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Biosynthesis of the Na,K-ATPase in MDCK Cells
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19
Biosynthesis of the Na,K-ATPase in MDCK Cells J . SHERMAN, T. MORIMOTO,
AND D.D.SABATIIW
Department of Cell Biology New York University School of Medicine New York, New York
I.
INTRODUCTION
P l a s m a membrane p r o t e i n s m e d i a t e t h e i n t e r a c t i o n s of c e l l s w i t h t h e i r environment and p l a y a c e n t r a l r o l e i n r e g u l a t i n g t h e composition of t h e i n t r a c e l l u l a r m i l i e u . B i o g e n e t i c mechanisms which govern t h e i n c o r p o r a t i o n o f s p e c i f i c p o l y p e p t i d e s i n t o plasma membranes and d e t e r m i n e t h e i r d i s p o s i t i o n w i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r a r e , t h e r e f o r e , of p a r t i c u l a r i n terest t o c e l l b i o l o g i s t s . The plasma membrane A T P a s e , which i n e u k a r y o t i c c e l l s e x t r u d e s Na+ and e s t a b l i s h e s t h e h i g h i n t r a c e l l u l a r K+ c o n c e n t r a t i o n , c o n s i s t s of c a t a l y t i c o r a( 1 0 0 , 0 0 0 d a l t o n s ) and g l y c o p r o t e i n o r B- ( a b o u t 6 0 , 0 0 0 d a l t o n s ) s u b u n i t s ( c f . Sweadner and G o l d i n , 1 9 8 0 ; Stekhoven and B o n t i n g , 1981) which are t h o u g h t t o form a l a r g e i n t e g r a l membrane p r o t e i n complex. The a-subu n i t a p p e a r s t o s p a n t h e membrane, s i n c e it c o n t a i n s a o u a b a i n - b i n d i n g s i t e which i s exposed on t h e e x t r a c e l l u l a r s u r f a c e ( P e r r o n e and B l o s t e i n , 1973; Ruoho and Kyte, 753
Copynght 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. ISBN 012-153319-0
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1 9 7 4 ) and an amino a c i d r e s i d u e a c c e s s i b l e t o phosphoryl a t i o n on t h e c y t o p l a s m i c s i d e of t h e membrane (Whittam, 1 9 6 2 ; Uesugi e t al., 1 9 7 1 ) . The e x a c t d i s p o s i t i o n of t h e @ - s u b u n i tw i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r has n o t been e s t a b l i s h e d , a l t h o u g h t h i s p o l y p e p t i d e a l s o can be l a b e l e d by a o u a b a i n a n a l o g a p p l i e d t o t h e c e l l s u r f a c e ( H a l l and Ruoho, 1 9 8 0 ) . C e l l s of r e n a l e p i t h e l i a , which a r e a c t i v e l y engaged i n i o n t r a n s p o r t , c o n t a i n h i g h c o n c e n t r a t i o n s of t h e Na,K-ATPase and t h e r e f o r e p r o v i d e a u s e f u l system f o r s t u d i e s on t h e b i o s y n t h e s i s of t h i s enzyme. The ATPase i s c o n f i n e d t o t h e b a s o l a t e r a l domains of t h e plasma membranes ( F u j i t a e t a l . , 1 9 7 2 ; Kyte, 1 9 7 6 1 , and t h i s r e s t r i c t e d l o c a l i z a t i o n of t h e enzyme i s thought t o p l a y an i m p o r t a n t r o l e i n e s t a b l i s h i n g t h e f u n c t i o n a l p o l a r i z a t i o n of t h e c e l l s . I n t h i s a r t i c l e w e r e p o r t s t u d i e s on t h e b i o s y n t h e sis of t h e ATPase c a r r i e d o u t employing t h e MDCK e p i t h e l i a l c e l l l i n e , which e x h i b i t s many of t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of p o l a r i z e d r e n a l e p i t h e l i a (Misfeldt e t a l . , 1976; Cereijido e t a l . , 1 9 7 8 ) , includi n g t h e development of j u n c t i o n a l complexes, which s e a l i n t e r c e l l u l a r s p a c e s and d e f i n e luminal and b a s o l a t e r a l c e l l s u r f a c e domains.
11.
METHODS
The p r o c e d u r e of J6rgensen ( 1 9 7 4 ) was used t o i s o l a t e t h e Na,K-ATPase from dog kidney. The i n d i v i d u a l s u b u n i t s were s e p a r a t e d by p r e p a r a t i v e sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) and i n j e c t e d i n t o r a b b i t s t o p r e p a r e a n t i b o d i e s which were p u r i f i e d by a f f i n i t y chromatography t o t h e immob i l i z e d enzyme. A.
C U L T U R E D CELLS
MDCK c e l l s were grown a t 37OC i n d i s p o s a b l e p l a s t i c r o l l e r b o t t l e s w i t h E a g l e ' s m i n i m a l e s s e n t i a l medium (Gibco) c o n t a i n i n g 1 0 % c a l f serum. Monolayers were h a r v e s t e d by s c r a p i n g i n a phosphate-buffered s a l i n e s o l u t i o n , and c e l l s w e r e broken by homogenization i n 0 . 5 M s u c r o s e w i t h 50 s t r o k e s of a t i g h t - f i t t i n g Dounce homog e n i z e r . Rough microsomes and f r e e polysomes w e r e isol a t e d a s d e s c r i b e d elsewhere (Feldman e t a l . , 1 9 8 2 ) .
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F o r i n v i v o l a b e l i n g o f MDCK c e l l s , 6 0 mM d i s h e s of l i g h t l y c o n f l u e n t MDCK c e l l s were p r e i n c u b a t e d 15-30 min i n 2 m l o f s e r u m - f r e e , m e t h i o n i n e - f r e e RPMI medium ( G i b c o ) b e f o r e t h e a d d i t i o n o f 62.5 v C i / m l [35S]methionine (New England N u c l e a r ) . Labeled c e l l s were h a r v e s t e d , a f t e r removal of t h e l a b e l i n g medium and t h r e e washings o f t h e monolayer w i t h p h o s p h a t e - b u f f e r e d s a l i n e , by s c r a p i n g i n t o 1 m l / p l a t e of 1 0 mM T r i s - H C 1 (pH 7 . 4 ) , 1 0 mM KC1, 1 mM MgC12, c o n t a i n i n g 0.5% w/v T r i t o n X-100. The l y s e d c e l l s were homogenized by f o u r s t r o k e s o f a Dounce homogenizer, and t h e n u c l e i were removed by 1-min c e n t r i f u g a t i o n i n an Eppendorf c e n t r i f u g e . TCA p r e c i p i t a t e s o f t h e p o s t n u c l e a r s u p e r n a t a n t s were s o l u b i l i z e d and p r o c e s s e d f o r i m m u n o p r e c i p i t a t i o n as d e s c r i b e d below. B.
