Cytochrome Systems Molecular Biology and Bioenergetics Edited by S. Papa University of Bari Bari, Italy

B. Chance University of Pennsylvania Philadelphia, Pennsylvania

and L. Ernster University of Stockholm Stockholm, Sweden

Plenum Press • New York and London

Library of Congress Cataloging in Publication Data

U N E S C O International Workshop on Cytochrome Systems: Molecular Biology and Bioenergetics (1987: Bari , Italy) Cytochrome Systems.

"Proceedings of the U N E S C O International Workshop on Cytochrome Systems: Molecular Biology and Bioenergetics, which was IUB Symposium No. 159, held Apr i l 14-18, 1987, in Bari , I ta l y " -Verso t.p.

Includes bibliographical references and index. 1. Cytochrome-Congresses. I. Papa, S. II. Chance, Britton. III. Ernster, L . IV.

Title. QP671.C85U54 1987 574.19'218 87-25766 ISBN 0-306-42693-5

The Symposium was generously supported by the following organizations:

U N E S C O European Expert Committee on Biomaterials International Union of Biochemistry, Committee on Symposia International Union of Pure and Applied Biophysics Consiglio Nazionale delle Ricerche Universitä di Bari Regione Puglia Calabrese Veicoli Industriali S.p.A., Bari Sigma Chemical Company

Proceedings of the U N E S C O International Workshop on Cytochrome Systems: Molecular Biology and Bioenergetics, which was IUB Symposium No. 159, held Apr i l 14-18, 1987, in Bari , Italy

© 1987 Plenum Press, New York A Division of Plenum Publishing Corporation 233 Spring Street, New York, N .Y . 10013

A l l rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Printed in the United States of America

CONTENTS

INTRODUCTION

Cy t o c h r o m e S y s t e m s : f r o m D i s c o v e r y t o P r e s e n t D e v e l o p m e n t s E.C. S l a t e r 3

I . GENETICS

G e n e t i c s o f E . C o l i C y t o c h r o m e s w h i c h a r e Components o f t h e A e r o b i c R e s p i r a t o r y C h a i n

R.B. G e n n i s 15

M o l e c u l a r G e n e t i c A p p r o a c h e s t o S t u d y i n g t h e S t r u c t u r e and F u n c t i o n o f t h e C y t o c h r o m e c^ and t h e C y t o c h r o m e b c ^ Complex f r o m R h o d o b a c t e r C a p s u l a t u s

F. D a l d a l 23

S t r u c t u r a l H o m o l o g i e s i n t h e C y t o c h r o m e B/C^ Complex from R h o d o b a c t e r

N.A. G a b e l l i n i 35

Genes and Sequences f o r S u b u n i t s o f R e s p i r a t o r y Complexes i n P a r a c o c c u s d e n i t r i f i c a n s

B. L u d w i g , B. K u r o w s k i , G. P a n s k u s and P. Steinrücke.. 4 1

O r g a n i z a t i o n and E x p r e s s i o n o f N u c l e a r Genes f o r Y e a s t C y t o c h r o m e c_ O x i d a s e

R.M. W r i g h t , J.D. T r a w i c k , C.E. T r u e b l o o d , T.E. P a t t e r s o n and R.O. P o y t o n 49

The cDNA a S t r u c t u r a l Gene f o r Rat L i v e r C y t o c h r o m e c O x i d a s e S u b u n i t V I c

G. S u s k e , C. E n d e r s , T. Mengel and B. Kadenbach 57

G e n e t i c s o f Y e a s t Coenzyme QH^-Cytochrome c R e d u c t a s e M. C r i v e l l o n e , A. Gampel, I . M u r o f f , M. Wu and A. T z a g o l o f f 67

Sequence A n a l y s i s o f Mammalian Cyt o c h r o m e b M u t a n t s N. H o w e l l and K. G i l b e r t 79

P l a n t M i t o c h o n d r i a l G enes, Cy t o c h r o m e c O x i d a s e and C y t o p l a s m i c M a l e S t e r i l i t y

M.J. H a w k e s f o r d and C . J . L e a v e r 87

vii

A M u t a t i o n A f f e c t i n g L a r i a t F o r m a t i o n , b u t n o t S p l i c i n g o f a Y e a s t M i t o c h o n d r i a l Group I I I n t r o n

R. v a n d e r V e e n , J.H.J.M. Kwakman and L.A. G r i v e l l . . . . 97

The E v o l u t i o n o f M i t o c h o n d r i a l l y Coded C y t o c h r o m e Genes: A Q u a n t i t a t i v e E s t i m a t e

C. S a c c o n e , M. A t t i m o n e l l i , C. L a n a v e , R. G a l l e r a n i and G. P e s o l e 103

I n v o l v e m e n t o f N u c l e a r Genes i n S p l i c i n g o f t h e M i t o c h o n d r i a l COB T r a n s c r i p t i n S. C e r e v i s i a e

J . K r e i k e , G. Krummeck, T. Söllner, C. S c h m i d t and R . J . Schweyen I I I

I n t r o n s as Key E l e m e n t s i n t h e E v o l u t i o n o f M i t o c h o n d r i a l Genomes i n Lower E u k a r y o t e s

K. W o l f , A. Ahne, L. D e l G i u d i c e , G. O r a l e r , F. K anbay, A.M. M e r l o s - L a n g e , F. W e l s e r and M. Zimmer 119

GENETICS: S h o r t R e p o r t s

C o r e P r o t e i n D e f i c i e n c y i n Complex I I I o f t h e R e s p i r a t o r y C h a i n , i n a M i s s e n c e E x o n i c M i t o c h o n d r i a l Y e a s t M u t a n t o f C y t o c h r o m e b Gene

P. C h e v i l l o t t e - B r i v e t , G. S a l o u and D. M e u n i e r - L e m e s l e 13 1

L o c a l i z a t i o n and P a r t i a l S e q u e n c i n g A n a l y s i s o f M i t o c h o n d r i a l Gene f o r A p o c y t o c h r o m e b i n S u n f l o w e r

D. P a c o d a , A.S. T r e g l i a , L. S i c u l e l l a , C. P e r r o t t a and R. G a l l e r a n i 133

M o l e c u l a r B a s i s f o r R e s i s t a n c e t o I n h i b i t o r s o f t h e M i t o ­c h o n d r i a l U b i q u i n o l - C y t o c h r o m e c R e d u c t a s e i n S a c c h a r m o y c e s C e r e v i s i a e

