Identification and Functional Analysis of the Nuclear 1991

7/31/2019 Identification and Functional Analysis of the Nuclear 1991

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J . M ol . Bio l . (1991) 221, 225-237

Identification and Functional Analysis of the NuclearLocalization Signals of Ribosomal Protein L25 from

Saccharomyces cerevisiaePeter J. Schaap, Jan vant Riet, Conrad L. Woldringh* and Hend rik A. Raubt

B io c h e m is c h L a b o r a to r iu m , Vr i j e Un iv e r s i t ei t , d e Bo e lela a n 1 0 8 31 0 8 1 H V A m s t er d a m , T h e N e t h e rl a n d s

S e&e M ol. Cy to logie , V ak groep M ol. Celbio logie, U nivers i t e i t va n A m sterdamPlant age Muidergracht 14 , 1018 TV Am sterdam , The N ether lands

(Received 4 M arch 1991; accepted 14 Ma y 1 9 9 1 )

T h e r e g i on s o f t h e l a r g e s u b u n i t r i b o s om a l p r o t e i n L 2 5 f r o m S a c ch a r o m y c es c e re vi s ia er e s p o n s i b l e f or n u c le a r l oc a l iz a t i o n o f t h e p r o t e i n w e r e i d e n t i f ie d b y c on s t r u c t i n g f u s i ong e n e s e n c od i n g v a r i o u s s e g m e n t s o f L 2 5 li n k e d t o t h e a m i n o t e r m i n u s o f / I -g a l a c t o s id a s e .I n d i r e c t i m m u n o fl u o r e s c e n c e o f y e a s t c e ll s e x p r e s s i n g t h e f u s i on s d e m o n s t r a t e d t h a t a m i n oa c i d r e s i d u e s 1 t o 1 7 a s w e l l a s 1 8 t o 4 1 o f L 2 5 p r o m o t e i m p o r t o f t h e r e p o r t e r p r o t e i n i n t ot h e n u c le u s . B o t h n u c le a r l o ca l i z a t i on s i g n a l (N L S ) s e q u e n c e s a p p e a r t o c on s i s t o f t w od i s t i n c t f u n c t i o n a l p a r t s : o n e s h o w e d r e l a t i v e l y w e a k n u c le a r t a r g e t i n g a c t i v it y , w h e r e a s t h eo t h e r co n s i d e r a b l y e n h a n c e s t h i s a c t i v i t y b u t d o e s n o t p r o m o t e n u c le a r i m p o r t b y i t s e lf .Mic ro in je c t ion of in vitro p r e p a r e d i n t a c t a n d N - t e r m i n a l ly t r u n c a t e d L 2 5 i n t o X e n o p u sl aev i s o oc y t e s d e m o n s t r a t e d t h a t t h e r e g i on c on t a i n i n g t h e t w o N L S s e q u e n c e s i s i n d e e dr e q u i r e d f or e f fi ci e n t n u c le a r l o c a li z a t i on o f t h e r i b o s om a l p r o t e i n . T h i s co n c lu s i o n w a sc on f i r m e d b y c om p l e m e n t a t i o n e x p e r i m e n t s u s i n g a y e a s t s t r a i n t h a t c on d i t i o n a l l ye x p r e s s e s w i l d -t y p e L 2 5 . T h e l a t t e r e x p e r i m e n t s a l s o i n d i c a t e d t h a t a m i n o a c id r e s i d u e s 1 t o4 1 o f L 2 5 a r e r e q u i r e d f or fu l l f u n c t i o n a l a c t i v it y o f y e a s t 6 0 S r i b o s om a l s u b u n i t s . Y e a s tc e ll s e x p r e s s i n g f or m s o f L 2 5 t h a t l a c k t h i s r e g i o n a r e v i a b l e , b u t s h o w i m p a i r e d g r o w t h a n da h i g h l y a b n o r m a l c e ll m o r p h o l o g y .