IMMUNEPRECIPITATION AND S D S - P A G E
A T P a s e p o l y p e p t i d e s l a b e l e d i n v i v o or s y n t h e s i z e d i n v i t r o were r e c o v e r e d by i n d i r e c t i m m u n o p r e c i p i t a t i o n from SDS-solubilized samples employing p r o t e i n A Sephar o s e . TCA p r e c i p i t a t e s o f l a b e l e d c e l l s w e r e S Q l u b i l i z e d i n 1 M T r i s and SDS ( 1 0 0 mM T r i s (pH 8 . 5 ) and 2 % S D S f i -
nal c o n c e n t r a t i o n ) and b o i l e d f o r 2 min. T r a n s l a t i o n m i x t u r e s w e r e s i m i l a r l y t r e a t e d w i t h SDS and h e a t e d . A l l samples were d i l u t e d 4 - f o l d w i t h a b u f f e r c o n t a i n i n g 150 m M NaC1, 50 mM T r i s - H C 1 (pH 7 . 4 1 , 5 mM EDTA, 2.5% w/v T r i t o n X-100, and 1 0 0 u n i t s / m l t r a s y l o l , and a s u i t a b l e amount o f a f f i n i t y - p u r i f i e d c a t a l y t i c o r g l y c o p r o t e i n IgG w a s added. The i m m u n o p r e c i p i t a t i o n m i x t u r e s were i h c u b a t e d f o r 2 h r a t room t e m p e r a t u r e , o r o v e r n i g h t a t 4 O C , and t h e n 2 h r a t room t e m p e r a t u r e w i t h p r o t e i n A Sepharose 4 B (Pharmacia , Bromma , Sweden) t o a d s o r b t h e a n t i b o d y - a n t i g e n complexes. A f t e r r e p e a t i n g 'washings o f t h e Sepharose b e a d s , t h e a d s o r b e d p r o t e i n s were s o l u b i l i z e d by h e a t i n g f o r 2 min a t 1 0 0 O C i n 5% SDS c o n t a i n i n g 50 mM T r i s - H C 1 (pH 8 . 5 ) , 5 mM EDTA, and 1 0 mM d i t h i o Samples were a n a l y z e d by e l e c t r o p h o r e t h r e i t o l (DTT) s i s on SDS-polyacrylamide g e l s ( 1 0 % o r 6-12% g r a d i e n t ) f o l l o w e d by f l u o r o g r a p h y o f d r i e d g e l s u s i n g Kodak (XR-5) f i l m (Laskey and M i l l s , 1 9 7 5 ) .
.
C.
PREPARATION O F mRNA
T o t a l RNA was e x t r a c t e d from c u l t u r e d c e l l s u s i n g g u a n i d i n e h y d r o c h l o r i d e ( p r a c t i c a l g r a d e , Sigma) (Cox , 1 9 6 8 ) . mRNA w a s p u r i f i e d by two c y c l e s of oligo-dT
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c e l l u l o s e ( C o l l a b o r a t i v e Research, Waltham, Massac h u s t t s ) chromatography (Aviv and Leder, 1 9 7 2 ) . D.
In v i t r o P R O T E I N S Y N T H E S I S
Poly(A) + mRNA (0.10 OD 6 /50 111 t r a n s l a t i o n v o l ume) o r polysomes ( 1 . 0 OD260758 p 1 t r a n s l a t i o n volume) were used t o program n u c l e a s e - t r e a t e d r a b b i t r e t i c u l o c y t e l y s a t e s (Pelham and Jackson, 1 9 7 6 ) o r wheat germ e x t r a c t s (Roman e t a l . , 1976) which were i n c u b a t e d a t 28' o r 25OC, r e s p e c t i v e l y . [35S]Methionine used i n t h e t r a n s l a t i o n s w a s o b t a i n e d from N e w England Nuclear. E.
SPECIAL MATERIALS
Tunicamycin was a g i f t from D r . F. Tomita (Kyowa Hakko Kogyo Co., L t d . , Tokyo, J a p a n ) . Monensin was g e n e r o u s l y s u p p l i e d by E l i L i l l y Co., I n d i a n a p o l i s , Indiana.
111.
RESULTS
In v i t r o t r a n s l a t i o n experiments w i t h f r e e and bound polysomes o b t a i n e d from MDCK c e l l s were c a r r i e d o u t t o d e t e r m i n e t h e s u b c e l l u l a r s i t e of s y n t h e s i s of t h e ATPase p o l y p e p t i d e s . I t was found t h a t bound, b u t n o t f r e e , polysomes s y n t h e s i z e d a 3 8 , 0 0 0 M y p o l y p e p t i d e which was immunoprecipitated w i t h a n t i b o d i e s a g a i n s t t h e B-subunit ( F i g . l a and b ) . The e l e c t r o p h o r e t i c mob i l i t y of t h i s p o l y p e p t i d e was much g r e a t e r t h a n t h a t of t h e mature 8-subunit ( 6 0 , 0 0 0 M ~ ) , which w a s immunop r e c i p i t a t e d from c e l l s l a b e l e d f o r 4 h r ( F i g . l e ) , b u t was i n d i s t i n g u i s h a b l e i n s i z e from t h e primary t r a n s l a t i o n p r o d u c t r e c o v e r e d from t r a n s l a t i o n m i x t u r e s programmed w i t h t o t a l mRNA from MDCK c e l l s ( F i g . l c ) . The f i n d i n g t h a t t h e 8-subunit of t h e ATPase i s s y n t h e s i z e d i n bound polysomes i m p l i e s t h a t t h e polypept i d e i s c o t r a n s l a t i o n a l l y i n s e r t e d i n t o t h e endoplasmic r e t i c u l u m (ER) membranes. To determine i f t h i s i n s e r t i o n i s accompanied by t h e concomitant c l e a v a g e of an amino t e r m i n a l i n s e r t i o n s i g n a l , t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e primary t r a n s l a t i o n p r o d u c t was compared t o t h a t of t h e p o l y p e p t i d e s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a drug which i n h i b i t s c o t r a n s l a t i o n a l
BIOSYNTHESIS OF Na,K-ATPase IN MDCK CELLS
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c
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F i g . 1 . S y n t h e s i s o f the 6 - s u b u n i t o f the Na,K-ATPase on membrane-bound p o l y s o m e s . I m m u n o p r e c i p i t a t e s f r o m r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( a ) and bound ( b ) p o l y s o m e s and tot a l mRNA ( c ) from MDCK c e l l s w e r e compared t o the g l y c o p r o t e i n s u b u n i t i s o l a t e d f r o m MDCK c e l l s l a b e l e d w i t h [ 3 5 S ] m e t h i o n i n e ( 1 2 5 p C i / p l a t e ) i n the p r e s e n c e ( a ) o r a b s e n c e ( e ) o f t u n i c a m y c i n ( 3 p g / m l , 2-hr p r e t r e a t m e n t ) . S a m p l e s were a n a l y z e d b y electrop h o r e s i s on a 1 0 % p o l y a c r y l a m i d e g e l .
g l y c o s y l a t i o n ( T r a c z and Lampen, 1975; S t r u c k and L e n n a r z , 1 9 7 7 ) . N o d i f f e r e n c e in a p p a r e n t m o l e c u l a r w e i g h t w a s d e t e c t e d ( F i g . I d ) , which s u g g e s t s t h a t , u n l i k e m o s t s e c r e t o r y and several membrane p r o t e i n s ( c f . K r e i b i c h e t a l . , 1980a; E m r e t a l . , 1980; L o d i s h
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F i g . 2 . Co- 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 o f the 8-subunit. Dishes of l i g h t l y c o n f l u e n t MDCK c e l l s w e r e p r e i n c u b a t e d w i t h either no d r u g (control) I t u n i c a m y c i n (3 v g / m l 2 hr) , or mnensin M, 1 h r ) i n c o m p l e t e medium. The preincubation medium was r e p l a c e d w i t h l a b e l i n g medium, and f o l l o w i n g a 30-min i n c u b a t i o n , [ 3 5 S ] m e t h i o n i n e was added t o e a c h p l a t e . T h e monol a y e r s were h a r v e s t e d 2 hr l a t e r , and S D S - s o l u b i l i z e d TCA p r e c i p i t a t e s o f the p o s t n u c l e a r s u p e r n a t a n t s w e r e a s s a y e d for l a b e l e d 8 - s u b u n i t b y i m m u n o p r e c i p i t a t i o n and e l e c t r o p h o r e s i s on a 6-12% p o l y a c r y l a m i d e g e l : ( a ) control , ( b ) t u n i c a m y c i n , (c) monensin.