A.M. C o l s o n , B. M e u n i e r and J . P . d i Rago 135

S t r u c t u r e o f t h e P r o t e i n s and Genes f o r t h e N u c l e a r - E n c o d e d S u b u n i t s o f N. C r a s s a C y tochrome O x i d a s e

M.D. S u a r e z , M. S a c h s , M. D a v i d and U.L. R a j B h a n d a r y . . 137

P a r t i a l Sequence o f Gene f o r C y t o c h r o m e Oxydase S u b u n i t I I o f S u n f l o w e r M i t o c h o n d r i a l DNA

L.R. C e c i , C. De B e n e d e t t o , C M . P e r r o t t a , L. S i c u l e l l a and R. G a l l e r a n i 139

U n u s u a l F e a t u r e s o f K i n e t o p l a s t DNA f r o m T r y p a n o s o m e s : N o v e l M e c h a n i s m o f Gene E x p r e s s i o n ( R N A - E d i t i n g ) , t h e A b s e n c e o f tRNA Genes and t h e V a r i a b l e P r e s e n c e o f a C y t o c h r o m e O x i d a s e S u b u n i t Gene

P. S l o o f , R. Benne, B. De V r i e s , T. H a k v o o r t and A. M u i j s e r s 14 1

I I . BIOSYNTHESIS

H e t e r o g e n e o u s E f f i c i e n c i e s o f mRNA T r a n s l a t i o n i n Human M i t o c h o n d r i a

A. Chomyn and G. A t t a r d i 145

R e g u l a t i o n o f t h e E x p r e s s i o n o f COI and C O I I I mRNAs i n Rat L i v e r M i t o c h o n d r i a

P. C a n t a t o r e , F. F r a c a s s o , A.M.S. L e z z a and M.N. G a d a l e t a 153

Two C o n s e n s u s Sequences f o r RNA P r o c e s s i n g i n N e u r o s p o r a C r a s s a M i t o c h o n d r i a

G. M a c i n o and M.A. N e l s o n 161

P r i m a r y S t r u c t u r e o f t h e Y e a s t N u c l e a r CBS 1 Gene P r o d u c t , N e c e s s a r y t o A c t i v a t e T r a n s l a t i o n o f M i t o c h o n d r i a l COB mRNA

V. F o r s b a c h and G. Rödel 169

A C a l m o d u l i n - L i k e P r o t e i n i n t h e Cy t o c h r o m e bc Complex R e q u i r e d f o r S y n t h e s i s o f B o t h C y t o c h r o m e bc and Cy t o c h r o m e c O x i d a s e Complexes i n Y e a s t M i t o c h o n d r i a

M.E . S c h m i t t and B.L. Trumpower 177

Im p o r t o f C y t o c h r o m e s b^ and c^ i n t o M i t o c h o n d r i a i s Dependent on B o t h Membrane P o t e n t i a l and N u c l e o s i d e T r i p h o s p h a t e s

F.U. H a r t l , J . O s t e r m a n n , N. P f a n n e r , M. T r o p s c h u g , B. G u i a r d and W. N e u p e r t 189

NADH: A Common R e q u i r e m e n t f o r t h e Import and M a t u r a t i o n o f C y t o c h r o m e s c and c ^

D.W. N i c h o l s o n , J . Ost e r m a n n and W. N e u p e r t 197

Imp o r t o f P r o t e i n s i n t o M i t o c h o n d r i a : S t r u c t u r a l and F u n c t i o n a l R o l e o f t h e P r e p e p t i d e

D. R o i s e 209

I n t e r a c t i o n o f t h e M i t o c h o n d r i a l P r e c u r s o r P r o t e i n A p o c y -t o c h r o m e c w i t h M o d e l Membranes and i t s I m p l i c a t i o n s f o r P r o t e i n T r a n s l o c a t i o n

B. de K r u i j f f , A. R i e t v e l d , W. J o r d i , T.A. B e r k h o u t , R.A. Demel, H. Görrissen and D. Mars h 2 15

I n f l u e n c e o f T h y r o i d Hormones on t h e E x p r e s s i o n o f N u c l e a r Genes E n c o d i n g S u b u n i t s o f t h e Cy t o c h r o m e bc Complex

V. J o s t e and B.D. N e l s o n 225

I I . BIOSYNTHESIS: S h o r t R e p o r t

I m p o r t o f C y t o c h r o m e c and Cy t o c h r o m e b^ i n t o M i t o c h o n d r i a J . O s t e r m a n n , F.U. H a r t l , M. T r o p s c h u n g , B. G u i a r d and W. N e u p e r t 235

ix

I I I . PROTEIN STRUCTURE

Cyt o c h r o m e c O x i d a s e : P a s t , P r e s e n t and F u t u r e M. Müller, N. L a b o n i a , B. Schläpfer and A. A z z i 23^

U n i v e r s a l F e a t u r e s i n C y t o c h r o m e O x i d a s e M. F i n e l , T. H a l t i a , L. Holm, T. J a l l i , T. M e t s o , A. P u u s t i n e n , M. R a i t i o , M. S a r a s t e and M. Wikström 24"

P o l y p e p t i d e S u b u n i t s Encoded by N u c l e a r Genes a r e E s s e n t i a l Components o f E u k a r y o t i c C y t o c h r o m e c_ O x i d a s e

T.E. P a t t e r s o n , C.E. T r u e b l o o d , R.M. W r i g h t and R.O. P o y t o n 25)

R e s p i r a t o r y Complex IV and C y t o c h r o m e a , a ^ G. B u s e , G.C.M. S t e f f e n s , R. B i e w a l d , B. B r u c h and S. H e n s e l 26

An Amino A c i d Sequence R e g i o n o f S u b u n i t I I o f C y t o c h r o m e O x i d a s e w h i c h may be R e s p o n s i b l e f o r E v o l u t i o n a r y Changes i n R e a c t i v i t y w i t h D i f f e r e n t C y t o c h r o m e s c

T.L. L u n t z and E. M a r g o l i a s h 27

E v i d e n c e f o r a F u n c t i o n a l R o l e o f N u c l e a r E n c o d e d S u b u n i t s i n M i t o c h o n d r i a l C y t o c h r o m e c O x i d a s e

R. B i s s o n and B. B a c c i 28!

S t r u c t u r a l A n a l y s i s o f t h e bc^ Complex f r o m B e e f H e a r t M i t o c h o n d r i a by t h e S i d e d H y d r o p a t h y P l o t and by C o m p a r i s o n w i t h O t h e r bc ^ C omplexes