K e y w o r d s : n u c le u s ; n u c le a r t a r g e t i n g ; r i b o s o m a l p r o t e i n ; y e a s t ; Xe n o p u s la e v i s

1 . In t roduc t ionT h e b i og e n e s i s o f e u k a r y o t i c r i b o s o m e s i s a

c om p l e x p r o c e s s w h i ch i n y e a s t r e q u i r e s t h e c o-o r d i n a t e p r o d u c t i o n a n d a s s e m b l y o f a b o u t 8 0d i f fe r e n t r i b o s o m a l p r o t e i n s ( r - p r o t e i n s t ) a n d f ou rr R N A s p e c i es ( fo r a r e v i e w , s e e R a u e & P l a n t a ,1991). W h e r e a s t h e m e c h a n i s m s r e s p o n s ib l e f or t h ec o- or d i n a t e s y n t h e s i s o f t h e n u m e r o u s r i b o s om a lc om p o n e n t s h a v e b e e n e x t e n s i v e l y s t u d i e d , m o s t o ft h e p a t h w a y o f r i b os o m e a s s e m b l y i n e u k a r y o t e sr e m a i n s t o b e c h a r t e d . A s s e m b l y o f e u k a r y o t i c r i b o -s o m e s i s p r e d o m i n a n t l y a n u c le a r p r o ce s s . T h u s ,m o s t n e w ly s y n t h e s i z ed r - p r ot e i n s h a v e t o b e

t A u t h o r t o w h o m co r r e sp o n d e n c e s h o u l d b ea d d r e s s e d .

$ A b b r e v i a ti o n s u s e d : r -p r o t e i n , r i b o s o m a l p r o t e i n ;F I T C , f l u o r e s c e i n i s o t h i o c y a n a t e ; D A P I , 4 , 6 -d i a m i d i n o -2 - p h e n y l i n d o l e ; C S L M , c o n f o c a l s c a n n i n g l a s e rm i c r o s c o p y ; N L S , n u c l e a r l o c a l i z a t i o n s i g n a l .

i m p o r t e d i n t o t h e n u c l eu s , w h e r e t h e y a s s oc ia t es to ic h iom e t r i c a l ly w i t h p r e c u r s o r -r R N A ( p r e -r R N A ), w h i c h i s c on c o m i t a n t l y p r o c e s s e d i n t om a t u r e r R N A m o l ec u le s . T h e n e w l y fo r m e d 4 0 Sa n d 6 0 S r i b os o m a l s u b u n i t s a r e t h e n t r a n s l oc a t e di n t o t h e c y t op l a s m t o f u n c t i o n i n t r a n s l a t i o n . T h ef or m a t i o n o f e u k a r y o t i c r i b o s o m e s , t h e r e f o r e ,e n t a i l s i n t e n s i v e t r a f fi c o f p r o t e i n s a n d r i b o n u c l e o -p r o t e i n p a r t i cl e s i n b o t h d i r e c t i o n s a c r o s s t h e d o u b l em e m b r a n e s e p a r a t i n g t h e n u c l ea r co m p a r t m e n tf r o m t h e c y t o p la s m . T h i s t r a f fi c a p p e a r s t o b e c h a n -n e l e d e x c lu s i v e l y t h r o u g h t h e n u c le a r p o r e s( F e l d h e r r e t a l . , 1 9 8 4 ; D w o r e t z k y & F e l d h e r r , 1 9 8 8 ;B a t a i l l e e t a l . , 1 9 9 0 ), g r o m m e t - l ik e s t r u c t u r e s t h a ts p a n t h e n u c le a r e n v e l o p e ( Al l en & D o u g l a s , 1 9 8 9 ;Ake y, 1990) .

