e c i p i t a t e d w i t h a n t i b o d y a g a i n s t t h e whole cytochrome o x i d a s e .

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I n o r d e r t o a n a l y s e t h e n a t u r e o f t h i s f a c t o r , i s o l a t e d m i t o c h o n d r i a were i n c u b a t e d w i t h c y t o p l a s m i c s u p e r n a t a n t which was i s o l a t e d from c e l l s k e p t f o r 2 h r s i n t h e p r e s e n c e o f c y c l o h e x i m i d e . T h i s c y t o p l a s m i c s u p e r n a t a n t s t i m u l a t e s p r o t e i n s y n t h e s i s t o a l m o s t t h e same e x t e n t a s does t h e s u p e r n a t a n t from c o n t r o l c e l l s . However, s u b u n i t s 1 and 2 were n o t l a b e l e d i n t h e i m m u n o p r e c i p i t a t e . T h e s e o b s e r v a t i o n s s u g g e s t t h a t t h e f a c t o r i n the c y t o p l a s m i c s u p e r n a t a n t c o n s i s t s o f one o r more p r o t e i n s , t h e p o o l s of which a r e ex­h a u s t e d d u r i n g c y c l o h e x i m i d e t r e a t m e n t of t h e c e l l s .

1500

1000-

5000-

4000

3000

2000-

1000-

o a

F i g . 3. G e l e l e c t r o p h o r e t i c a n a l y s i s of i m m u n o p r e c i p i t a -t e d components of c y t o ­chrome o x i d a s e from i s o l a t 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 a b e l e d a s de­s c r i b e d i n F i g . 2 were d i s ­s o l v e d w i t h T r i t o n X-100 and t r e a t e d w i t h an a n t i b o d y a g a i n s t whole cytochrome o x i d a s e . The p r e c i p i t a t e was d i s s o l v e d i n SDS c o n t a i n i n g b u f f e r and a n a l y s e d by SDS g e l e l e c t r o p h o r e s i s .

A: I m m u n o p r e c i p i t a t e from m i t o c h o n d r i a l a b e l e d a s i n F i g . 2A

B: I m m u n o p r e c i p i t a t e from m i t o c h o n d r i a l a b e l e d a s i n F i g . 2B

10 20 30 4) 50 60 Number of gel slice

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T h e r e a r e m a i n l y two p o s s i b i l i t i e s t o e x p l a i n t h e n a t u r e of s u c h a f a c t o r : a) i t may have some unknown c a t a l y t i c f u n c t i o n i n t h e a s s e m b l y of cytochrome o x i d a s e ; b) i t may r e p r e s e n t (a) component (s ) of cytochrome o x i d a s e , i . e. c y t o p l a s m i c a l l y made s u b u n i t s of t h e enzyme

The s e c o n d p o s s i b i l i t y i s i n a c c o r d a n c e w i t h t h e f i n d i n g of Schwab e t a l . (6) t h a t c y t o p l a s m i c a l l y made s u b u n i t s a r e p r e ­s e n t i n p r e c u r s o r p o o l s of d i f f e r e n t s i z e s i n N e u r o s p o r a . F u r t h e r m o r e , i t i s s u p p o r t e d by t h e f o l l o w i n g e x p e r i m e n t .

F i g . 4. G e l e l e c t r o p h o r e t i c a n a l y s i s of cytochrome o x i ­d a s e components l a b e l e d i n v i t r o i n t h e p r e s e n c e of c y t o p l a s m i c s u p e r n a t a n t c o n t a i n i n g 1 4 - C - l a b e l e d p r o t e i n s

I s o l a t e d m i t o c h o n d r i a were i n c u b a t e d w i t h 3 - H - l e u c i n e i n t he p r e s e n c e of c y t o ­p l a s m i c s u p e r n a t a n t f o r 30 min. The c y t o p l a s m i c s u p e r ­n a t a n t was i s o l a t e d from c e l l s l a b e l e d w i t h 14-C-l e u c i n e f o r 10 min. P o s i ­t i o n s of s u b u n i t s 1 - 7 were d e t e r m i n e d by s e p a r a ­t i n g homogeneously l a b e l e d cytochrome o x i d a s e on t h e same g e l s l a b .

