Molecular Dynamics of the 32,000-Dalton Photosystem II Herbicide

Chapter 1 8

Molecular

Dynamics

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Photosystem

of

II

the

32,000-Dalton

Herbicide-Binding

1

Protein

1

2,3

Autar K . Mattoo , Franklin E . Callahan , Bruce M. Greenberg , Pierre Goloubinoff , and Marvin Edelman 2,4

1,2

1

2

Plant Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, M D 20705 Plant Genetics Department, The Weizmann Institute of Science, Rehovot, 76-100, Israel The ubiquitous 32kDa thylakoid protein, the psbA gene product, i s a major product of the p l a s t i d t r a n s l a t i o n a l machinery and an i n t e g r a l component of the photosystem I I reaction center. The pro t e i n i s also the target s i t e for t r i a z i n e and urea -type herbicides. It i s first synthesized as a precursor on stromal lamellae where it is processed to the mature 32kDa form. Following processing, the 32kDa protein i s translocated to the t o p o l o g i c a l l y d i s t i n c t granal lamellae. Sometime during its translocation and/or integration within the granal photosystem II, the protein undergoes covalent modification with palmitic a c i d . This modification occurs i n the amino-terminal h a l f of the protein and i s i n h i b i t e d by atrazine and diuron h e r b i c i d e s . L i g h t - i n t e n s i t y dependent degradation of the pro t e i n occurs after i t s assembly i n the granal lamel l a e , and r e s u l t s i n a membrane-associated 23.5kDa product derived from the amino terminal two-thirds of the parent protein. The cleavage s i t e i s local ized to a functionally active and phylogenetically conserved region between amino acid residues 238 and 248. Contiguous with the proposed cleavage domain i s an α-helix-destabilizing region, border ed by arginine residues 225 and 238. This region, c h a r a c t e r i s t i c of r a p i d l y catabolized proteins, seems to have evolved along with oxygenic photo system II, since it is absent from the analogous protein i n the non-oxygenic photosynthetic b a c t e r i a .

3

Current address: Department of Biology, University of Waterloo, Ontario N2L 3G1, Canada Current address: Central R&D Department, E. I. du Pont de Nemours and Company, Wilmington, DE 19898

4

c

0097-6156/88/0379-0248$06.00/0 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

18.

MATTOO ET AL.