T.A. L i n k , H. Schägger and G. v o n Jagow 289

C r y s t a l l i s a t i o n o f W a t e r - S o l u b l e P r e p a r a t i o n s o f t h e I r o n -S u l f u r - P r o t e i n and C y t o c h r o m e c^ o f U b i q u i n o l : C y t o c h r o m e R e d u c t a s e f r o m N e u r o s p o r a M i t o c h o n d r i a

J . Römisch, U. H a r n i s c h , U. S c h u l t e and H. W e i s s 303

I r o n C l u s t e r S i t e s o f C a r d i a c I p - S u b u n i t o f S u c c i n a t e D e h y d r o g e n a s e

T.E. K i n g , N.S. R e i m e r , M.T. Seaman, L.Q. Sun, Q.W. Wang, K.T. Y a s u n o b u and S.H. Ho 309

P o l y p e p t i d e s i n M i t o c h o n d r i a l E l e c t r o n - T r a n s f e r C o m p l e x e s T. Ozawa, M. N i s h i k i m i , H. S u z u k i , M. T a n a k a and Y. Shimomura 315

The F a t t y A c i d - A n c h o r e d F o u r Herne C y t o c h r o m e o f t h e P h o t o -s y n t h e t i c R e a c t i o n C e n t e r f r o m t h e P u r p l e B a c t e r i u m Rhodopseudomonas V i r i d i s

K.A. Weyer, F. L o t t s p e i c h , W. Schäfer and H. M i c h e l 325

x

I I I . PROTEIN STRUCTURE: S h o r t R e p o r t s

Membrane P r o t e i n i n P a r a c o c c u s D e n i t r i f i c a n s R e s e m b l e s S u b u n i t I I I o f B e e f H e a r t C y t o c h r o m e O x i d a s e

T. H a l t i a and M. F i n e l 335

The C i r c u l a r D i c h r o i s m P r o p e r t i e s o f t h e R i e s k e P r o t e i n and t h e b and c C y t o c h r o m e s o f t h e M i t o c h o n d r i a l b c | Complex

G. S o l a i n i , M. C r i m i , F. B a l l e s t e r , M. D e g l i E s p o s t i and G. L e n a z 337

M o n o m e r i z a t i o n o f C y t o c h r o m e O x i d a s e M i g h t Be E s s e n t i a l f o r S u b u n i t I I I Removal

M. F i n e l 339

F o u r i e r - T r a n s f o r m I n f r a - R e d S t u d i e s o f C y t o c h r o m e c O x i d a s e M.F. G r a h n , P . I . K a r i s , J.M. W r i g g l e s w o r t h and D. Chapman 34 1

I s o e n z y m e s o f Human C y t o c h r o m e c O x i d a s e T.B.M. H a k v o o r t , G.J.C. R u y t e r , K.M.C. S i n j o r g o and A.O. M u i j s e r s 343

F o l d i n g o f I n t e g r a l Membrane P r o t e i n s : R e n a t u r a t i o n E x p e r i ­ments w i t h B a c t e r i o r h o d o s p i n S u p p o r t a Two-Stage M e c h a n i s m

J . L . P o p o t and D.M. Engelman 345

I V . REACTION DOMAINS AND OXIDO-REDUCTION MECHANISMS

The N a t u r e o f F e r r y l , F e ( l V ) = 0 , Haem i n P e r o x i d a s e s A. J . Thomson, C. Greenwood, P.M.A. Gadsby and N. F o o t e 349

P o l a r i z a t i o n i n Herne P r o t e i n s and Cyto c h r o m e c O x i d a s e J.O. A l b e n 36 1

E f f e c t s o f F r e e z i n g on t h e C o o r d i n a t i o n S t a t e and L i g a n d O r i e n t a t i o n i n H e m o p r o t e i n s

H. A n n i and T. Y o n e t a n i 37 1

C a t a l y t i c M e c h a n i s m o f 0^ R e d u c t i o n by C y t o c h r o m e O x i d a s e M. Wikström 377

The E l e c t r o n - T r a n s f e r R e a c t i o n s i n and S u b s t r a t e B i n d i n g t o C y t o c h r o m e c_ O x i d a s e

B. F. v a n G e l d e r , A.C.F. G o r r e n , L. V l e g e l s and R. Wever 385

C o n t r o l o f C y t o c h r o m e O x i d a s e i n P r o t e o l i p o s o m e s : F l u x and S t o i c h i o m e t r y

P. N i c h o l l s , S. S h a u g h n e s s y and A.P. S i n g h 391

Cy t o c h r o m e c O x i d a s e A c t i v i t y i s R e g u l a t e d by N u c l e o t i d e s and A n i o n s

B. K a d e n b a c h , A. S t r o h , F . J . Hüther and J . B e r d e n . . . . 399

xi

Cytochrome O x i d a s e and N e u r o m u s c u l a r D i s e a s e s C.P. L e e , M.E. M a r t e n s , P.L. P e t e r s o n and J. S . H a t f i e l d 407

E l e c t r o n T r a n s f e r Between C a r d i a c C y t o c h r o m e c^ and c C.H. K i m , C. B a l n y and T.E. K i n g 415

Herne C l u s t e r S t r u c t u r e s and E l e c t r o n T r a n s f e r i n M u l t i h e m e C y t o c h r o m e s C^

R. H a s e r and J . Mosse 423

IV. REACTION DOMAINS AND OXIDO-REDUCTION MECHANISMS: S h o r t R e p o r t s

E l e c t r o n Exchange Between C y t o c h r o m e s a. P. S a r t i , F. M a l a t e s t a , G. A n t o n i n i , M.T. W i l s o n and M. B r u n o r i 433

P u l s e d , R e s t i n g and P e r o x y Forms o f C y t o c h r o m e O x i d a s e G. A n t o n i n i , F. M a l a t e s t a , P. S a r t i , M.T. W i l s o n and M. B r u n o r i 435

E f f e c t o f pH and T e m p e r a t u r e on E l e c t r o n T r a n s f e r i n M i x e d -V a l e n c e C y t o c h r o m e c O x i d a s e

P. B r e z e z i n s k i and Bo.G. Malmström 437

The pH-Dependence o f t h e R e d u c t i o n a l L e v e l s o f C y t o c h r o m e c and C y t o c h r o m e a Düring T u r n o v e r o f C y t o c h r o m e c O x i d a s e

P.E. Thörnstrom, B. M a i s o n - P e t e r i , P. B r z e z i n s k i , L. A r v i d s s o n , P.O. F r e d r i k s s o n and Bo.G. Malmström.... 439