D e l e t i o n a n a l y s i s a n d s i t e -s p e c if ic m u t a g e n e s i s o fa n u m b e r o f v e r t e b r a t e a n d y e a s t n u c le a r p r o t e in s ,a s w e l l a s s t u d i e s u s i n g f u s i o n s o f v a r i o u s p a r t s o fs u c h p r o t e i n s t o r e p o r t e r p o l y p e p t i d e s n o r m a l l y

0 1991 Academ ic P r es s L im i ted022-2836/91/170225-13 $03.00/O 22 5


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P . J . S c h a a p et a l.

r e t a i n e d i n t h e c yt o p la s m , h a v e es t a b l is h e d t h a tn u c le a r i m p o r t d e p e n d s u p o n t h e p r e s e n c e o f o n e o rm o r e s p e c if ic s h o r t a m i n o a c id s e q u e n c e s ( fo rr e v i e w s , s e e R o b e r t s , 1 9 8 9 ; S i l v e r , 1 9 9 1 ; G a r c i a -B u s t o s e t al. , 1991) . The se nuc le a r loc a l i z a t ions i g n a l s ( N L S ) , w h i c h m a y b e l o ca t e d a t a n y p os i t i onw i t h i n t h e p o l y p e p t i d e c h a i n . a p p e a r t o i n t e r a c tw i t h r e ce p t o r s t h a t a r e p a r t o f t h e n u c l e a r p o r ec om p l e x ( L i & T h o m a s , 1 9 8 9 ; L e e & M e l e s e , 1 9 8 9 ;S i lve r et al . , 1 9 8 9 ; B e n d i t t e t al. , 1989) , a f t e r whic ht h e p r o t e in i s t r a n s l oc a t e d t h r o u g h t h e p o r e b y a ne n e r g y -d e p e n d e n t m e c h a n i s m (R i ch a r d s o n et al . ,1 9 8 8 : N e w m e y e r & F o r b e s , 1 9 8 8 ; K a l i n i ch &D o u g l a s . 1 9 8 9 : A k e y & , Goldfa rb , 1989) .F u r t h e r m o r e , c y toso l i c p ro te in s th a t spe c i f ic a l lyr e c o g n i z e N L S s e q u e n c e s h a v e b e e n d e s c r i b e d(Ada m et al. , 1 9 8 9 ; A d a m , 1 9 8 9 ; Y a m a s a k i et al . ,1 9 8 9 ), s u g g e s t i n g t h a t n u c le a r p r o t e i n s m a y b eg u i d e d t o t h e p o r e b y t h e s e p r o t e i n s .

T h e m o s t e x t e n s i v e ly s t u d i e d N L S i s a s e q u e n c eo f s e v e n a m i n o a c id r e s id u e s w i t h i n t h e s i m i a n v i r u s4 0 ( S V 4 0 ) la r g e T - a n t i g e n ( K a l d e r o n et al. , 19843) .A l t h o u g h s p e c if ic r e s i d u e s c r i t i ca l f o r n u c le a r i m p o r th a v e b e e n i d e n t i f ie d i n t h i s p r o t o t y p a l N L S( K a l d e r o n et al. . 1 9 8 4 a ) a s w e l l a s i n s e v e r a l o t h e r s( M o r e l a n d e t al . , 1 9 8 7 ; W y c h o w s k i et al . . 1 9 8 7 ;S h a u l s k y rt al. . 1990; U n d e r w o o d & F r i e d , 1 9 9 0 ) ,t h e r e a p p e a r t o b e f e w c on s t r a i n t s u p o n t h e s p e c if ica m i n o a c i d s eq u e n c e o f a N L S . a p a r t f r o m a p r e -d o m i n a n c e o f b a s i c a m i n o a c i d r e s i d u e s . H a l l e t a l .( 1 9 8 4 ) h a v e p r o p o s e d a s e c on d t y p e o f N L S o n t h eb a s i s o f s t u d i e s o n n u c l e a r l o ca l i z a t i on o f t h e y e a s tM A T c r 2 p r o t e i n . I n t h i s t y p e , a m o t i f c on s i s t i n g o ft w o b a s i c a m i n o a c id r e s i d u e s f l a n k i n g a s t r e t c h o ft h r e e h y d r o p h o b i c r e s i d u e s . o n e o f w h i c h i s ap r o li n e . i s t h o u g h t t o b e t h e co m m o n s t r u c t u r a lt h e m e .