1 9 8 1 ) , t h e s m a l l s u b u n i t of t h e A T P a s e does n o t c o n t a i n a t r a n s i e n t amino t e r m i n a l i n s e r t i o n s i g n a l . Had a s i g n a l been removed from t h e 8-subunit s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a d e t e c t a b l e increase i n t h e e l e c t r o p h o r e t i c m o b i l i t y of t h i s r e l a t i v e s m a l l p o l y p e p t i d e would have r e s u l t e d . I t a p p e a r s , t h e r e f o r e , t h a t t h e 8-subunit should be added t o t h e growing l i s t of membrane p r o t e i n s t h a t a r e s y n t h e s i z e d i n bound polysomes, b u t a r e n o t p r o c e s s e d p r o t e o l y t i c a l l y during t h e i r cotr an slatio n al i n s e r t i o n et al.,
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i n t o t h e membranes ( B o n a t t i and B l o b e l , 1979; Chyn e t a l . , 1979; S c h e c h t e r et a l . , 1979; Bar-Nun e t a l . , 1980; c f . K r e i b i c h e t a1 1980b; Okada e t a l . , 1 9 8 2 ) . A n a l y s i s of 3 S & l a b e l e d 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 normal and t u n i c a m y c i n - t r e a t e d c e l l s a l s o d e m o n s t r a t e d t h a t e x t e n s i v e co- and p o s t t r a n s l a t i o n a l g l y c o s y l a t i o n of t h e 8 - s u b u n i t a c c o u n t s f o r t h e l a r g e d i f f e r e n c e i n app a r e n t m o l e c u l a r w e i g h t between t h e p r i m a r y t r a n s l a t i o n p r o d u c t and t h e mature s u b u n i t . I n b r i e f l y l a b e l e d MDCK c e l l s (Fig. 2a) it w a s p o s s i b l e t o i d e n t i f y , i n a d d i t i o n t o t h e mature glycoprotein ( 6 0 , 0 0 0 M r ) , a polypeptide (45 ,0 0 0 M r ) which r e p r e s e n t e d an i n t e r m e d i a t e s t a g e i n t h e g l y c o s y l a t i o n p r o c e s s . T h i s p r o d u c t must r e s u l t from t h e c o t r a n s l a t i o n a l t r a n s f e r of o l i g o s a c c h a r i d e s t o t h e n a s c e n t c h a i n , s i n c e it w a s n o t p r e s e n t i n t h e tunicamyc i n - t r e a t e d c e l l s , which c o n t a i n e d o n l y an u n g l y c o s y l a t e d p o l y p e p t i d e o f M r 38,000 ( F i g . 2 b ) . Direct e v i d e n c e f o r t h e c o t r a n s l a t i o n a l g l y c o s y l a t i o n o f t h e B-subunit w a s o b t a i n e d i n c o l l a b o r a t i o n w i t h D r . D. Colman u s i n g t r a n s l a t i o n s y s t e m s programmed w i t h t o t a l r a t b r a i n mRNA and supplemented w i t h microsomal membranes. Under t h e s e cond i t i o n s a membrane a s s o c i a t e d p o l y p e p t i d e of M r 45,000 was o b t a i n e d . The second g l y c o s y l a t i o n s t a g e d u r i n g t h e m a t u r a t i o n of t h e 8 - s u b u n i t , which i n c r e a s e d t h e a p p a r e n t m o l e c u l a r w e i g h t compared t o t h a t c h a r a c t e r i s t i c o f t h e m a t u r e p o l y p e p t i d e ( M 6~ 0 , 0 0 0 ) , a p p e a r s t o t a k e p l a c e i n t h e G o l g i a p p a r a t u s , s i n c e i t w a s p a r t i a l l y b l o c k e d by t r e a t ment o f c e l l s w i t h t h e sodium ionophore monensin ( F i g . 2 c ) . This drug is thought t o impair i n t r a c e l l u l a r t r a f f i c o f s e c r e t o r y ( T a r t a k o f f and V a s s a l l i , 1978; Uchida e t a l . , 1980) and membrane p r o t e i n s (Johnson and S c h l e s i n g e r , 1980; Ktitiriainen et a l . , 1980; S t r o u s and L o d i s h , 1980) a f t e r t h e y e x i t from t h e ER, b u t b e f o r e t h e y r e a c h t h e plasma membrane. The c o n c l u s i o n t h a t t h e 45,000 M~ p o l y p e p t i d e det e c t e d i n MDCK c e l l s r e p r e s e n t s an immature m i C r O S O m a 1 form o f t h e f3-subunit, which must t r a v e r s e t h e G o l g i app a r a t u s f o r i t s m a t u r a t i o n and t r a n s f e r t o t h e plasma membrane, has been s u b s t a n t i a t e d by p u l s e c h a s e e x p e r i ments i n which t h e d i s t r i b u t i o n of newly s y n t h e s i z e d $ - s u b u n i t w a s examined i n s u b c e l l u l a r f r a c t i o n s o f MDCK c e l l 9 l a b e l e d w i t h 35s m e t h i o n i n e . Five minutes a f t e r a d m i n i s t r a t i o n of t h e l a b e l , a 45,000 M r . 8 - s u b u n i t p o l y p e p t i d e was d e t e c t e d i n p u r i f i e d rough microsomes. T h i r t y m i n u t e s l a t e r , however, t h i s p o l y p e p t i d e w a s no l o n g e r p r e s e n t i n t h i s f r a c t i o n , a s e x p e c t e d of a p r o d u c t i n t r a n s i t t o a d i f f e r e n t c e l l u l a r l o c a t i o n , presumably t h e plasma membrane. A t t h a t t i m e t h e 45,000 M r p o l y p e p t i d e w a s s t i l l d e t e c t e d i n a f r a c t i o n of smooth memb r a n e s where a t a b o u t 4 5 min t h e m a t u r e form w a s found.
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a
b
F i g . 3 . In vivo and i n v i t r o s y n t h e s i s o f the a - s u b u n i t o f the Na,K-ATPase. The a - s u b u n i t o f the ATPase was i m m u n o p r e c i p i t a t e d from MDCK c e l l s l a b e l e d f o r 30 m i n w i t h [ 3 5 S ] m e t h i o n i n e i n the a b s e n c e ( a ) or p r e s e n c e (b) o f t u n i c a m y c i n . These i n vivo s y n t h e s i z e d p o l y p e p t i d e s were compared t o those o b t a i n e d from r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( c ) and bound (a) p o l y somes p r e p a r e d from MDCK c e l l s . An i m m u n o p r e c i p i t a t e d p r o d u c t o f t o t a l p o l y s o m e s i s shown i n l a n e ( e ) . T r a n s l a t i o n s of MDCK t o t a l mRNA i n a r e t i c u l o c y t e l y s a t e (f) and wheat germ l y s a t e s ( 9 ) and ( h ) y i e l d e d s i m i l a r r e s u l t s . S a m p l e s w e r e a n a l y z e d on a 10% g e l .