V. Fe-S CENTERS

S t u d i e s o f B a c t e r i a l NADH-Ubiquinone ( o r M e n a q u i n o n e ) O x i d o r e d u c t a s e Systems

T. O h n i s h i , S.W. M e i n h a r d t and K. M a t s u s h i t a 443

L o c a l i z e d V a l e n c e S t a t e s i n I r o n - S u l f u r C l u s t e r s and T h e i r P o s s i b l e R e l a t i o n s h i p t o t h e A b i l i t y o f I r o n - S u l f u r C l u s t e r s t o C a t a l y z e R e a c t i o n s O t h e r Than E l e c t r o n T r a n s f e r

H. B e i n e r t 451

P r o b i n g t h e Fe/S Domain W i t h EPR: P a n d o r a ' s BOX A j a r W.R. Hagen 459

A s p e c t s o f S p i n C o u p l i n g Between Even and Odd E l e c t r o n S y s t e m s : A p p l i c a t i o n s t o S u c c i n a t e : Q R e d u c t a s e

J.C. S a l e r n o and Xu Yan 467

The I r o n - S u l f u r C l u s t e r s i n S u c c i n a t e D e h y d r o g e n a s e M.K. J o h n s o n , J . E . M o r n i n g s t a r , E.B. K e a r n e y , G. C e c c h i n i and B.A.C. A c k r e l l 473

Mechanisms o f E l e c t r o n T r a n s f e r i n S u c c i n a t e D e h y d r o g e n a s e and F u m a r a t e R e d u c t a s e : P o s s i b l e F u n c t i o n s f o r I r o n - S u l p h u r C e n t r e 2 and C y t o c h r o m e 1}

R. Cammack, J . M a g u i r e and B.A.C. A c k r e l l 485

xii

V I . QUINONE BINDING SITES:STRUCTURE AND FUNCTION

S t r u c t u r a l and M e c h a n i s t i c A s p e c t s o f t h e Q u inone B i n d i n g S i t e s o f t h e bc Complexes

P.R. R i e h 495

QP-S-The E l e c t r o n A c c e p t o r o f S u c c i n a t e D e h y d r o g e n a s e T.E. K i n g and Y. Xu 503

C h a r a c t e r i s t i c s o f QP-C and R e c o n s t i t u t i o n o f t h e QH^-Cyt c r e d u c t a s e

T.Y. Wang, Z.P. Zhang and T.E. K i n g 509

The K i n e t i c s o f O x i d a t i o n o f C y t o c h r o m e b a r e i n Agreement w i t h t h e Q - C y c l e H y p o t h e s i s

S. De V r i e s , A.N. v a n Hoek, A. t e n Bookum and J.A. B e r d e n 517

The Q - R e a c t i o n Domains o f M i t o c h o n d r i a l QH^' C y t o c h r o m e c_ O x i d o r e d u c t a s e

J.A. B e r d e n , A.N. v a n Hoek, S. de V r i e s and P . J . S c h o p p i n k 523

EPR S t u d i e s o f t h e Q u i n o n e R e a c t i o n S i t e s i n B a c t e r i a S.W. M e i n h a r d t , X. Y a n g , B.L. Trumpower and T. O h n i s h i 533

EPR S i g n a l I I i n P h o t o s y s t e m I I : Redox and P a r a m a g n e t i c I n t e r a c t i o n s w i t h t h e 0^ E v o l v i n g Enzyme

A.W. R u t h e r f o r d and S. S t y r i n g 541

S t u d i e s on t h e U b i q u i n o n e P o o l i n M i t o c h o n d r i a l E l e c t r o n T r a n s f e r

G. L e n a z , R. F a t o and G. P a r e n t i C a s t e l l i 549

V I . QUINONE BINDING S I T E S : STRUCTURE AND FUNCTION: S h o r t R e p o r t s

E f f e c t s o f P r o t e o l y t i c D i g e s t i o n and C h e m i c a l M o d i f i c a t i o n o f b-c C o m p l e x on t h e Fe-S C e n t e r and t h e S t a b l e U b i s e m i -q u i n o n e (SQc)

T. C o c c o , S. M e i n h a r d t , D. G a t t i , M. L o r u s s o , T. O h n i s h i and S. Papa 559

K i n e t i c s o f U b i q u i n o l C y t o c h r o m e c R e d u c t a s e : L a c k o f a D i f f u s i o n - L i m i t e d S t e p w i t h S h o r t C h a i n U b i q u i n o l s as S u b s t r a t e s

R. F a t o , C. C a s t e l l u c c i o , G. L e n a z , M. B a t t i n o and G. P a r e n t i C a s t e l l i 561

V I I . S P E C I F I C REDOX SYSTEMS

B i o c h e m i s t r y and M o l e c u l a r B i o l o g y o f t h e E s c h e r i c h i a C o l i A e r o b i c R e s p i r a t o r y C h a i n

Y. A n r a k u 565

xiii

The I n t e r p l a y Between P h o t o s y n t h e s i s and R e s p i r a t i o n i n F a c u l t a t i v e A n o x y g e n i c P h o t o t r o p h i c B a c t e r i a

D. Z a n n o n i 575

The U b i q u i n o l - C y t o c h r o m e c O x i d o r e d u c t a s e o f P h o t o t r o p h i c B a c t e r i a

M. S n o z z i and J . Kälin 585

The Redox R e a c t i o n Between C y t o c h r o m e and t h e S e m i q u i -n o n e - Q u i n o l C o u p l e o f Q i s E l e c t r o g e n i c i n U b i q u i n o l -

c Cyt c^ O x i d o r e d u c t a s e

D.E. R o b e r t s o n and P.L. D u t t o n 593

C o n s t r u c t i o n and C h a r a c t e r i z a t i o n o f M o n o l a y e r F i l m s o f t h e R e a c t i o n C e n t e r Cytochrome-c_ P r o t e i n f r o m Rhodopseudomonas V i r i d i s

G. A l e g r i a and P.L. D u t t o n

S t r u c t u r e - F u n c t i o n R e l a t i o n s h i p s i n t h e Rhodopseudomonas V i r i d i s R e a c t i o n C e n t e r Complex: E l e c t r o g e n i c S t e p s C o n t r i -b u t i n g t o F o r m a t i o n

V.P. S k u l a c h e v , L.A. D r a c h e v , S.M. D r a c h e v a , A.A. K o n s t a n t i n o v , A.Yu. Semenov and V.A. S h u v a l o v . .