Y e a s t r - p r o t e i n s . r a n g e i n s i z e f r o m a b o u t 3 . 5 t oa b o u t 4 4 k D a ( cf . R a u B et al . , 1990), we l l below thelimit of 60 to 70 k D a a b o v e w h i c h f r e e p a s s a g et h r o u g h t h e n u c le a r p o r e l )y p a s s i v e d i f fu s i o nb e c om e s s e v e r e l y r e s t r i c t e d ( fo r a r e v i e w . s e eR o b e r t s . 1 9 8 9 ). T h u s , r i b o s om a l p r o t e i n s c o u l ds i m p l y d i f fu s e i n t o t h e n u c le u s a n d a cc u m u l a t et h e r e b y b i n d i n g t o a n u c le a r c om p o n e n t ( e .g . p r e -r R N A ). N e v e r t h e l e s s , t w o p r o t e i n s o f t h e l a r g e s u b -u n i t o f y e a s t r i b o s om e s ( I ,3 (M o r e l a n d ct al. , 1 9 8 5 )a n d L 2 9 ( U n d e r w o o d $ F r i e d , 1 9 9 0 ) ), a s w e l l a s o n es m a l l s u b u n i t p r o t e i n ( r p 5 1 ( G r i t z et al . , . 1985)) h a v eb e e n s h o w n t o c on t a i n s p e c if ic s e q u e n c e s t h a t a r ea b l e t o m e d i a t e e f fi ci e n t n u c le a r i m p o r t o f a nEscher ich ia co l i P - g a l a c t o s id a s e r e p o r t e r t h a t i sn o r m a l l y d e n i e d e n t r y i n t o t h e y e a s t n u c l e u s .I n a c t i v a t i o n o f t h e N L S s e q u e n c e s o f L 2 9 r e s u l t e di n i m p a i r e d g r o w t h o f t h e y e a s t c e ll s c a r r y i n g t h em u t a t i o n . w h i c h f u r t h e r s u p p o r t s t h e i r r o le i nn u c l e a r i m p o r t o f t h i s p r o t e i n ( U n d e r w o o d & F r i e d ,1 9 9 0 ) . W h i l e t h e e x p e r i m e n t a l d a t a d o n o t f or m a l l ye x c lu d e t h e p o s s i b il i t y t h a t t h e r - p r o t e i n s e g m e n t si n q u e s t i o n c a u s e s p e ci fi c n u c l e a r r e t e n t i o n o f t h ep r o t e i n o f w h i ch t h e y a r e p a r t : r a t h e r t h a np r o m o t i n g i t s n u c l e a r u p t a k e , s e v e r a l i n d i r e ct a r g u -m e n t s s u p p o r t t h e v i ew t h a t i m p o r t o f t h e t h r e e

y e a s t r - p r o t e i n s i n q u e s t i o n i n t o t h e n u c le u s i s a na c t iv e p r o ce s s t h a t d e p e n d s o n t h e s e s eg m e n t ) sa c t i n g a s N L S s e q u e n c e s ( s e e d i s c u s s i on i nU n d e r w o o d & F r i e d , 1 9 9 0 ).