S t u d i e s on t h e b i o s y n t h e s i s of t h e A T P a s e a - s u b u n i t were a l s o c a r r i e d o u t w i t h c u l t u r e d MDCK c e l l s , a s w e l l as c e l l - f r e e s y s t e m s programmed w i t h polysomes o r p u r i f i e d mRNA. The a - s u b u n i t immunoprecipitated from c e l l s l a b e l e d w i t h [35S]methionine f o r 30 min had t h e same s i z e a s t h e m a t u r e s u b u n i t p u r i f i e d from dog k i d n e y . A p o l y p e p t i d e of t h e same m o b i l i t y w a s o b t a i n e d from t u n i c a m y c i n - t r e a t e d c e l l s ( F i g . 3a and b) a s e x p e c t e d f o r a product t h a t does n o t c o n t a i n asparagine-linked oligosaccharide chains.
BIOSYNTHESIS OF Na.K-ATPase IN MDCK CELLS
76 1
A s was t h e case f o r t h e 8-subunit t h e newly s y n t h e s i z e d a - s u b u n i t l a b e l e d d u r i n g s h o r t (5-min) p u l s e s w i t h [35S]methionine w a s found i n t r a c e l l u l a r l y i n a s s o c i a t i o n w i t h rough microsomal membranes, b e f o r e i t a p p e a r e d i n a f r a c t i o n o f smooth membranes which c o n t a i n e d plasma memb r a n e f r a g m e n t s . A d i r e c t d e t e r m i n a t i o n o f t h e s i t e of s y n t h e s i s of t h e a - s u b u n i t w a s hampered by d i f f i c u l t i e s i n o b t a i n i n g a n 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 of t h e exp e c t e d s i z e , when f r e e o r bound polysomes ( F i g . 3c and d ) o r p u r i f i e d mRNAs ( F i g . 3 f , g , and h ) from MDCK c e l l s were u s e d t o program r e t i c u l o c y t e o r wheat germ t r a n s l a t i o n s y s t e m s . Although o c c a s i o n a l l y a 1 0 0 , 0 0 0 Mr-pyod u c t w a s r e c o v e r e d ( F i g . 3e and h ) , an i m m u n o p r e c i p i t a b l e p r o d u c t of a p p r o x i m a t e l y 85,000 M~ w a s g e n e r a l l y o b t a i n e d and f r e q u e n t l y t h e y i e l d o f t h i s p r o d u c t w a s h i g h e r when It appears f r e e r a t h e r t h a n bound polysomes were used. t h a t t h i s p r o d u c t r e s u l t e d from i n c o m p l e t e t r a n s l a t i o n r a t h e r t h a n from p r o t e o l y t i c d e g r a d a t i o n , as it w a s a l s o found when a combination o f p r o t e a s e i n h i b i t o r s were added t o t h e t r a n s l a t i o n s y s t e m s . R e c e n t l y , i n c o l l a b o r a t i o n with,N. Nabi, w e have found t h a t i n c o n t r a s t t o t h e s i t u a t i o n w i t h polysomes from MDCK c e l l s , i n v i t r o t r a n s l a t i o n o f b r a i n polysomes y i e l d s a p r o d u c t of t h e s i z e e x p e c t e d f o r t h e l a r g e s t ( a + ) o f t h e two forms of t h e a - s u b u n i t c h a r a c t e r i s t i c of b r a i n A T P a s e (Sweadner, 1 9 7 9 ) . I n t h i s i n s t a n c e t h e t r a n s l a t a b l e mRNA w a s o n l y c o n t a i n e d i n bound polysomes. Taken t o g e t h e r , o u r o b s e r v a t i o n s s u g g e s t t h a t , a l t h o u g h a s i z a b l e f r a c t i o n of mRNA f o r a - s u b u n i t may be found i n f r e e polysomes o f MDCK c e l l s where p a r t i a l s y n t h e s i s may t a k e p l a c e , i n s e r t i o n of t h e p o l y p e p t i d e i n t o t h e ER membrane a c t u a l l y i s a c o t r a n s l a t i o n a l e v e n t . Using i n v i t r o systems c o n t a i n i n g microsomal memb r a n e s w e are c u r r e n t l y a t t e m p t i n g t o e l u c i d a t e t h e sequence o f e v e n t s i n v o l v e d i n t h e s y n t h e s i s and i n c o r p o r a t i o n o f t h e a - s u b n i t i n t o t h e membrane and t h e s i t e and mode of a s s o c i a t i o n of t h e two s u b u n i t s i n t o a f u n c t i o n a l complex. The MDCK c u l t u r e c e l l system a l s o a p p e a r s t o p r o v i d e a s u i t a b l e model t o s t u d y t h e mechanisms t h a t est a b l i s h and m a i n t a i n t h e r e s t r i c t e d l o c a l i z a t i o n o f t h e enzyme t o t h e b a s o l a t e r a l domains of e p i t h e l i a l c e l l s and thus determine t h e i r functional p o l a r i t y .
ACKNOWLEDGMENT
W e thank M r . B i l l Dolan, M s . Susan Malamet, and M s . Harriet S n i t k i n € o r t i s s u e c u l t u r e work. We are g r a t e f u l to M r s . Myrna Chung, M r . B r i a n Z i e t l o w , and Ms. Jody C u l k i n € o r a s s i s t a n c e i n t h e The work w a s s u p p o r t e d by N I H g r a n t s p r e p a r a t i o n of t h e m a n u s c r i p t . t o D r . S a b a t i n i AG 01461, GM 2 0 2 7 7 , and AG 00378.
762
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Stekhoven, F. S., and Bonting, S. L. (1981). T r a n s p o r t adenosine t r i p h o s p h a t a s e s : p r o p e r t i e s and f u n c t i o n s . P h y s i o l . R e v . 6 1 , 1-76. S t r o u s , E. J. A. M . , and Lodish, M. F. (1980). I n t r a c e l l u l a r t r a n s p o r t of s e c r e t o r y and membrane p r o t e i n s i n hepatoma Cell 22, c e l l s i n f e c t e d by v e s i c u l a r s t o m a t i t i s v i r u s . 709-717. S t r u c k , D. K . , and Lennarz, W. J. (1977). Evidence f o r t h e p a r t i cipation of saccharide-lipids i n the synthesis of the oligos a c c h a r i d e c h a i n o f ovalbumin. J. B i o l . Chem. 2 5 2 , 10071113. Sweadner, K. J. (1979). Two m o l e c u l a r forms of ( N a + + K+)J . B i o l . Chem. 2 5 4 , 6060-6067. s t i m u l a t e d ATPase i n b r a i n . Sweadner, K. J . , and Goldin, S. M. (1980). Active t r a n s p o r t of sodium and potassium i o n s . N. E n g l . J . Med. 302, 777-783. T a r t a k o f f , A . , and V a s s a l l i , P. (1978). Comparative s t u d i e s of J. Cell i n t r a c e l l u l a r transport of secretory proteins. B i o l . 7 9 , 694-707. Tracz, J. S . , and Lampen, 0. (1975). Tunicamycin i n h i b i t i o n of p o l y i s o p r e n y l N-acetylglucosaminyl phosphate formation i n B i o c h e m . B i o p h y s . R e s . Commun. 6 5 , c a l f l i v e r microsomes. 248-257. uchida, N . , Smilowitz, H . , Ledger, P. W., and Tanzer, M. L. (1980). K i n e t i c s t u d i e s of t h e i n t r a c e l l u l a r t r a n s p o r t of p r o c o l l a g e n and f i b r o n e c t i n i n human f i b r o b l a s t s - - E f f e c t s of t h e monoval e n t ionophore monensin. J . B i o l . Chem. 2 5 5 , 8638-8644. Uesugi, S . , Dulak, N . C . , Dixon, J. F . , Hexum, T D . , Dahl, J. L . , Perdue, J. F., and Hokin, L. E. (1971). S t u d i e s on t h e c h a r a c t e r i z a t i o n of t h e sodium-potassium t r a n s p o r t adenosine J. B i o l . Chem. 2 4 6 , 531-543. triphosphatase. Whittam, R. (1962). The asymmetrical s t i m u l a t i o n o f a membrane adenosine t r i p h o s p h a t a s e i n r e l a t i o n t o a c t i v e c a t i o n t r a n s port. B i o c h e m . J . 8 4 , 110-118.