Dynamics ofHerbicide-Binding Protein

249

C h l o r o p l a s t s encode one o f the most s t u d i e d o f p l a n t gene p r o d u c t s , a unique p r o t e i n w i t h an apparent m o l e c u l a r weight o f 32,000 on SDSpolyacrylamide g e l s . The 32kDa p r o t e i n has a c o n s e r v e d p r i m a r y s t r u c t u r e (1) i n the algae and h i g h e r p l a n t s t e s t e d ( 2 ) . The r e c o g n i t i o n o f t h i s p l a s t i d p r o t e i n as a monogenic t a r g e t f o r the most commonly used photosystem I I h e r b i c i d e s , a t r a z i n e (3) and d i u r o n ( 4 ) , and as one o f the most r a p i d l y t u r n i n g over p l a n t p r o t e i n s (5) has generated c o n s i d e r a b l e i n t e r e s t among m o l e c u l a r b i o l o g i s t s and b i o t e c h n o l o g i s t s because o f the p o t e n t i a l f o r g e n e t i c e n g i n e e r i n g . In a d d i t i o n , the r e c e n t d e m o n s t r a t i o n o f the 32kDa p r o t e i n as an e s s e n t i a l component o f the h i g h e r p l a n t photosystem I I r e a c t i o n c e n t e r ( 6 , 7 ) has added a new d i m e n s i o n t o t h i s i n t e r e s t w i t h s t i l l b r o a d e r i m p l i c a t i o n s f o r c r o p improvement i n a g r i c u l t u r e . B i o s y n t h e s i s , T r a n s l o c a t i o n and P o s t t r a n s l a t i o n a l M o d i f i c a t i o n s S y n t h e s i s . The 32kDa p r o t e i n i s coded f o r by the psbA gene l o c a t e d i n the p l a s t i d genome ( 8 ) . The psbA gene has been sequenced from s e v e r a l h i g h e r p l a n t c h l o r o p l a s t s , a l g a e and c y a n o b a c t e r i a and, l i k e i t s p r o t e i n p r o d u c t ( 2 ) , i s h i g h l y c o n s e r v e d ( F i g . 1 ) . The c o d i n g o f the h i g h e r p l a n t psbA gene sequence p r e d i c t s a h y d r o p h o b i c p r o t e i n w i t h 353 amino a c i d r e s i d u e s and a m o l e c u l a r weight o f about 39,000 (8). The p r o t e i n i s , i n f a c t , s y n t h e s i z e d on membrane-bound ribosomes ( 9 ) . I t i s f i r s t s y n t h e s i z e d as a p r e c u r s o r and has been i d e n t i f i e d as such in v i t r o , i n o r g a n e l l o and j l n v i v o . Processing of the p r e c u r s o r to the mature 32kDa form was shown in v i v o and i n o r g a n e l l o by p u l s e - c h a s e experiments ( 1 2 ) . The apparent s i z e o f the p r e c u r s o r i s about 3 3 . 5 - 3 4 . 5 k D a on SDSp o l y a c r y l a m i d e g e l s which i s somewhat d i f f e r e n t from the m o l e c u l a r weight p r e d i c t e d from the deduced open r e a d i n g frame. This d i s c r e p a n c y c o u l d r e s u l t i f the p r o t e i n behaved i n an anomalous manner on SDS-PAGE. A l t e r n a t i v e l y , i t was suggested t h a t t r a n s l a t i o n o f 32kDa mRNA i n i t i a t e s a t the second m e t h i o n i n e i n the open r e a d i n g frame at p o s i t i o n 37 (met37) i n s t e a d o f the one at p o s i t i o n 1 ( m e t l ) . T h i s would r e s u l t i n a p r o t e i n w i t h a m o l e c u l a r weight o f 34.5kDa (15-17). The l a t t e r p o s s i b i l i t y has been e x p e r i m e n t a l l y d i s c o u n t e d by a r e c e n t study t h a t used f u l l l e n g t h , t r u n c a t e d and mutated c o n s t r u c t s o f psbA gene from Solanum n i g r u m as t e m p l a t e s i n an i n v i t r o t r a n s c r i p t i o n / t r a n s l a t i o n system ( 1 8 ) . I t was found t h a t when the met37 codon, ATG, was mutagenized t o AAG (which codes f o r l y s i n e ) o r t o AGG ( w h i c h codes f o r a r g i n i n e ) , w h i l e s t i l l r e t a i n i n g the f i r s t ATG codon ( m e t l ) , the t r a n s l a t i o n product had the same m o b i l i t y on SDS-PAGE as the 33.5kDa p r e c u r s o r p r o t e i n . On the o t h e r hand, d e l e t i o n at the 5 ' - e n d o f the gene to remove the codon f o r m e t l w i t h o u t a f f e c t i n g the met37 codon produced a t r u n c a t e d , 29kDa protein. Thus, t r a n s l a t i o n o f mRNA f o r the 32kDa p r o t e i n i n i t i a t e s a t m e t l . Recent i m m u n o l o g i c a l e v i d e n c e showing m e t l and met37 as p a r t o f the mature p r o t e i n ( 1 9 ) , and l o c a l i z a t i o n o f a p o s t t r a n s l a t i o n a l p h o s p h o r y l a t i o n at p o s i t i o n 2 o f the open r e a d i n g frame w i t h the g e n e r a t i o n o f a c e t y l - 0 - p h o s p h o t h r e o n i n e as the N - t e r m i n a l r e s i d u e i n the mature 32kDa p r o t e i n ( 2 0 ) , support the c o n t e n t i o n t h a t i n i t i a t i o n o f t r a n s l a t i o n o c c u r s at m e t l . I t would seem then t h a t , as i s the case w i t h o t h e r h y d r o p h o b i c p h o t o s y n t h e t i c membrane p r o t e i n s ( 3 0 ) , the 32kDa p r o t e i n behaves anomalously on SDS-PAGE.