I n o r d e r t o e x t e n d o u r k n o w l e d g e o f t h e w a y i nw h i c h e ff ic ie n t n u c le a r i m p o r t o f e u k a r y o t i c r - p r o -t e i n s is a s s u r e d . w e h a v e a n a l y z e d s e v e r a l a d d i t i on a ly e a s t r - p r o t e i n s f or t h e p r e s e n c e o f N L S s e q u e n c e su s i n g / J - g a l a ct o s id a s e f u s i on p r o t e i n s . H e r e w er e p o r t t h e i d e n t i f ic a t i on . a s w e l l a s t , h e p a r t i a l f u n c -t i o n a l ch a r a c t e r i z a t i o n , o f t w o s u c h N L S s e q u e n c e si n t h e r - p r o t e i n L 2 5 . a l a r g e s u b u n i t p r o t e i n t h a t b i n d s t o a n r v o l u t i o n a r i l p c on s e r v e d r e g i o n w i t h i nd o t n a i n T I T o f 2 6 S r R N A ( E l -B a r a d i rt al . . 1 9 8 5 )a n d h a s h o m o l og s i n b o t h b a c t e r i a a n d c h l or o p l a s t s( R a u P e t al. , 1 9 8 9 ) . O u r d a t , a c le a r l y s u p p o r t t h ec on c lu s i on t h a t t h e s e N L S s e q u e n c es a r e r e q u i r e df or e ff ic ie n t t r a n s p o r t o f I, 2 5 i n t o t h e n u c le u s .F u r t h e r m o r e . b o t h a r e l oc a t e d i n a s e g m e n t o f t h ep r o t e i n c om p l e t e l y s e p a r a t e f r o m t h e d o m a i nr e c e n t l y d e f in e d b y u s ( R u t g e r s rt al. . 1990, 1991) a sb e i n g b o t h n e c e s s a r y a n d s u f i ci e n t f or b i n d i n g o fI , 2 5 t o 2 6 8 r R N A .

2 . Mater ials and Methods

Escherichia coli DHl (F-. re ci l l . e ndA l , y y rA M, t h i - I .h sdR 17 ( r ; . m:), supE44. reldl, I- (Hanahan. 1983)) wasused for plasmid transfo rmation and propagation. E. colist,rain JMlOl (thi. A(luc-~ToAB). sup!Z. (F : traD36.p r o A B , la0ZAM 15)) was used for propagation of Ml3(Yanish-Perron e t al . . 1985 ) .

S ac c harom y c es c e re oi s iae KWGI-7X (MATa. h i s G 1 . Y .Led&3, l e u2 - 112 . u , de l - 100 . ~ ~ 3- 55 ) or its derivativeYC R61 were used as host skains in all experiments.YCR 61 (formerly called BW GI-7A/42) is a derivative ofBWG l-7A in which the chromosotnal L2.5 gene has beenplaced under control of the (iAL 10 up&ream activatingsequence (Rutgers of ccl.. 1990 ). Yeast transfortnationswere carried out according to the method described by Ttoe t a l . (1983). Transformant)s were grown on O.7?,, (w/v)yeast nitrogen base without amino acids (YXR: Difco)containing 2 O;, (w/v) glucose and 50 pg of each of therequired amino acids/l.

(b) EnzywLes a n d r e co m b i n a n t I )L V A t u c h n i y w sRestriction enzymes and Ba/3 1 exonuclease were

purchased from B R L ( G a i t h e r s b u r g , M L ) ) a n d u s e daccording to the suppliers instructions. DKA manipu la-tions were carried out essentially as described by Man iatise t al . (1982) . Oligonucleotidrs were synthesized in ou rlaboratory using an Applied Biosystems 38lA 1)X-Asynthesizer.

(c) C lons t v uc t ion qf 1acZ f u s ion y e ne . rAll ZacZ fusion genes were constructed using the

Bum HI site located close to the 5-end of the la& codingregion present in t,he multicopy yeast shuttle vectorpLGSD 5 (Guarente rt al.. 1982: Fig. l(a)). The varioustypes of fusion employed are depicted schemat,ically inFig, 1. All fusion penes were u nder t,he control of the


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N u c l ea r I m p o r t o f L Z 5 2 2 1

(a ) pLGSD.5

pLGSac

(b )pBMCY138

MU-L25

ML%L25Nco

(c)M13-N

M13.I

(d )