Biosynthesis of the Na,K-ATPase in MDCK Cells J . SHERMAN, T. MORIMOTO,
AND D.D.SABATIIW
Department of Cell Biology New York University School of Medicine New York, New York
I.
INTRODUCTION
P l a s m a membrane p r o t e i n s m e d i a t e t h e i n t e r a c t i o n s of c e l l s w i t h t h e i r environment and p l a y a c e n t r a l r o l e i n r e g u l a t i n g t h e composition of t h e i n t r a c e l l u l a r m i l i e u . B i o g e n e t i c mechanisms which govern t h e i n c o r p o r a t i o n o f s p e c i f i c p o l y p e p t i d e s i n t o plasma membranes and d e t e r m i n e t h e i r d i s p o s i t i o n w i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r a r e , t h e r e f o r e , of p a r t i c u l a r i n terest t o c e l l b i o l o g i s t s . The plasma membrane A T P a s e , which i n e u k a r y o t i c c e l l s e x t r u d e s Na+ and e s t a b l i s h e s t h e h i g h i n t r a c e l l u l a r K+ c o n c e n t r a t i o n , c o n s i s t s of c a t a l y t i c o r a( 1 0 0 , 0 0 0 d a l t o n s ) and g l y c o p r o t e i n o r B- ( a b o u t 6 0 , 0 0 0 d a l t o n s ) s u b u n i t s ( c f . Sweadner and G o l d i n , 1 9 8 0 ; Stekhoven and B o n t i n g , 1981) which are t h o u g h t t o form a l a r g e i n t e g r a l membrane p r o t e i n complex. The a-subu n i t a p p e a r s t o s p a n t h e membrane, s i n c e it c o n t a i n s a o u a b a i n - b i n d i n g s i t e which i s exposed on t h e e x t r a c e l l u l a r s u r f a c e ( P e r r o n e and B l o s t e i n , 1973; Ruoho and Kyte, 753
Copynght 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. ISBN 012-153319-0
J. SHERMAN eta/.
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1 9 7 4 ) and an amino a c i d r e s i d u e a c c e s s i b l e t o phosphoryl a t i o n on t h e c y t o p l a s m i c s i d e of t h e membrane (Whittam, 1 9 6 2 ; Uesugi e t al., 1 9 7 1 ) . The e x a c t d i s p o s i t i o n of t h e @ - s u b u n i tw i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r has n o t been e s t a b l i s h e d , a l t h o u g h t h i s p o l y p e p t i d e a l s o can be l a b e l e d by a o u a b a i n a n a l o g a p p l i e d t o t h e c e l l s u r f a c e ( H a l l and Ruoho, 1 9 8 0 ) . C e l l s of r e n a l e p i t h e l i a , which a r e a c t i v e l y engaged i n i o n t r a n s p o r t , c o n t a i n h i g h c o n c e n t r a t i o n s of t h e Na,K-ATPase and t h e r e f o r e p r o v i d e a u s e f u l system f o r s t u d i e s on t h e b i o s y n t h e s i s of t h i s enzyme. The ATPase i s c o n f i n e d t o t h e b a s o l a t e r a l domains of t h e plasma membranes ( F u j i t a e t a l . , 1 9 7 2 ; Kyte, 1 9 7 6 1 , and t h i s r e s t r i c t e d l o c a l i z a t i o n of t h e enzyme i s thought t o p l a y an i m p o r t a n t r o l e i n e s t a b l i s h i n g t h e f u n c t i o n a l p o l a r i z a t i o n of t h e c e l l s . I n t h i s a r t i c l e w e r e p o r t s t u d i e s on t h e b i o s y n t h e sis of t h e ATPase c a r r i e d o u t employing t h e MDCK e p i t h e l i a l c e l l l i n e , which e x h i b i t s many of t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of p o l a r i z e d r e n a l e p i t h e l i a (Misfeldt e t a l . , 1976; Cereijido e t a l . , 1 9 7 8 ) , includi n g t h e development of j u n c t i o n a l complexes, which s e a l i n t e r c e l l u l a r s p a c e s and d e f i n e luminal and b a s o l a t e r a l c e l l s u r f a c e domains.
11.
METHODS
The p r o c e d u r e of J6rgensen ( 1 9 7 4 ) was used t o i s o l a t e t h e Na,K-ATPase from dog kidney. The i n d i v i d u a l s u b u n i t s were s e p a r a t e d by p r e p a r a t i v e sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) and i n j e c t e d i n t o r a b b i t s t o p r e p a r e a n t i b o d i e s which were p u r i f i e d by a f f i n i t y chromatography t o t h e immob i l i z e d enzyme. A.
C U L T U R E D CELLS
MDCK c e l l s were grown a t 37OC i n d i s p o s a b l e p l a s t i c r o l l e r b o t t l e s w i t h E a g l e ' s m i n i m a l e s s e n t i a l medium (Gibco) c o n t a i n i n g 1 0 % c a l f serum. Monolayers were h a r v e s t e d by s c r a p i n g i n a phosphate-buffered s a l i n e s o l u t i o n , and c e l l s w e r e broken by homogenization i n 0 . 5 M s u c r o s e w i t h 50 s t r o k e s of a t i g h t - f i t t i n g Dounce homog e n i z e r . Rough microsomes and f r e e polysomes w e r e isol a t e d a s d e s c r i b e d elsewhere (Feldman e t a l . , 1 9 8 2 ) .