C a t a l y t i c S i t e s f o r R e d u c t i o n and O x i d a t i o n o f Q u i n o n e s A. C r o f t s , H. R o b i n s o n , K. An d r e w s , S. v a n Dören and Ed B e r r y 617

S t u d i e s o f t h e E l e c t r o g e n i c i t y o f t h e R e d u c t i o n o f C y t o ­chrome b t h r o u g h t h e A n t i m y c i n - S e n s i t i v e S i t e o f t h e U b i q u i n o 5 - ( i y t o c h r o m e c ^ O x i d o r e d u c t a s e Complex o f R h o d o b a c t e r S p h a e r o i d e s

E. G l a s e r and A.R. C r o f t s 625

M u t a n t s A f f e c t i n g t h e Q u i n o l O x i d a t i o n S i t e o f U b i q u i n o l -Cyt c ^ - O x i d o r e d u c t a s e

D.E. R o b e r t s o n , F. D a l d a l and P.L. D u t t o n 633

Cy t o c h r o m e o f B a c i l l u s S u b t i l i s H. F r i d e n and L. H e d e r s t e d t 641

M i t o c h o n d r i a l C y t o c h r o m e b C A . Yu and L. Yu 649

I s o l a t i o n o f t h e P h o t o s y s t e m Two R e a c t i o n C e n t r e and t h e l o c a t i o n and F u n c t i o n o f Cy t o c h r o m e D ^ . ^

J . B a r b e r , K. G o u n a r i s and D.J. Chapman 657

V I I . S P E C I F I C REDOX SYSTEMS: S h o r t R e p o r t s

E f f e c t o f A n t i m y c i n on t h e K i n e t i c s o f R e d u c t i o n o f b and c j C y t o c h r o m e s i n S i n g l e T u r n o v e r o f t h e M i t o c h o n d r i a l b - c ^ Complex

D. B o f f o l i , M. L o r u s s o and S. Papa 669

The b-Q C y c l e , a new model f o r t h e Pathways o f E l e c t r o n s and P r o t o n s t h r o u g h t h e b c ^ Complex! A c t i o n o f A n t i m y c i n A at C e n t e r 0

D. M e u n i e r - L e m e s l e and P. C h e v i l l o t t e - B r i v e t 673

xiv

601

. 609

R a p i d O x i d a t i o n K i n e t i c s o f Q u i n o l C y t o c h r o m e c O x i d o r e d u c t a s e : Q u i n o n e o r S e m i q u i n o n e C y c l e ?

A.N. v a n Hoek, J.A. B e r d e n and S. De V r i e s 675

E l e c t r o n C o n d u c t i o n Between C y t o c h r o m e s o f t h e M i t o c h o n ­d r i a l R e s p i r a t o r y C h a i n i n t h e P r e s e n c e o f A n t i m y c i n p l u s M y x o t h i a z o l

I.C. West 679

The R e l e a s e o f NAD* R a d i c a l i s t h e L i m i t i n g S t e p i n t h e Redox R e a c t i o n Between F e ( I I l ) C y t o c h r o m e c and NADH

A. F e r r i , P. C h i o z z i , M.E. C a t t o z z o and D. P a t t i 681

P r o p e r t i e s o f C y t o c h r o m e b i n Complex I I o f A s c a r i s 558 . .

M u s c l e M i t o c h o n d r i a and l t s F u n c t i o n i n A n a e r o b i c R e s p i r a ­t i o n

K. K i t a , S. T a k a m i y a , R. F u r u s h i m a , Y. Ma and H. Oya 683

V I I I . COOPERATIVITY AND ALLOSTERIC TRANSITIONS

P u l s e d and R e s t i n g C y t o c h r o m e O x i d a s e : an U p d a t e o f O p t i c a l and M o l e c u l a r P r o p e r t i e s

M. B r u n o r i , P. S a r t i , F. M a l a t e s t a , G. A n t o n i n i and M.T. W i l s o n 689

I s H O I n v o l v e d i n E l e c t r o n T r a n s p o r t Between C y t o c h r o m e s a_ and a_^ i n C y t o c h r o m e O x i d a s e ?

J.M. W r i g g l e s w o r t h , M.F. G r a h n , J . E l s d e n and H. Baum 697

C o n f o r m a t i o n a l T r a n s i t i o n s o f C y t o c h r o m e c_ O x i d a s e I n d u c e d by P a r t i a l R e d u c t i o n

C.P. S c h o l e s , C. F a n , J . Bank, R. D o r r and Bo.G. Malmström 705

R e d o x - L i n k e d C o n f o r m a t i o n a l Changes i n C y t o c h r o m e £ O x i d a s e T. A l l e y n e and M.T. W i l s o n 713

I X . PROTON-MOTIVE ACTIVITY

P r o t o n m o t i v e A c t i v i t y o f t h e C y t o c h r o m e C h a i n o f M i t o c h o n ­d r i a : M o d e l s and F e a t u r e s

S. Papa and N. C a p i t a n i o 723

The M e c h a n i s m o f E l e c t r o n G a t i n g i n C y t o c h r o m e c O x i d a s e Bo.G. Malmström 733

P r o t o n Pump A c t i v i t y i n B a c t e r i a l C y t o c h r o m e O x i d a s e s N. Sone 743

T o p o g r a p h y and P r o t o n m o t i v e M e c h a n i s m o f M i t o c h o n d r i a l C o u p l i n g S i t e 2

A.A. K o n s t a n t i n o v and E. Popova 751

xv

The Q-Gated P r o t o n Pump Model o f U b i q u i n o l C y t o c h r o m e c R e d u c t a s e : E l e c t r o n and P r o t o n T r a n s f e r P a t h ways and R o l e o f P o l y p e p t i d e s

M. L o r u s s o , D. B o f f o l i , T. C o c c o , D. G a t t i and S. Papa 767

P h o t o s y n t h e t i c C o n t r o l and A T P / E l e c t r o n R a t i o i n B a c t e r i a l P h o t o p h o s p h o r y l a t i o n

V. Adam, M. V i r g i l i , G. V e n t u r o l i , D. P i e t r o b o n and B.A. M e l a n d r i 777

I X . PROTON-MOTIVE ACTIVITY: S h o r t R e p o r t

P r o t o n m o t i v e A c t i v i t y o f M i t o c h o n d r i a l C y t o c h r o m e c O x i d a s e

N. C a p i t a n i o , E. De N i t t o and S. Papa 787

APPENDIX

P r o p o s a l f o r a N o v e l N o m e n c l a t u r e f o r t h e S u b u n i t s o f Cyto c h r o m e c O x i d a s e