UASG

B SC B

B c W e x S m

E Sl B

F i g u r e 1 . C o n s t r u c t i o n o f g e n e s e n c o d i n g v a r i o u s L 2 5 - /I - g a l a c t o s i d a s e f u s i o n p r o t e i n s a n d o f c h i m e r i c L 2 5 g e n e s . B e n ta r r o w s c o r r e s p o n d t o t r a n s c r ip t i o n i n i t i a t i on s i t e s . T h i n l in e s i n d i ca t e v e ct o r s e q u e n c e s . D i ff e r e n t m o d u l e s u s e d i n t h ec o n s t r u c t i o n a r e s h o w n i n d i f fe r e n t s h a d i n g . R e s t r i c t i o n s i t e s a r e i n d i c a t e d a s f o ll o w s : B , B a m H I ; E , E c o R I ; H , H i n d I I I ;H p , B p a I ; K , K p n I ; N s , N & I ; N C , N c o I ; P , P & I ; R , R s a I ; S C , S a c I ; S l , S u E I ; S m , S m a I . T h e p r e f i x e x - i n d i c a t e s ac l e a v a g e s i t e l o s t b y m o d i f i ca t i o n . ( a ) P a r t i a l r e s t r i c t i o n m a p o f t h e U A S o - C Y C l - Zu c Z i n s e r t o f p l a s m i d p L G S D 5( G u a r e n t e e t a l . , 1 9 8 2 ) . T h e o l i g on u c l e ot i d e i n s e r t e d t o o b t a i n p l a s m i d p L G S a c i s s h o w n . ( b ) P a r t i a l r e s t r i c t i o n m a p s oft h e L 2 5 in s e r t s o f p l a s m i d p B M C Y 1 3 8 ( L e er e t a l ., 19 8 4 ) a n d M 1 3 -L 2 5 . R i n d i ca t e s t h e 2 u p s t r e a m a c t i v a t in g s e q u e n c e s( R P G b o x e s ) o f t h e L 2 5 g e n e . ( c) G e n e r a l s t r u c t u r e o f t h e i n t e r m e d i a t e s u s e d i n c on s t r u c t i n g Z u c Z f u s i on g e n e s ca r r y i n gN - t e r m i n a l ( M 1 3 - N ) o r i n t e r n a l ( M 1 3 - I ) p o r t i o n s o f t h e L 2 5 c o d i n g r e g i o n . ( d ) G e n e r a l s t r u c t u r e o f t h e in s e r t o f p l a s m i dY C p A L 2 5 .

G A L l -1 0 u p s t r e a m a c t i v a t i n g r e g i on ( U A S ,) , t h u sa l l o w i n g c o n d i t i o n a l e x p r e s s i o n i n o r d e r t o a v o i d p o s s i b l ep r o b l e m s d u e t o t o x i c it y o f t h e f u s i o n p r o t e i n s .