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F o r i n v i v o l a b e l i n g o f MDCK c e l l s , 6 0 mM d i s h e s of l i g h t l y c o n f l u e n t MDCK c e l l s were p r e i n c u b a t e d 15-30 min i n 2 m l o f s e r u m - f r e e , m e t h i o n i n e - f r e e RPMI medium ( G i b c o ) b e f o r e t h e a d d i t i o n o f 62.5 v C i / m l [35S]methionine (New England N u c l e a r ) . Labeled c e l l s were h a r v e s t e d , a f t e r removal of t h e l a b e l i n g medium and t h r e e washings o f t h e monolayer w i t h p h o s p h a t e - b u f f e r e d s a l i n e , by s c r a p i n g i n t o 1 m l / p l a t e of 1 0 mM T r i s - H C 1 (pH 7 . 4 ) , 1 0 mM KC1, 1 mM MgC12, c o n t a i n i n g 0.5% w/v T r i t o n X-100. The l y s e d c e l l s were homogenized by f o u r s t r o k e s o f a Dounce homogenizer, and t h e n u c l e i were removed by 1-min c e n t r i f u g a t i o n i n an Eppendorf c e n t r i f u g e . TCA p r e c i p i t a t e s o f t h e p o s t n u c l e a r s u p e r n a t a n t s were s o l u b i l i z e d and p r o c e s s e d f o r i m m u n o p r e c i p i t a t i o n as d e s c r i b e d below. B.
IMMUNEPRECIPITATION AND S D S - P A G E
A T P a s e p o l y p e p t i d e s l a b e l e d i n v i v o or s y n t h e s i z e d i n v i t r o were r e c o v e r e d by i n d i r e c t i m m u n o p r e c i p i t a t i o n from SDS-solubilized samples employing p r o t e i n A Sephar o s e . TCA p r e c i p i t a t e s o f l a b e l e d c e l l s w e r e S Q l u b i l i z e d i n 1 M T r i s and SDS ( 1 0 0 mM T r i s (pH 8 . 5 ) and 2 % S D S f i -
nal c o n c e n t r a t i o n ) and b o i l e d f o r 2 min. T r a n s l a t i o n m i x t u r e s w e r e s i m i l a r l y t r e a t e d w i t h SDS and h e a t e d . A l l samples were d i l u t e d 4 - f o l d w i t h a b u f f e r c o n t a i n i n g 150 m M NaC1, 50 mM T r i s - H C 1 (pH 7 . 4 1 , 5 mM EDTA, 2.5% w/v T r i t o n X-100, and 1 0 0 u n i t s / m l t r a s y l o l , and a s u i t a b l e amount o f a f f i n i t y - p u r i f i e d c a t a l y t i c o r g l y c o p r o t e i n IgG w a s added. The i m m u n o p r e c i p i t a t i o n m i x t u r e s were i h c u b a t e d f o r 2 h r a t room t e m p e r a t u r e , o r o v e r n i g h t a t 4 O C , and t h e n 2 h r a t room t e m p e r a t u r e w i t h p r o t e i n A Sepharose 4 B (Pharmacia , Bromma , Sweden) t o a d s o r b t h e a n t i b o d y - a n t i g e n complexes. A f t e r r e p e a t i n g 'washings o f t h e Sepharose b e a d s , t h e a d s o r b e d p r o t e i n s were s o l u b i l i z e d by h e a t i n g f o r 2 min a t 1 0 0 O C i n 5% SDS c o n t a i n i n g 50 mM T r i s - H C 1 (pH 8 . 5 ) , 5 mM EDTA, and 1 0 mM d i t h i o Samples were a n a l y z e d by e l e c t r o p h o r e t h r e i t o l (DTT) s i s on SDS-polyacrylamide g e l s ( 1 0 % o r 6-12% g r a d i e n t ) f o l l o w e d by f l u o r o g r a p h y o f d r i e d g e l s u s i n g Kodak (XR-5) f i l m (Laskey and M i l l s , 1 9 7 5 ) .
.
C.
PREPARATION O F mRNA
T o t a l RNA was e x t r a c t e d from c u l t u r e d c e l l s u s i n g g u a n i d i n e h y d r o c h l o r i d e ( p r a c t i c a l g r a d e , Sigma) (Cox , 1 9 6 8 ) . mRNA w a s p u r i f i e d by two c y c l e s of oligo-dT
J. SHERMANeta/.
756
c e l l u l o s e ( C o l l a b o r a t i v e Research, Waltham, Massac h u s t t s ) chromatography (Aviv and Leder, 1 9 7 2 ) . D.
In v i t r o P R O T E I N S Y N T H E S I S
Poly(A) + mRNA (0.10 OD 6 /50 111 t r a n s l a t i o n v o l ume) o r polysomes ( 1 . 0 OD260758 p 1 t r a n s l a t i o n volume) were used t o program n u c l e a s e - t r e a t e d r a b b i t r e t i c u l o c y t e l y s a t e s (Pelham and Jackson, 1 9 7 6 ) o r wheat germ e x t r a c t s (Roman e t a l . , 1976) which were i n c u b a t e d a t 28' o r 25OC, r e s p e c t i v e l y . [35S]Methionine used i n t h e t r a n s l a t i o n s w a s o b t a i n e d from N e w England Nuclear. E.
SPECIAL MATERIALS
Tunicamycin was a g i f t from D r . F. Tomita (Kyowa Hakko Kogyo Co., L t d . , Tokyo, J a p a n ) . Monensin was g e n e r o u s l y s u p p l i e d by E l i L i l l y Co., I n d i a n a p o l i s , Indiana.
111.
RESULTS
In v i t r o t r a n s l a t i o n experiments w i t h f r e e and bound polysomes o b t a i n e d from MDCK c e l l s were c a r r i e d o u t t o d e t e r m i n e t h e s u b c e l l u l a r s i t e of s y n t h e s i s of t h e ATPase p o l y p e p t i d e s . I t was found t h a t bound, b u t n o t f r e e , polysomes s y n t h e s i z e d a 3 8 , 0 0 0 M y p o l y p e p t i d e which was immunoprecipitated w i t h a n t i b o d i e s a g a i n s t t h e B-subunit ( F i g . l a and b ) . The e l e c t r o p h o r e t i c mob i l i t y of t h i s p o l y p e p t i d e was much g r e a t e r t h a n t h a t of t h e mature 8-subunit ( 6 0 , 0 0 0 M ~ ) , which w a s immunop r e c i p i t a t e d from c e l l s l a b e l e d f o r 4 h r ( F i g . l e ) , b u t was i n d i s t i n g u i s h a b l e i n s i z e from t h e primary t r a n s l a t i o n p r o d u c t r e c o v e r e d from t r a n s l a t i o n m i x t u r e s programmed w i t h t o t a l mRNA from MDCK c e l l s ( F i g . l c ) . The f i n d i n g t h a t t h e 8-subunit of t h e ATPase i s s y n t h e s i z e d i n bound polysomes i m p l i e s t h a t t h e polypept i d e i s c o t r a n s l a t i o n a l l y i n s e r t e d i n t o t h e endoplasmic r e t i c u l u m (ER) membranes. To determine i f t h i s i n s e r t i o n i s accompanied by t h e concomitant c l e a v a g e of an amino t e r m i n a l i n s e r t i o n s i g n a l , t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e primary t r a n s l a t i o n p r o d u c t was compared t o t h a t of t h e p o l y p e p t i d e s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a drug which i n h i b i t s c o t r a n s l a t i o n a l
BIOSYNTHESIS OF Na,K-ATPase IN MDCK CELLS
a
b
c
757
d
e
F i g . 1 . S y n t h e s i s o f the 6 - s u b u n i t o f the Na,K-ATPase on membrane-bound p o l y s o m e s . I m m u n o p r e c i p i t a t e s f r o m r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( a ) and bound ( b ) p o l y s o m e s and tot a l mRNA ( c ) from MDCK c e l l s w e r e compared t o the g l y c o p r o t e i n s u b u n i t i s o l a t e d f r o m MDCK c e l l s l a b e l e d w i t h [ 3 5 S ] m e t h i o n i n e ( 1 2 5 p C i / p l a t e ) i n the p r e s e n c e ( a ) o r a b s e n c e ( e ) o f t u n i c a m y c i n ( 3 p g / m l , 2-hr p r e t r e a t m e n t ) . S a m p l e s were a n a l y z e d b y electrop h o r e s i s on a 1 0 % p o l y a c r y l a m i d e g e l .