G. B u s e , G.C.M. S t e f f e n s , R. B i e w a l d , B. B r u c h and S. H e n s e l 791

A u t h o r I n d e x 795

S u b j e c t I n d e x 799

xvi

IMPORT OF CYTOCHROMES t>2 AND c 1 INTO MITOCHONDRIA IS DEPENDENT

ON BOTH MEMBRANE POTENTIAL AND NUCLEOSIDE TRIPHOSPHATES

Franz-Ulrich H a r t l , Joachim Ostermann, Nikolaus Pfanner, Maximilian Tropschug, Bernard Guiard* and Walter Neupert

I n s t i t u t für Physiologische Chemie, Goethestr. 33 8000 München 2, FRG, and * Centre de Genetique Moleculaire, 91190 Gif-sur-Yvette France

SUMMARY

Import of precursors of cytochromes and c.j into mito­chondria requires a mitochondrial membrane pote n t i a l . We show here that i n addition to AY> nucleoside triphosphates (NTPs) are necessary for protein translocation. At low concentrations of NTPs, intermediate-sized cytochrome b^ was accumulated spanning the outer and inner membranes at contact S i t e s . For complete translocation into mitochondria, higher concentrations of NTPs were necessary. We conclude that d i f f e r e n t levels of NTPs are required f o r d i s t i n c t steps i n the import pathway.

INTR0DUCTI0N

Most mitochondrial proteins are coded for by the nucleus and are synthesized as precursors on cytoplasmic polysomes. Transport of proteins into mitochondria can be subdivided into several steps (for reviews see: Harmey and Neupert, 1985; Pfanner and Neupert, 1987; Hartl et a l . , 1987): ( i ) s p e c i f i c i n t e r a c t i o n with receptors on the mitochondrial surface; ( i i ) transport i n t o mitochondria at translocation contact s i t e s between outer and inner membranes; ( i i i ) processing of precursors having N-terminal extensions by the processing peptidase i n the matrix; (iv) additional modifications, including covalent or non-covalent attachment of cofactors or, i n ce r t a i n cases, a second prote o l y t i c processing step; and (v) assembly i n t o active supramolecular protein complexes.

Transport into or translocation across the inner membrane i s dependent on an energized inner membrane (Hallermayer and Neupert, 1976; Nelson and Schatz, 1979; Zimmermann et a l . , 1981; Schleyer et a l . , 1982; Gasser et a l . , 1982; Kolanski et a l . , 1982). Energy i s required i n the form of the e l e c t r i c a l component AY of t o t a l protonmotive force (Pfanner and Neupert, 198$). For long, however, i t could not be decided whether high energy phosphate Compounds, such as ATP, are necessary i n a d d i t i o n toAY. Indeed, t h i s was recently shown for the import of the ß subunit of F QF^-ATPase ( F ß) (Pfanner and Neupert, 1986), the ADP/ATP-translocator of the inner membrane, and fusion proteins between F F -ATPase subunit 9 and dihydrofolate reductase (Pfanner et a l . ,

In the present report we investigated whether the import of

189

cytochromes b^ and was dependent on NTPs, Cytochrome b^ i s a soluble component of the intermembrane space (Daum et a l . , 1982a). Cytochrome c. i s anchored to the inner membrane but contains a large hydrophilic aomain which protrudes into the intermembrane space ( L i et a l . , 1981). Compared to ß subunit of F F^-ATPase t h e i r import and sorting pathways are more complex i n that both precursor proteins are p r o t e o l y t i c a l l y processed i n two steps (Daum et a l . , 1982b; Ohashi et a l . , 1982; Teintze et a l . , 1982). The f i r s t processing step i s performed by the matrix peptidase, i . e . precursors have to be translocated either completely or at least p a r t i a l l y across the inner membrane. The second processing event occurs at the outer surface of the inner membrane by a so f a r uncharacterized protease(s) (Pratje and Guiard, 1986). On addition both proteins have to acquire heme, which i s covalently attached i n case of cytochrome c^.

Selective and independent manipulation of NTP levels and the membrane Potential showed that import of cytochromes b~ and c^ required both NTPs and&Y. Interestingly, at low levels of NTPs precursors were only p a r t i a l l y translocated into mitochondria: they accumulated i n translocation contact s i t e s (Schleyer and Neupert, 198$).

MATERIALS AND METHODS

Growth of Neurospora crassa (wild type l/+k) (Schleyer et a l . , 1982) and Is o l a t i o n of mitochondria by P e r c o l l (Pharmacia) density gradient centrifugation was done as described (Hartl et a l . , 1986). Yeast c e l l s of wild type Saccharomyces cerevisiae (D273-10B) were grown on 2% lactate and mitochondria were isolated according to Daum et a l . (1982a). Mitochondria were f i n a l l y suspended i n SEM buffer (2$0 mM sucrose, 1 mM EDTA, 10 mM M0PS/K0H, pH 7.2) at a protein concentration of 2.$ mg/ml.

Precursor proteins were synthesized by coupled t r a n s c r i p t i o n / t r a n s l a t i o n . For cytochrome c^, a füll length cDNA was iso l a t e d from a N. crassa l i b r a r y and cloned into pGEM4.. For cytochrome b^, the genomic clone described previously (Guiard, 198$) was used. Transcription/translation of the cloned sequences followed the methods of Krieg and Melton (1984) and Stueber et a l . (1984), respectively. Postribosomal supernatants were prepared and supplemented as published (Schleyer et a l . , 1982).

Labelled reticulocyte lysates and isolated mitochondria were treated with apyrase (Sigma, grade VIII) e s s e n t i a l l y as described before (Pfanner and Neupert, 1986). Afterwards reticulocyte lysates were cooled to 0°C and diluted with BSA buffer (2$0 mM sucrose, 80 mM KCl, $ mM MgCl 2, 10 mM MOPS, 3% (w/v) BSA, pH 7.2). Antimycin A, oligomycin or valinomycin (8 tiM, 20 nM and 1 uM, respectively) were added from 100-fold concentrated stock Solutions i n ethanol when indicated. Mixtures for import into N. crassa mitochondria contained 8 mM potassiura ascorbate and 0.2 mM N,N,N',N'-tetramethylphenylenediamine (TMPD) as an energy source, whereas mixtures for yeast mitochondria included 20 mM potassium succinate. Then mitochondria ($0 iig of protein) were added. In order to Supplement NTPs, either ATP or GTP (8 mM f i n a l concentration) were added from 200 mM stock S o l u t i o n s i n water. For neutralization s u f f i c i e n t amounts of 1 M MOPS/NaOH, pH 7.2, were included. Incubation was for 30 min at 2$°C i n a t o t a l volume of 100 u l . Mitochondria were then reisolated by centrifugation (1$ min 27,000 x g), resuspended i n SEM buffer and treated with Proteinase K (1$ ug/ml f i n a l concentration) as described (Hartl et a l . , 1986). Reisolated mitochondria were lysed i n SDS sample buffer. SDS Polyacrylamide electrophoresis and fluorography were carried out according to published methods (Laemmli, 1970; Hartl et a l . , 1986).