Whole c e l l s were p u l s e l a b e l e d w i t h 1 4 - C - l e u c i n e and t h e p o s t - r i b o s o m a l c y t o p l a s m i c s u p e r n a t a n t was i s o l a t e d . M i t o ­c h o n d r i a from u n l a b e l e d c e l l s were i n c u b a t e d a f t e r a d d i t i o n of t h e 1 4 - C - l a b e l e d s u p e r n a t a n t i n t h e p r e s e n c e of 3-H-leu-

1 2 3 4 5 6 7

I I I I I I !

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c i n e . A f t e r i n c u b a t i o n i m m u n o p r e c i p i t a t i o n was c a r r i e d out. The g e l e l e c t r o p h o r e t i c s e p a r a t i o n o f t h e i m m u n o p r e c i p i t a t e i s shown i n F i g . 4. The 3 - H - l a b e l d i s p l a y s t h e i n t e g r a t i o n o f s u b u n i t s 1, 2 and 3 i n t o an i m m u n o p r e c i p i t a b l e complex, a s e x p e c t e d from t h e d a t a i n F i g . 3. The 1 4 - C - r a d i o a c t i v i t y i s found i n a double band. T h i s double band c o i n c i d e s w i t h t h e p o s i t i o n s o f s u b u n i t s 6 and 7. I n a c o n t r o l e x p e r i m e n t i t was found t h a t no 1 4 - C - l a b e l was p r e c i p i t a t e d when i s o l a t e d m i t o c h o n d r i a were mixed but not i n c u b a t e d w i t h 1 4 - C - l a b e l e d c y t o p l a s m i c s u p e r n a t a n t .

T h e s e o b s e r v a t i o n s may be i n t e r p r e t e d i n t h e f o l l o w i n g way : S u b u n i t s 6 and 7 a r e p r e s e n t i n t h e c y t o p l a s m i c s u p e r n a t a n t as p r e c u r s o r s and t h e i r i n t e g r a t i o n i n t o an as s e m b l y complex i s n e c e s s a r y f o r t h e f u r t h e r i n t e g r a t i o n o f s u b u n i t s 1 and 2 i n an i m m u n o p r e c i p i t a b l e complex.

C o n c l u s i o n s •

C e l l s a p components s t i m u l a t e t h e f o r m a t i o n of m i t o c h o n d r i a l l y s y n t h e s i z e d p o l y p e p t i d e s i n i s o l a t e d m i t o c h o n d r i a . C y t o p l a s ­m i c a l l y s y n t h e s i z e d p r o t e i n s appear t o be n e c e s s a r y f o r t h e i n t e g r a t i o n of m i t o c h o n d r i a l l y made s u b u n i t s 1 and 2 of cytochrome o x i d a s e i n t o a complex w h i c h c a n be immunoprecipi­t a t e d w i t h a n t i b o d y a g a i n s t whole cytochrome o x i d a s e . T h i s complex may be t h e r e s u l t of a p a r t i a l a s s e m b l y r e a c t i o n . M i t o c h o n d r i a c o n t a i n a s p e c i f i c p r o t e i n a s e w h i c h a f f e c t s newly formed i n v i t r o t r a n s l a t i o n p r o d u c t s but n o t p r o p e r l y i n t e ­g r a t e d p r o t e i n s of t h e i n n e r m i t o c h o n d r i a l membrane. I t i s supposed t h a t t h i s p r o t e i n a s e becomes a c t i v e when m i t o c h o n d r i a l t r a n s l a t i o n p r o d u c t s a c c u m u l a t e w h i c h c a n n o t be p r o p e r l y i n t e ­g r a t e d i n t o m i t o c h o n d r i a l membrane complexes. Thus, e x p e r i ­ments w i t h i s o l a t e d m i t o c h o n d r i a a r e c o n s i d e r e d t o be a u s e f u l approach t o t h e u n d e r s t a n d i n g o f t h e mechanism and t h e r e g u ­l a t i o n o f t h e a s s e m b l y o f m i t o c h o n d r i a l membrane complexes.

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Acknowledgements. T h i s work was s u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , We 107/1 and Ne 101/11 (Schwer-punktprogramm "Biochemie d e r Morphogenese").

R e f e r e n c e s

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2) T z a g o l o f f , Α., Rubin, M. S. and S i e r r a , M. F . (1973) B i o c h i m . B i o p h y s . A c t a 301 , 17 - 104

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FEBS LETTERS 47, 290 - 294 5) Werner, S., Schwab, A. and N e u p e r t , W. (1974) E u r . J .

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