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Figure 1 Comparison o f deduced amino a c i d sequences from psbA g e n e s . Note the c o n s e r v a t i o n o f the P E S T - l i k e r e g i o n c o n t i g u o u s w i t h t h e a - h e l i x d e s t a b i l i z i n g , p r i m a r y c l e a v a g e domain i n t h e 32kDa p r o t e i n . The arrow i n d i c a t e s t h e p r o b a b l e s i t e where t h e p r e c u r s o r p i e c e on t h e c a r b o x y - t e r m i n u s i s c l e a v e d ( 2 2 ) . psbA sequences were c o m p i l e d as f o l l o w s : ( 1 ) A n a c y s t i s n i d u l a n s copy I , and (2) copy I I / I I I ( 4 1 ) . (3) Anabaena 7120 copy I , and ( 4 ) Anabaena 7120 copy I I ( 4 2 ) . ( 5 ) F r e m y e l l a d i p l o s i p h o n ( 4 3 ) . ( 6 ) E u g l e n a g r a c i l i s ( 4 4 ) . (7) Chlamydomonas r e i n h a r d t i i ( 4 5 ) . ( 8 ) M a r c h a n t i a polymorpha ( 4 6 ) . ( 9 ) Zea mays ( 4 7 ) . ( 1 0 ) S i n a p i s a l b a ( 4 8 ) . ( 1 1 ) B r a s s i c a napus T 4 9 ) . (12) Medico s a t i v a (50T. ( l 3 ) Pisum s a t i v u m ( 5 1 ) . ( 1 4 ) G l y c i n e max ( 5 2 ) . (15) P e t u n i a h y b r i d a ( 5 ^ . (16) Amaranthus h y b r i d u s ( 1 5 ) . TT7) Solanum n i g r u m i s t h e r e f e r e n c e sequence ( 5 4 ; and i s i d e n t i c a l t o t h e S p i n a c i a o l e r a c e a , N i c o t i a n a d e b n e y i (8) and N i c o t i a n a tabacum (55) psbA sequences. (Adapted from r e f . 5 6 ) .


18.

MATTOOETAL.


251

P r e c u r s o r P r o c e s s i n g . The p r e c u r s o r p r o t e i n i s s y n t h e s i z e d on s t r o m a l l a m e l l a e and i s processed t h e r e t o the 32kDa mature form (21). P r o c e s s i n g o f the p r e c u r s o r i s a p o s t t r a n s l a t i o n a l event (12) and t a k e s p l a c e at the c a r b o x y t e r m i n u s ( 2 2 ) . Following p r o c e s s i n g , the mature p r o t e i n t r a n s l o c a t e s t o s p a t i a l l y - d i s t i n c t c h l o r o p l a s t membranes, the g r a n a , where f u n c t i o n a l photosystem I I r e a c t i o n centers are mainly l o c a t e d (21, 2 3 ) . The mechanism o f t r a n s l o c a t i o n i s not u n d e r s t o o d . Any mechanism put forward t o e x p l a i n the l a t e r a l d i f f u s i o n o f the p r o t e i n has to r e c o n c i l e the f a c t t h a t the 32kDa p r o t e i n on s t r o m a l l a m e l l a e i s i n t e g r a t e d i n an o r i e n t a t i o n t h a t seems s i m i l a r t o the p r o t e i n i n t e g r a t e d w i t h i n the g r a n a l l a m e l l a e . T h i s i s shown by experiments i n v o l v i n g t r y p s i n i z a t i o n o f the two membrane types f o l l o w i n g t h e i r i s o l a t i o n from p u l s e - l a b e l e d S p i r o d e l a p l a n t s . P r e v i o u s l y i t was e s t a b l i s h e d t h a t the membrane-bound 32kDa p r o t e i n y i e l d s , upon t r y p s i n d i g e s t i o n , two membrane-associated fragments o f 22kDa and 20kDa on SDS-PAGE ( 2 4 ) . U s i n g t h i s t e c h n i q u e , we have found i d e n t i c a l i n s i t u t r y p s i n i z a t i o n p a t t e r n s o f the s t r o m a l l a m e l l a e v e r s u s g r a n a l - a s s o c i a t e d 32kDa p r o t e i n ( F i g . 