190

RESULTS

Import of cytochromes and c^ requires nucleoside triphosphates ~-

Reticulocyte lysates containing the S-methionine lab e l l e d precursor of cytochrome b^ and freshly i s o l a t e d yeast mitochondria were pretreated with differ e n t concentrations of apyrase, an adenosine 5 !-triphosphatase and adenosine 5'-diphosphatase which caused rapid depletion of endogenous ATP and ADP. Mitochondria and reticulocyte lysate were mixed i n the presence of succinate (as a respiratory Substrate) and oligomycin (which i n h i b i t s the F F^-ATPase, Wikstrom and Krab, 1982). The l a t t e r was included to prevent reduction of the mitochondrial membrane potential by ATPase a c t i v i t y . Following incubation for 30 min at 25°C, the samples were treated with Proteinase K to digest cytochrome b^ that had not been.imported into mitochondria. Then mitochondria were reisolated and dissolved i n SDS-containing buffer for subsequent

1 2 3 4

Apyrase ++ +

Fig. 1. Import of cytochrome b^ into yeast mitochondria i s i n h i b i t e d by apyrase treatment. Reticulocyte lysates containing l a b e l l e d precursor of cytochrome b^ were incubated for 1$ min at 30°C and 15 min at 2$°C with apyrase: reactions 1 and 3 received 1 and 0.25 U/ml, respectively; reactions 2 and 4- received corresponding amounts of inactivated apyrase. Mitochondria were added to the lysate i n presence of succinate and oligomycin. Incubation for import was for 30 min at 25°C. Afterwards the samples were cooled to 0°C and diluted 1:2 with SEM. Proteinase K treatment was then performed (30 min at 0°C). Protease a c t i v i t y was stopped by adding PMSF to 1 mM. Then mitochondria were reisolated by centrifugation and dissociated i n SDS-containing buffer. The samples were analyzed by electrophoresis and fluorography. Abbreviations: i , interraediate; m, mature cytochrome bv>.

electrophoresis and fluorography. A fluorograph of the dried gel i s shown i n Fig.1. Control samples received an apyrase preparation which had been inactivated by heating to 95°C for 10 min. In these reactions, cytochrome b"2 precursor was imported into a protease protected location and processed to the mature form. Besides mature cytochrome b^, a small quantity of the intermediate sized form (about 20% of to t a l ) was observed which was also protected against externally added protease (Fig.1, lanes 2 and O • When lysate and mitochondria had been pretreated with apyrase, import was cl e a r l y diminished (Fig.1, lanes 1 and 3)» Apyrase per se did reduce neither the protease resistance of endogenous mitochondrial proteins nor the amount of precursor proteins i n the reticulocyte lysate (Pfanner and Neupert, 1986; Pfanner et a l . , 1987). We conclude that pretreatment with apyrase causes i n h i b i t i o n of import of cytochrome b^ into mitochondria.

191

To test whether the apyrase effect was due to depletion of NTPs, experiments were performed where ATP or GTP were included during import (Fig.2).

— I

m

Apyrase + + + + + + - + + + -Av

+ + + + + + + + -ATP G1P AIP GTP ATP

Fig. 2. Import of cytochrome from apyrase treated r e t i c u l o c y t e lysate i s restored by addition of ATP or GTP. Reticulocyte lysates and mitochondria were incubated with the following concentrations of apyrase: reactions 1- 3, U U/ml; reactions 5-7, 1 U/ml; reaction U received U U inactivated apyrase/ral and reactions 8 and 9 1 U/ml. Import and protease treatment was performed as described i n legend to Fig.1, except that i n reactions 2,6 and 9, 8 mM ATP and i n reactions 3 and 7, 8 mM GTP were added during import. In reaction 9, 1 nM valinomycin was added prior to ATP. A fluorograph of the dried gel i s shown. Abbreviations as i n Fig.1. p, precursor of cytochrome b^.

Under these conditions the import of cytochrome b^ could be f u l l y restored (Fig.2, lanes 2,3 and 6,7). This import, however, was completely abolished when valinomycin plus potassium ions were added to destroy the membrane potential across the inner membrane (Fig.2, lane 9). In t h i s case, addition of ATP did not restore import.

The same re s u l t was obtained with import of cytochrome c 1 into mitochondria of N. crassa. Experimental conditions were e s s e n i i a l l y as described for import of cytochrome b^, except that ascorbate/TMPD was used to establish a membrane potential and NADH was included, which i s required for the second maturation step of cytochrome c^ (Schleyer and Neupert, 1985| see accompanying a r t i c l e by Nicholson et a l . ) . A fluorograph corresponding to the experimental design presented i n Fig.2 was quantified by densitometry (Fig.3).

Again apyrase treatment d r a s t i c a l l y reduced import and processing (Fig.3, lanes 1,5) which could be restored by addition of ATP or GTP (Fig.3, lanes 2,3,6,7). Import was completely blocked a f t e r i n h i b i t i o n of the membrane potential with antimycin A and oligomycin and could not be restored by including ATP i n the reaction (Fig.3, lane 9). Consequently, the presence of NTPs and a membrane potential are two separate requirements. NTPs cannot S u b s t i t u t e for the requirement ofZVY» At present,

192

however, i t i s unknown which form of high energy phosphate i s actually needed since nucleoside phosphate kinases present i n mitochondria can lead to formation of various nucleoside triphosphates.

150-,

=100-

S ° 50-

0J

Apyrase

1 u I

1 2 3 4 5 6 7 8 9

ATP GTP ATP GTP ATP

F i g . 3. Import of cytochrome c. i s i n h i b i t e d by apyrase treatment and restored by ATP or GTP. The experimental design was es s e n t i a l l y as described i n the legend to Fig.2, except that mitochondria from N. crassa were used and import was performed i n the presence of ascorbate/TMPD and NADH instead of succinate. Antimycin A was included i n addition to oligomycin. Formation of mature cytochrome c^ was quantified by densitometry of the fluorograph. The amount of mature c. formed i n controls (inactivated apyrase) was set at 100%.