g l y c o s y l a t i o n ( T r a c z and Lampen, 1975; S t r u c k and L e n n a r z , 1 9 7 7 ) . N o d i f f e r e n c e in a p p a r e n t m o l e c u l a r w e i g h t w a s d e t e c t e d ( F i g . I d ) , which s u g g e s t s t h a t , u n l i k e m o s t s e c r e t o r y and several membrane p r o t e i n s ( c f . K r e i b i c h e t a l . , 1980a; E m r e t a l . , 1980; L o d i s h
758
J. SHERMAN et el.
F i g . 2 . Co- 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 o f the 8-subunit. Dishes of l i g h t l y c o n f l u e n t MDCK c e l l s w e r e p r e i n c u b a t e d w i t h either no d r u g (control) I t u n i c a m y c i n (3 v g / m l 2 hr) , or mnensin M, 1 h r ) i n c o m p l e t e medium. The preincubation medium was r e p l a c e d w i t h l a b e l i n g medium, and f o l l o w i n g a 30-min i n c u b a t i o n , [ 3 5 S ] m e t h i o n i n e was added t o e a c h p l a t e . T h e monol a y e r s were h a r v e s t e d 2 hr l a t e r , and S D S - s o l u b i l i z e d TCA p r e c i p i t a t e s o f the p o s t n u c l e a r s u p e r n a t a n t s w e r e a s s a y e d for l a b e l e d 8 - s u b u n i t b y i m m u n o p r e c i p i t a t i o n and e l e c t r o p h o r e s i s on a 6-12% p o l y a c r y l a m i d e g e l : ( a ) control , ( b ) t u n i c a m y c i n , (c) monensin.
1 9 8 1 ) , t h e s m a l l s u b u n i t of t h e A T P a s e does n o t c o n t a i n a t r a n s i e n t amino t e r m i n a l i n s e r t i o n s i g n a l . Had a s i g n a l been removed from t h e 8-subunit s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a d e t e c t a b l e increase i n t h e e l e c t r o p h o r e t i c m o b i l i t y of t h i s r e l a t i v e s m a l l p o l y p e p t i d e would have r e s u l t e d . I t a p p e a r s , t h e r e f o r e , t h a t t h e 8-subunit should be added t o t h e growing l i s t of membrane p r o t e i n s t h a t a r e s y n t h e s i z e d i n bound polysomes, b u t a r e n o t p r o c e s s e d p r o t e o l y t i c a l l y during t h e i r cotr an slatio n al i n s e r t i o n et al.,
BIOSYNTHESIS OF Na,K-ATPaseIN MDCK CELLS
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i n t o t h e membranes ( B o n a t t i and B l o b e l , 1979; Chyn e t a l . , 1979; S c h e c h t e r et a l . , 1979; Bar-Nun e t a l . , 1980; c f . K r e i b i c h e t a1 1980b; Okada e t a l . , 1 9 8 2 ) . A n a l y s i s of 3 S & l a b e l e d 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 normal and t u n i c a m y c i n - t r e a t e d c e l l s a l s o d e m o n s t r a t e d t h a t e x t e n s i v e co- and p o s t t r a n s l a t i o n a l g l y c o s y l a t i o n of t h e 8 - s u b u n i t a c c o u n t s f o r t h e l a r g e d i f f e r e n c e i n app a r e n t m o l e c u l a r w e i g h t between t h e p r i m a r y t r a n s l a t i o n p r o d u c t and t h e mature s u b u n i t . I n b r i e f l y l a b e l e d MDCK c e l l s (Fig. 2a) it w a s p o s s i b l e t o i d e n t i f y , i n a d d i t i o n t o t h e mature glycoprotein ( 6 0 , 0 0 0 M r ) , a polypeptide (45 ,0 0 0 M r ) which r e p r e s e n t e d an i n t e r m e d i a t e s t a g e i n t h e g l y c o s y l a t i o n p r o c e s s . T h i s p r o d u c t must r e s u l t from t h e c o t r a n s l a t i o n a l t r a n s f e r of o l i g o s a c c h a r i d e s t o t h e n a s c e n t c h a i n , s i n c e it w a s n o t p r e s e n t i n t h e tunicamyc i n - t r e a t e d c e l l s , which c o n t a i n e d o n l y an u n g l y c o s y l a t e d p o l y p e p t i d e o f M r 38,000 ( F i g . 2 b ) . Direct e v i d e n c e f o r t h e c o t r a n s l a t i o n a l g l y c o s y l a t i o n o f t h e B-subunit w a s o b t a i n e d i n c o l l a b o r a t i o n w i t h D r . D. Colman u s i n g t r a n s l a t i o n s y s t e m s programmed w i t h t o t a l r a t b r a i n mRNA and supplemented w i t h microsomal membranes. Under t h e s e cond i t i o n s a membrane a s s o c i a t e d p o l y p e p t i d e of M r 45,000 was o b t a i n e d . The second g l y c o s y l a t i o n s t a g e d u r i n g t h e m a t u r a t i o n of t h e 8 - s u b u n i t , which i n c r e a s e d t h e a p p a r e n t m o l e c u l a r w e i g h t compared t o t h a t c h a r a c t e r i s t i c o f t h e m a t u r e p o l y p e p t i d e ( M 6~ 0 , 0 0 0 ) , a p p e a r s t o t a k e p l a c e i n t h e G o l g i a p p a r a t u s , s i n c e i t w a s p a r t i a l l y b l o c k e d by t r e a t ment o f c e l l s w i t h t h e sodium ionophore monensin ( F i g . 2 c ) . This drug is thought t o impair i n t r a c e l l u l a r t r a f f i c o f s e c r e t o r y ( T a r t a k o f f and V a s s a l l i , 1978; Uchida e t a l . , 1980) and membrane p r o t e i n s (Johnson and S c h l e s i n g e r , 1980; Ktitiriainen et a l . , 1980; S t r o u s and L o d i s h , 1980) a f t e r t h e y e x i t from t h e ER, b u t b e f o r e t h e y r e a c h t h e plasma membrane. The c o n c l u s i o n t h a t t h e 45,000 M~ p o l y p e p t i d e det e c t e d i n MDCK c e l l s r e p r e s e n t s an immature m i C r O S O m a 1 form o f t h e f3-subunit, which must t r a v e r s e t h e G o l g i app a r a t u s f o r i t s m a t u r a t i o n and t r a n s f e r t o t h e plasma membrane, has been s u b s t a n t i a t e d by p u l s e c h a s e e x p e r i ments i n which t h e d i s t r i b u t i o n of newly s y n t h e s i z e d $ - s u b u n i t w a s examined i n s u b c e l l u l a r f r a c t i o n s o f MDCK c e l l 9 l a b e l e d w i t h 35s m e t h i o n i n e . Five minutes a f t e r a d m i n i s t r a t i o n of t h e l a b e l , a 45,000 M r . 8 - s u b u n i t p o l y p e p t i d e was d e t e c t e d i n p u r i f i e d rough microsomes. T h i r t y m i n u t e s l a t e r , however, t h i s p o l y p e p t i d e w a s no l o n g e r p r e s e n t i n t h i s f r a c t i o n , a s e x p e c t e d of a p r o d u c t i n t r a n s i t t o a d i f f e r e n t c e l l u l a r l o c a t i o n , presumably t h e plasma membrane. A t t h a t t i m e t h e 45,000 M r p o l y p e p t i d e w a s s t i l l d e t e c t e d i n a f r a c t i o n of smooth memb r a n e s where a t a b o u t 4 5 min t h e m a t u r e form w a s found.