2 ) . M o r e o v e r , even i n the p r e c u r s o r s t a t e , the s t r o m a l - l a m e l l a e a s s o c i a t e d p r o t e i n shows s i m i l a r t r y p s i n - s e n s i t i v e domains ( P . Goloubinoff, unpublished). Thus, t h e s e d a t a r a i s e the p o s s i b i l i t y t h a t membrane i n t e g r a t i o n and o r i e n t a t i o n are independent o f p r o c e s s i n g and t r a n s l o c a t i o n o f the 32kDa p r o t e i n . Membrane I n t e g r a t i o n . What i s the d r i v i n g f o r c e b e h i n d the i n t e g r a t i o n process? An a n a l y s i s o f s e v e r a l decoded amino a c i d sequences o f the p r o t e i n shows c o n s i d e r a b l e v a r i a b i l i t y i n the carboxy-terminal region. Notwithstanding t h i s v a r i a b i l i t y , a n e g a t i v e charge o f a t l e a s t 1 i s m a i n t a i n e d i n the p r e c u r s o r p i e c e o f the s p e c i e s examined. Asp342 i s c o n s e r v e d i n a l l t h e s e c a s e s w h i l e a h i g h frequency o f g l u o r asp a t p o s i t i o n 347 i s observed (25). The i n t e g r a t i o n event c o u l d be d r i v e n by the n e g a t i v e charge on the p r e c u r s o r p i e c e o f the 33.5kDa p r o t e i n . Such a model f o r i n t e g r a t i o n o f the mature 32kDa p r o t e i n w i t h i n s t r o m a l l a m e l l a e i s c o n s i s t e n t w i t h the membrane t r i g g e r h y p o t h e s i s ( 2 6 ) , i n the p r e s e n t c a s e , b e i n g a p p l i e d t o a p r o t e i n t h a t undergoes p r o c e s s i n g at the c a r b o x y t e r m i n u s . The i n t e r a c t i o n o f the p r o t e i n w i t h s t r o m a l membrane l i p i d s c o u l d p o s s i b l y h e l p i n t h i s i n t e g r a t i o n process. In t h i s c o n t e x t , i t i s w o r t h n o t i n g t h a t s t r o m a l membranes have a d i f f e r e n t complement o f g l y c o l i p i d m o l e c u l a r s p e c i e s as compared t o t h a t found i n the g r a n a l membranes ( 2 7 ) . Whether t h i s d i f f e r e n c e i s f a v o r a b l e f o r i n t e g r a t i o n o f the 32kDa p r o t e i n i n s t r o m a l membranes i s not known. P o s s i b l e Signals for T r a n s l o c a t i o n . The q u e s t i o n a r i s e s : What s i g n a l s e n a b l e s t r o m a l 32kDa p r o t e i n t o move to the g r a n a l l a m e l l a e ? At 30 /xmole.m"^.s"^ o f w h i t e l i g h t , the half l i f e o f the n e w l y - s y n t h e s i z e d 32kDa p r o t e i n on s t r o m a l membranes i s between 9-18 min w h i l e the newly t r a n s l o c a t e d g r a n a l 32kDa p r o t e i n has a h a l f l i f e o f 6-12 h o u r s ( 2 1 , 2 8 ) . T h i s d i f f e r e n c e i n the l i f e t i m e s o f i t s presence i n s t r o m a l v e r s u s g r a n a l membranes i s p a r t i a l l y r e f l e c t e d i n western b l o t a n a l y s i s o f steady s t a t e l e v e l s o f p r o t e i n at t h e s e two d i s t i n c t l o c a t i o n s , v i z . , the amount o f s t r o m a l 32kDa p r o t e i n i s o n e - f i f t h t o o n e - t e n t h o f t h a t found i n