Intermediate-sized cytochrome spanning outer and inner membranes at contact sites can be accumulated at low levels of NTPs

For precursors of ß subunit of F F^-ATPase, cytochrome c^ and the Fe/S-protein of complex I I I , i t has been previously shown that import into mitochondria performed at lower temperatures results i n the formation of so called "contact s i t e intermediates" (Schleyer and Neupert, 1985; Hartl et a l . , 1986). Such an intermediate i s characterized as follows: the N-terminal presequence of the precursor has been translocated across both membranes i n aAydependent manner and i s cleaved off by the processing peptidase i n the raatrix. A large part of the Polypeptide, however,is s t i l l outside the mitochondrion where i t can be digested by externally added protease. I t follows that these features can be only f u l f i l l e d at regions where outer and inner membranes are close enough together to be spanned by a S i n g l e Polypeptide chain.

Experiments with apyrase pretreatment indicated that af t e r depletion of ATP the formation of intermediate-sized cytochrome b^ was not reduced to the same extent as was processing to the mature form. In contrast to mature b^, more than 85% of which were protease-resistent, t h i s intermediate was largel y sensitive to externally added protease. This effect could be cl e a r l y demonstrated when apyrase concentrations were t i t r a t e d over a wider ränge (0 to 100 U apyrase/ml of lysate or mitochondrial Suspension). After import, one half of each reaction was treated with Proteinase K. Following electrophoresis and fluorography the

193

amounts of intermediate-sized b p formed were quantified by densitometry (Fig. 4).

Ol l 10 20~"loO Apyrase [U/ml]

Fig 4» Intermediate sized cytochrome b^ i s accumulated i n contact si t e s at low levels of NTPs. Reticulocyte lysates and mitochondria were incubated with apyrase (0-100 U/ml). Controls received corresponding amounts of inactivated apyrase. Import and protease treatment were performed as described i n the legend to Fig.1, except that only h a l f of each sample received protease. Formation of intermediate cytochrome b^ was quantified by densitometry of the fluorograph. The amount of intermediate b^ formed i n controls (inactivated apyrase) was set at 100%.

At apyrase concentrations between U and 20 U/ml, the formation of intermediate b^ was reduced by 10 to 4-0% compared to controls that had received inactivated apyrase. Most of t h i s intermediate sized b p, however, was digested by externally added Proteinase K thus f u l f i l l i n g the c r i t e r i a for a contact s i t e intermediate described above. Only with very low concentrations of apyrase was intermediate-sized b^ protected against externally added protease. Within a narrow ränge of 1 to U U apyrase/ml, protease protection decreased from 95 to 30% i n d i c a t i n g that only above a d i s t i n c t l e v e l of NTPs import into protease protected Position did take place. When the membrane potential was dissipated, no processing to intermediate b^ was observed. We conclude that p a r t i a l translocation of the precursor can occur at low levels of NTPs; t h i s step depends on the potential across the inner membrane. For complete translocation, however, higher levels of NTPs are required.

DISCUSSION

The import of precursors of cytochromes b^ and c^ into mitochondria needs NTPs i n addition to &Y« At low levels of NTPs, a translocational intermediate of cytochrome b^ could be accumulated i n contact s i t e s .

Owing to the presence of nucleoside phosphate kinases i t i s unclear so far which form of high energy phosphate Compound (eg. ATP or GTP) i s the

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d i r e c t energy source. Non-hydrolyzable ATP-analogues could not restore import after depletion of NTPs with apyrase (Pfanner and Neupert, 1986) indicating'that the mechanism of action of NTPs involves the hydrolysis of high energy phosphate bonds. The dependence of protein import into mitochondria on NTPs seems to be a general phenomenon. Similar effects have been recently observed with the precursors of ß subunit of F F 1-ATPase (Pfanner and Neupert, 1986) and the ADP/ATP-transloca?or (Pfanner et a l . , 1987). Interestingly, the i n s e r t i o n of porin into the outer membrane (which i s independent o f & Y ) also seems to need NTPs (Kleene et a l * , i n preparation).

What could be the role of NTPs? Previous results had already suggested that NTPs modify the conformation of cyto s o l i c mitochondrial precursor proteins (Pfanner et a l . , 1987). E i l e r s and Schatz (1986) have demonstrated that lack of t e r t i a r y structure i s a prerequisite for protein import into mitochondria. Taking the resistance against digestion by protease as a measure for the degree of t e r t i a r y structure i t could be shown that the presence of NTPs results i n the unfolding of precursor molecules (Pfanner et a l . , 1987) thus rendering them competent for translocation across the mitochondrial membranes. In the present study,AYdependent p a r t i a l translocation of the precursor of cytochrome b^ was possible at very low concentrations of NTPs res u l t i n g i n an intermediate reaching into the matrix with i t s aminoterminus but having a large part of the molecule s t i l l outside the mitochondrion. Complete translocation into a protease protected Position was then achieved by adding ATP or GTP and was independent of (Hartl and Neupert, unpublished). These findings are consistent with the idea that the membrane potential i s only necessary for the translocation of the aminoterminal part of the precursor across the inner membrane. I t i s assumed to exert an electrophoretic effect on the positive charges contained i n the presequence (Pfanner and Neupert, 198$; Roise et a l . , 1986). Complete translocation of the precursor across both membranes i s only possible when the Polypeptide i s kept i n an unfolded state, the energy source for the unfolding reaction being NTPs. Specific binding of the precursor to receptors on the surface of mitochondria, i n s e r t i o n into and p a r t i a l translocation across the mitochondrial membranes i s possible at very low levels of NTPs; thus, i t seems l i k e l y that the presequence folds independently of the mature part of the precursor and can be recognized by the mitochondrial import machinery. On the other hand i t cannot be excluded that besides the unfolding of cytosolic precursors the role of NTPs includes functions such as the modification of mitochondrial membranes e.g. by phosphorylation of mitochondrial transport coraponents.

The existence of "unfolding proteins" i n the cytosol has been proposed (Rothman and Kornberg, 1986; Zimmermann and Meyer, 1986) that could bind to precursor proteins and whose action would involve the hydrolysis of high energy phosphate bonds. The role of NTPs i n preventing (mis)folding of precursor proteins into an import incompetent conformation could explain the general importance of NTPs for the translocation of proteins across membranes i n both procaryotic and eucaryotic Systems (for review see Zimmermann and Meyer, 1986).

ACKNOWLEDGEMENTS The authors wish to thank S. Meier for expert technical assistance and

Dr. D. Nicholson for c r i t i c a l l y reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 184, B2).

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