760
J. SHERMAN eta/.
ur
a
b
F i g . 3 . In vivo and i n v i t r o s y n t h e s i s o f the a - s u b u n i t o f the Na,K-ATPase. The a - s u b u n i t o f the ATPase was i m m u n o p r e c i p i t a t e d from MDCK c e l l s l a b e l e d f o r 30 m i n w i t h [ 3 5 S ] m e t h i o n i n e i n the a b s e n c e ( a ) or p r e s e n c e (b) o f t u n i c a m y c i n . These i n vivo s y n t h e s i z e d p o l y p e p t i d e s were compared t o those o b t a i n e d from r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( c ) and bound (a) p o l y somes p r e p a r e d from MDCK c e l l s . An i m m u n o p r e c i p i t a t e d p r o d u c t o f t o t a l p o l y s o m e s i s shown i n l a n e ( e ) . T r a n s l a t i o n s of MDCK t o t a l mRNA i n a r e t i c u l o c y t e l y s a t e (f) and wheat germ l y s a t e s ( 9 ) and ( h ) y i e l d e d s i m i l a r r e s u l t s . S a m p l e s w e r e a n a l y z e d on a 10% g e l .
S t u d i e s on t h e b i o s y n t h e s i s of t h e A T P a s e a - s u b u n i t were a l s o c a r r i e d o u t w i t h c u l t u r e d MDCK c e l l s , a s w e l l as c e l l - f r e e s y s t e m s programmed w i t h polysomes o r p u r i f i e d mRNA. The a - s u b u n i t immunoprecipitated from c e l l s l a b e l e d w i t h [35S]methionine f o r 30 min had t h e same s i z e a s t h e m a t u r e s u b u n i t p u r i f i e d from dog k i d n e y . A p o l y p e p t i d e of t h e same m o b i l i t y w a s o b t a i n e d from t u n i c a m y c i n - t r e a t e d c e l l s ( F i g . 3a and b) a s e x p e c t e d f o r a product t h a t does n o t c o n t a i n asparagine-linked oligosaccharide chains.
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A s was t h e case f o r t h e 8-subunit t h e newly s y n t h e s i z e d a - s u b u n i t l a b e l e d d u r i n g s h o r t (5-min) p u l s e s w i t h [35S]methionine w a s found i n t r a c e l l u l a r l y i n a s s o c i a t i o n w i t h rough microsomal membranes, b e f o r e i t a p p e a r e d i n a f r a c t i o n o f smooth membranes which c o n t a i n e d plasma memb r a n e f r a g m e n t s . A d i r e c t d e t e r m i n a t i o n o f t h e s i t e of s y n t h e s i s of t h e a - s u b u n i t w a s hampered by d i f f i c u l t i e s i n o b t a i n i n g a n 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 of t h e exp e c t e d s i z e , when f r e e o r bound polysomes ( F i g . 3c and d ) o r p u r i f i e d mRNAs ( F i g . 3 f , g , and h ) from MDCK c e l l s were u s e d t o program r e t i c u l o c y t e o r wheat germ t r a n s l a t i o n s y s t e m s . Although o c c a s i o n a l l y a 1 0 0 , 0 0 0 Mr-pyod u c t w a s r e c o v e r e d ( F i g . 3e and h ) , an i m m u n o p r e c i p i t a b l e p r o d u c t of a p p r o x i m a t e l y 85,000 M~ w a s g e n e r a l l y o b t a i n e d and f r e q u e n t l y t h e y i e l d o f t h i s p r o d u c t w a s h i g h e r when It appears f r e e r a t h e r t h a n bound polysomes were used. t h a t t h i s p r o d u c t r e s u l t e d from i n c o m p l e t e t r a n s l a t i o n r a t h e r t h a n from p r o t e o l y t i c d e g r a d a t i o n , as it w a s a l s o found when a combination o f p r o t e a s e i n h i b i t o r s were added t o t h e t r a n s l a t i o n s y s t e m s . R e c e n t l y , i n c o l l a b o r a t i o n with,N. Nabi, w e have found t h a t i n c o n t r a s t t o t h e s i t u a t i o n w i t h polysomes from MDCK c e l l s , i n v i t r o t r a n s l a t i o n o f b r a i n polysomes y i e l d s a p r o d u c t of t h e s i z e e x p e c t e d f o r t h e l a r g e s t ( a + ) o f t h e two forms of t h e a - s u b u n i t c h a r a c t e r i s t i c of b r a i n A T P a s e (Sweadner, 1 9 7 9 ) . I n t h i s i n s t a n c e t h e t r a n s l a t a b l e mRNA w a s o n l y c o n t a i n e d i n bound polysomes. Taken t o g e t h e r , o u r o b s e r v a t i o n s s u g g e s t t h a t , a l t h o u g h a s i z a b l e f r a c t i o n of mRNA f o r a - s u b u n i t may be found i n f r e e polysomes o f MDCK c e l l s where p a r t i a l s y n t h e s i s may t a k e p l a c e , i n s e r t i o n of t h e p o l y p e p t i d e i n t o t h e ER membrane a c t u a l l y i s a c o t r a n s l a t i o n a l e v e n t . Using i n v i t r o systems c o n t a i n i n g microsomal memb r a n e s w e are c u r r e n t l y a t t e m p t i n g t o e l u c i d a t e t h e sequence o f e v e n t s i n v o l v e d i n t h e s y n t h e s i s and i n c o r p o r a t i o n o f t h e a - s u b n i t i n t o t h e membrane and t h e s i t e and mode of a s s o c i a t i o n of t h e two s u b u n i t s i n t o a f u n c t i o n a l complex. The MDCK c u l t u r e c e l l system a l s o a p p e a r s t o p r o v i d e a s u i t a b l e model t o s t u d y t h e mechanisms t h a t est a b l i s h and m a i n t a i n t h e r e s t r i c t e d l o c a l i z a t i o n o f t h e enzyme t o t h e b a s o l a t e r a l domains of e p i t h e l i a l c e l l s and thus determine t h e i r functional p o l a r i t y .
ACKNOWLEDGMENT
W e thank M r . B i l l Dolan, M s . Susan Malamet, and M s . Harriet S n i t k i n € o r t i s s u e c u l t u r e work. We are g r a t e f u l to M r s . Myrna Chung, M r . B r i a n Z i e t l o w , and Ms. Jody C u l k i n € o r a s s i s t a n c e i n t h e The work w a s s u p p o r t e d by N I H g r a n t s p r e p a r a t i o n of t h e m a n u s c r i p t . t o D r . S a b a t i n i AG 01461, GM 2 0 2 7 7 , and AG 00378.
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