252


the g r a n a l f r a c t i o n . In a d d i t i o n to i t s m o d i f i c a t i o n by phosphor y l a t i o n ( d i s c u s s e d a b o v e ) , the 32kDa p r o t e i n a l s o undergoes another p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n , v i z . , w i t h p a l m i t i c a c i d ( 2 1 , 2 8 ) . The a c y l l i n k a g e i s r e s i s t a n t t o a c i d i c h y d r o x y l a m i n e and t o c o n d i t i o n s o f d e n a t u r a t i o n , e l e c t r o p h o r e s i s and f i x a t i o n o f the p r o t e i n on S D S - p o l y a c r y l a m i d e g e l s , s u g g e s t i n g an e t h e r or amide linkage. The p a l m i t o y l a t i o n o f the p r o t e i n i s l i g h t s t i m u l a t e d and r a p i d , b e i n g observed w i t h i n 1 min o f r a d i o l a b e l i n g o f S p i r o d e l a plants with ^H-palmitic a c i d . Both a t r a z i n e and d i u r o n , t h a t b i n d the 32kDa p r o t e i n , i n h i b i t a c y l a t i o n o f the p r o t e i n ( 2 9 ) . These d a t a suggest t h a t such h e r b i c i d e s might compete f o r s i t e s on the p r o t e i n t h a t are p a l m i t o y l a t e d , o r cause a c o n f o r m a t i o n a l change i n the p r o t e i n such t h a t a c y l a t i o n s i t e s a r e no more a c c e s s i b l e t o the enzyme t h a t l i g a t e s p a l m i t i c a c i d t o the p r o t e i n . L i g h t , d i u r o n o r h e a t a r e known to cause c o n f o r m a t i o n a l changes i n the p r o t e i n p a r t i c u l a r l y i n the r e g i o n bounded by T22 and T20 ( 4 , 30). An a c y l a t i o n s i t e appears t o be a s s o c i a t e d w i t h the c h a r a c t e r i s t i c , t r y p s i n - d e r i v e d membrane-associated p r o t e i n fragments, T22 and T20 as w e l l ( 2 1 ) . Furthermore, a c y l a t i o n i s r e s t r i c t e d to the 32kDa p r o t e i n a s s o c i a t e d w i t h the g r a n a l lamellae. S p e c u l a t i o n s on the r o l e o f p a l m i t o y l a t i o n i n the dynamics o f the 32kDa p r o t e i n i n c l u d e : 1. Promotes proper f u n c t i o n a l i n t e g r a t i o n w i t h i n the g r a n a l photosystem I I r e a c t i o n c e n t e r ; and 2 . a c t s as a r a p i d t r a n s l o c a t i o n s i g n a l t h a t d e s t a b i l i z e s the p r o t e i n on s t r o m a l l a m e l l a e a l l o w i n g i t s l a t e r a l d i f f u s i o n to grana. Degradation Once the 32kDa p r o t e i n i s t r a n s l o c a t e d t o the g r a n a l l a m e l l a e i t undergoes a l i g h t - d e p e n d e n t d e g r a d a t i o n . I t s h a l f l i f e i n the g r a n a i s dependent on the l i g h t i n t e n s i t y a t which the p l a n t i s c u l t i v a t e d , w h i l e the p r o t e i n i s s t a b l e i n the d a r k ( 3 1 ) . D e g r a d a t i o n o c c u r s i n v i s i b l e l i g h t , where b o t h photosystem I and I I are f u n c t i o n a l as w e l l as i n f a r r e d l i g h t , where photosystem I i s predominantly a c t i v e ( 3 2 ) . Both a t r a z i n e and u r e a - t y p e h e r b i c i d e s b i n d to the p r o t e i n and c h a r a c t e r i s t i c a l l y i n h i b i t i t s d e g r a d a t i o n ( 3 1 , 3 2 ) . The l i g h t - i n t e n s i t y dependent d e g r a d a t i o n o f the 32kDa p r o t e i n r e s u l t s i n the p r o d u c t i o n o f a membrane-associated fragment o f 23.5kDa ( 3 3 ) . The p r i m a r y c l e a v a g e o c c u r s about 2kDa c a r b o x y - t e r m i n a l to a r g 2 2 5 , somewhere between arg238 and i l e 2 4 8 ( F i g . 1 ) . T h i s domain o f the 32kDa p r o t e i n i s l o c a l i z e d i n the f u n c t i o n a l l y - a c t i v e h y d r o p h i l i c l o o p between h e l i c e s IV and V ( 3 3 ) , i s r i c h i n glutamate r e s i d u e s , and has been conserved p h y l o g e n e t i c a l l y ( F i g . 1). I t i s the r e g i o n o f the p r o t e i n where quinone and h e r b i c i d e s a r e p o s t u l a t e d to b i n d and where s i n g l e amino a c i d changes t h a t are a l t e r e d i n h e r b i c i d e mutants o f h i g h e r p l a n t s and a l g a e e x i s t . I n t e r e s t i n g l y , adjacent to t h i s proposed s i t e o f p r i m a r y c l e a v a g e i s a 14 amino a c i d sequence b o r d e r e d by arg225 and arg238 and r i c h i n g l u t a m a t e , s e r i n e and t h r e o n i n e r e s i d u e s ( F i g s . 1 and 3 ) . T h i s r e g i o n resembles s i m i l a r - t y p e r e g i o n s c a l l e d ' P E S T (showing preponderance o f p r o l i n e - P , g l u t a m a t e - E , s e r i n e - S and t h r e o n i n e - T r e s i d u e s and bounded by p o s i t i v e l y charged amino a c i d s ) t h a t a r e commonly found i n r a p i d l y degraded p r o t e i n s ( 3 4 ) . PEST r e g i o n s are suggested t o 1


18.

MATTOOETAL.


253

Figure 2 In s i t u s t r y p s i n d i g e s t i o n p a t t e r n s o f 3 5 s - t h i o n i n e - l a b e l e d s t r o m a l and g r a n a l membranes. S p i r o d e l a p l a n t s were p u l s e l a b e l e d i n the l i g h t w i t h [ ^ ^ s j m e t h i o n i n e f o r 3 m i n , washed w i t h n o n - r a d i o a c t i v e growth medium and i n c u b a t e d f u r t h e r f o r 20 min w i t h n o n - r a d i o a c t i v e m e t h i o n i n e (ImM). The p l a n t s were h a r v e s t e d , homogenized and whole t h y l a k o i d s i s o l a t e d . F r a c t i o n a t i o n o f thylakoids into stromal (SL) and g r a n a l (GL) l a m e l l a e was a c h i e v e d by the method o f L e t o et a l . (37). Each membrane sample was d i g e s t e d w i t h ( + ) or w i t h o u t ( - T ~ t r y p s i n (24) and then prepared f o r SDS-PAGE. The p o s i t i o n s o f u n d i g e s t e d 32kDa p r o t e i n [p32] and t r y p s i n fragments [T22 and T20] are i n d i c a t e d . m e

Lc

P F H M L G I S L F FTTAWALpflFR3ALVLS

32kDa

EEHMLaVAQVFGGSLFSiAMIJQbLVTSSLIRETTENESANEGYRFGQEEET

AANPVKGKTMRTPDHEDT

PEST region

Cleavage domain

Figure 3 Comparison o f a l i g n e d sequences i n the P E S T - l i k e r e g i o n o f the 32kDa photosystem I I r e a c t i o n c e n t e r p r o t e i n w i t h t h o s e o f L s u b u n i t o f Khodopseudomonas c a p s u l a t a ( L c ) . Adapted from r e f . 3 8 .


254


Figure 4 D e g r a d a t i o n o f the 32kDa p r o t e i n i n i s o l a t e d S p i r o d e l a membranes. S p i r o d e l a p l a n t s were p u l s e l a b e l e d w i t h [^S]-methionine for 2 h and homogenized w i t h STN/lOmM Mg b u f f e r ( 4 ) . Membranes were c e n t r i f u g e d , washed w i t h lOmM t r i s - g l y c i n e , pH 8 . 5 , c o n t a i n i n g lOmM M g C l and suspended i n STN/lOmM Mg b u f f e r . I n c u b a t i o n s were c a r r i e d out i n the d a r k (D) o r a t 50/iM DCMU o r 50piM a t r a z i n e (AT) f o r the t i m e s i n d i c a t e d . Samples were then s u b j e c t e d t o SDS-PAGE. The p o s i t i o n s o f 32kDa p r o t e i n and the l i g h t h a r v e s t i n g c h l o r o p h y l l a/b a p o p r o t e i n (LHCP) are i n d i c a t e d . 2


18. MATTOOETAL.


255

function as primary determinants for degradation of the proteins harboring them. In the chloroplast genome, which has been fully sequenced (40), a PEST-like region is found only in the 32kDa protein (33). The close proximity of the proposed cleavage site with the PEST-like region in the 32kDa protein suggests that the latter may be involved in regulating the degradation of the 32kDa protein. The PEST-like region is absent from the analogous protein from the nonoxygenic bacterial photosynthetic reaction center (35) v i z . , the L subunit (Fig. 3). In contrast to the 32kDa protein which is rapidly degraded, the L subunit is relatively stable. This raises the possibility of a linkage between evolution of oxygenic photosystem II and the introduction of the PEST-like region in the 32kDa protein. Genetic or engineered alteration of the site of cleavage and the PEST-like region in the 32kDa protein should prove valuable in studying the structure-function relationships of this reaction center component. Mimicking In Vivo Degradation in Isolated Thylakoids An in vitro system is being developed to study degradation of the 32kDa protein in isolated thylakoids. The requirements for a successful in vitro system include: 1. the protein should be degraded without affecting other membrane components; 2. protein degradation should be light dependent; and 3. herbicides (atrazine and diuron) that bind to the 32kDa protein should inhibit the degradation. Results in Fig. 4 demonstrate degradation of the 32kDa protein in isolated thylakoids. The results meet the three c r i t e r i a mentioned above. The detection of the 23.5kDa product in this system is s t i l l under investigation. From the steady state ratio of 23.5kDa product to 32kDa protein found in vivo, the rate of 23.5kDa product formation was estimated to be 4-fold higher than the parent protein (33). However, since protein synthesis and reutilization of radioactivity do not occur ^in vitro during the chase (as they do in vivo), the absence of the 23.5kDa product in this system is not really remarkable. The data in F i g . 4 further suggest that the activity responsible for the degradation of the 32kDa protein is located in the thylakoids (36). Thus, this model system should now be useful to test inhibitors, activators, etc. on their effects on the 32kDa protein degradation and obtain a handle on the kind of protease or autocatalytic mechanism that leads to degradation. Acknowledgment s We thank Sudhir Sopory for comments on the manuscript. Results of experiments presented here were supported in part by a United States-Israel Binational Agricultural Research and Development (BARD) grant to AKM and ME. Literature Cited 1. Whitfeld, P.R.; Bottomley, W. Ann. Rev. Plant Physiol. 1983, 34, 279-310. 2. Hoffman-Falk, H . ; Mattoo, A.K.; Marder, J.B.; Edelman, M.; Ellis, R.J. J. B i o l . Chem. 1982, 257, 4583-7.


256


3. Steinback, K . E . ; McIntosh, L.; Bogord, L.; Arntzen, C.J. Proc. Natl. Acad. S c i . USA 1981, 78, 7463-7. 4. Mattoo, A.K.; Pick, U . ; Hoffman-Falk, H . ; Edelman, M. Proc. Natl. Acad. S c i . USA 1981, 78, 1572-6. 5. Mattoo, A.K.; Edelman, M. In Frontiers of Membrane Research in Agriculture; St. John, J . B . ; Berlin, E . ; Jackson, P.C., Eds.; Rowman and Allanheld: New Jersey, 1985; pp 23-34. 6. Nanba, O.; Satoh, K. Proc. Natl. Acad. S c i . USA 1987, 84, 109-12. 7. Marder, J.B.; Chapman, D. J.; Telfer, A.; Nixon, P.J.; Barber, J . Plant Mol. B i o l . 1987, 9, 325-33. 8. Zurawski, G.; Bohnert, H.-J.; Whitfeld, P.R.; Bottomley, W. Proc. Natl. Acad. S c i . USA 1982, 79, 7699-703. 9. Ellis, R.J. Biochim. Biophys. Acta. 1977, 463, 185-215. 10. Minami, E. -I.; Watanabe, A. Arch. Biochem. Biophys. 1984, 235, 562-70. 11. Herrin, D.; Michaelis, A. FEBS Lett. 1985, 184, 90-95. 12. Reisfeld, A . ; Mattoo, A.K.; Edelman, M. Eur. J. Biochem. 1982, 124, 125-129. 13. Ellis, R. J . Ann. Rev. Plant Physiol. 1981, 32, 117-37. 14. Grebanier, A . E . ; Coen, D.M.; Rich, A . ; Bogorad, L. J. Cell B i o l . 1978, 78, 734-46. 15. Hirschberg, J.; McIntosh, J . Science 1983, 222, 1346-9. 16. Hirschberg, J.; Bleecker, A.; Kyle, D.J.; McIntosh, L.; Arntzen, C.J. Z. Naturforsch. 1984, 39C, 412-20. 17. Cohen, B.N.; Coleman, T.A.; Schmitt, J.J.; Weissbach, H. Nucl. Acids Res 1984, 12, 6221-30. 18. Eyal, Y.; Goloubinoff, P.; Edelman, M. Plant Mol. B i o l . 1987, 8, 337-43. 19. Sayre, R.T.; Andersson, B . ; Bogorad, L. Cell 1986, 47, 601-8. 20. Michel, H . ; Hunt, D.F.; Shabanowitz, J.; Bennett, J . J. B i o l . Chem. 1988, 263, 1123-30. 21. Mattoo, A.K.; Edelman, M. Proc. Natl. Acad. S c i . USA 1987, 84, 1497-1501. 22. Marder, J.B.; Goloubinoff, P.; Edelman, M. J. B i o l . Chem. 1984, 259, 3900-8. 23. Wettern, M. Plant S c i . Lett. 1986, 43, 173-7. 24. Marder, J . B . ; Mattoo, A . K . , Edelman,M. Methods Enzymol. 1986, 118, 384-96. 25. Edelman, M . ; Goloubinoff, P . ; Marder, J . B . ; Fromm, H . ; Devic, M . ; Fluhr, R.; Mattoo, A.K. In Molecular Form and Function of the Plant Genome; van Vloten-Doting, L.; Groot, G.S.P.; Hall, T.C. Eds.; Plenum: New York, 1985; pp. 291-300. 26. Wickner, W. Ann. Rev. Biochem. 1979, 48, 23-45. 27. Norman, H . ; St. John, J . B . ; Callahan, F . E . ; Mattoo, A.K.; Wergin, W.P. In The Metabolism, Structure and Function of Plant Lipids; Stumpf, P.K.; Mudd, J.B.; Nes, W.D., Eds.; Plenum: New York, 1987, pp 193-5. 28. Callahan, F . E . ; Edelman, M . ; Mattoo, A.K. In Progress in Photosynthesis Research; Biggens, J . Ed.; Nijhoff: The Hague, 1987; V o l . 3; pp. 799-802. 29. Callahan, F . E . ; Mattoo, A.K. Plant Physiol. Suppl. 1987, 83, p. 113. 30. Wiest, S.C. Physiol. Plant. 1986, 66, 527-35.


18. MATTOO ET AL.


257

31. Mattoo,A.K.; Hoffman-Falk, H . ; Marder, J . B . ; Edelman, M. Proc. Natl. Acad. S c i . USA 1984, 81, 1380-4. 32. Gaba, V . ; Marder, J.B.,; Greenberg, B.M.; Mattoo, A.K.; Edelman, M. Plant Physiol. 1987, 84, 348-52. 33. Greenberg, B.M.; Gaba, V . ; Mattoo, A.K.; Edelman, M. EMBO J. 1987, 6, 2865-9. 34. Rogers, S.; Wells, R.; Rechsteiner, M. Science 1986, 234, 364-8. 35. Deisenhofer, J.; Epp, O.; Miki, K . ; Huber, R.; Michel, H. Nature 1985, 318, 618-24. 36. Reisman, S.; Ohad, I. Biochim. Biophys. Acta. 1986, 849, 51-61. 37. Leto, K.J.; B e l l , E . ; McIntosh, L. EMBO J. 1985, 4, 1645-53. 38. Youvan, D.C.; Bylina, E.J.; Alberti, M . ; Begusch, H . ; Hearst, J.E. Cell 1984, 37, 949-57. 39. Kirsch, W.; Seyer, P . ; Herrmann, R.G. Curr. Genet. 1986, 10, 843-55. 40. Shinozaki, K. et al. EMBO J. 1986. 5, 2043-9. 41. Golden, S.S.; Brusslan, J.; Haselkorn, R. EMBO J. 1986, 11, 2789-98. 42. Curtis, S.; Haselkorn, R. Plant Mol. B i o l . 1984, 3, 249-58. 43. Mulligan, B . ; Schulte, N . ; Chen, L.; Bogorad, L. Proc. Natl. Acad. S c i . USA 1984, 81, 2693-7. 44. Karabin, G.D.; Farley, M . ; Hallick, R.B. Nucl. Acids Res. 1984, 12, 5801-12. 45. Erickson, J.M.; Rahire, M . ; Rochaix, J.D. EMBO J. 1984, 3, 2753-62. 46. Ohyama, K. et al. Nature 1986, 322, 572-4. 47. Larrinua, I . M . ; McLaughlin, W.E. In Progress in Photosynthesis Research; Biggins, J . Ed.; Nijhoff: The Hague, 1987; Vol. 4; pp. 649-52. 48. Link, G.; Langridge, U. Nucl. Acids Res. 1984, 12, 945-58. 49. Reith, M . ; Straus, N.A. Theor. Appl. Genet. 1987, 73, 357-63. 50. Aldrich, J.; Cherney, B . ; Merlin, E. Nucl. Acids Res. 1986, 23, 9537. 51. Oishi, K.K.; Shapiro, D.R.; Tewari, K.K. Mol. Cell B i o l . 1984, 4, 2556-63. 52. Spielmann, A.; Stutz, E. Nucl. Acids Res. 1983, 11, 7157-67. 53. Aldrich, J.; Cherney, B.; Merlin, E . ; Christopherson, L . A . ; Williams, C. Nucl. Acids Res. 1986, 23, 9536. 54. Goloubinoff, P.; Edelman, M . ; Hallick, R.B. Nucl. Acids Res. 1984, 24, 9489-96. 55. Sugita, M . ; Suguira, M. Mol. Gen. Genet. 1984, 195, 308-13. 56. Goloubinoff, P. Ph.D. Thesis, The Weizmann Institute of Science, Rehovot, Israel, 1988. RECEIVED August 5, 1988


Molecular Dynamics of the 32,000-Dalton Photosystem II Herbicide

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