T h e N s i I - H i n d 1 1 1 f r a g m e n t o f p l a s m i d p B M C Y 1 3 8 ,c o n t a i n i n g t h e c o m p l e t e c o d i n g r e g i o n o f L 2 5 ( L e e r e t a l . ,1 9 8 4 ; F i g . l (b ) ), w a s cl o n e d b e t w e e n t h e P s t I a n d H i n d 1 1 1s i t e s o f t h e M l S m p l l p o l y l i n k e r ( M e s s i n g , 1 9 8 3 ) . T h er e s u l t i n g M 1 3 - L 25 p l a s m i d w a s l i n e a r i ze d b y c le a v i n g a te i t h e r theKpn1 o r t h e S a c 1 s i t e w it h i n t h e L 2 5 c od i n gr e g i o n , a n d t h i s w a s f o ll o w e d b y l i m i t e d d i g e s t i o n w i t hB u Z 3 1 e x o n u c l e a s e ( F i g . l (b ) ). F r a g m e n t s c o n t a i n i n g a5 - t e r m i n a l s e g m e n t o f t h e L 2 5 c od i n g r e g i o n w e r e l i b e r -a t e d b y c le a v a g e a t t h e E c o R I s i t e p r e s e n t i n t h e p ol y -l i n k e r 5 - d is t a l t o t h e L 2 5 s eq u e n c e . T h e s e f r a g m e n t s w e r er e c l on e d b e t w e e n t h e E co R I a n d S m u I s i t e s o f M 1 3 m p 1 8( Y a n i s h - P e r r o n e t u Z . , 1 9 8 5 ) , g i v i n g r i s e t o t h e M 1 3 - Ns e r i e s o f p l a s m i d s ( F i g . l (c )) . T h e p r e c i s e 3 - e n d p o i n t o ft h e L 2 5 s e q u e n c e i n e a c h s e le c t e d p l a s m i d w a s d e t e r m i n e db y s e q u e n c i n g a c co r d i n g t o t h e d i d e o x y c h a i n t e r m i n a t i o nm e t h o d ( S a n g e r e t u Z . , 1 9 7 7 ).

G e n e s e n c o d in g f u s i on p r o t e i n s t h a t c on t a i n v a r i o u sN - t e r m i n a l s e q u e n c e s o f L 2 5 w e r e c on s t r u c t e d b y r e p l a c -i n g t h e X h o I -B a m H I f r a g m e n t o f p L G S D 5 ( F i g. l (a ) )w i t h t h e S a Z I - B u m H I f r a g m e n t f r o m t h e a p p r o p r i a t eM 1 3 - N p l a s m i d . I n t h i s w a y , i n - fr a m e f u s i on s o f t h e5 - t e r m i n a , l r e g i o n o f t h e L 2 5 g e n e ( in c l u d i n g 5 - fl a n k i n g

s e q u e n c e s u p t o t h e N s i I s i t e ) t o t h e 3 r d c o d on o f t h e l a &g e n e w e r e o b t a i n e d .

G e n e s e n c o d i n g f u s i on p r o t e i n s t h a t c on t a i n v a r i o u si n t e r n a l s e q u e n c e s o f L 2 5 w e r e c on s t r u c t e d u s i n g p l a s m i dM 1 3 - L 2 5 N co . T h i s p l a s m i d w a s d e r i v e d f r o m M 1 3 - L 2 5 b yo l i g on u c l e ot i d e - d i r e c t e d m u t a g e n e s i s , w h i c h c r e a t e d a nN c o I s i t e a t t h e A T G i n i t i a t i o n c o d o n o f t h e L 2 5 c o d i n gr e g i on ( J . J . v a n d e n H e u v e l & H . A . R a u e , u n p u b l i s h e dr e s u l t s ; F i g . l (b ) ). A f t e r c l e a v a g e o f M1 3 - L 2 5 N c o w i t hN c o I a n d B u m H I , t h e N c o I s it e w a s m a d e b l u n t u s i n gp h a g e T 4 D N A p o l ym e r a s e . T h e p l a s m i d w a s t h e n li g a t e dw i t h t h e R s u I - B u m H I f r a g m e n t o f t h e a p p r o p r i a t eM1 3 - N p l a s m i d . T h e r e s u l t i n g M1 3 - I c on s t r u c t s ( F i g . l ( c) )w e r e c h e ck e d b y s e q u e n c i n g . F i n a l l y , t h e S a Z I I B u m H If r a g m e n t s o f M 1 3 - I p la s m i d s c on t a i n i n g t h e d e s i r e di n t e r n a l p o r t i o n o f t h e L 2 5 co d i n g r e g i on , p r e c e d e d b y a nA T G c o d on , w e r e u s e d t o r e p l a c e t h e X h o I -B a m H I f r a g -m e n t o f p L G S D 5 .

G e n e s e n c o d in g f u s i on s p r o t e i n s t h a t co n t a i n a m i n oa c i d r e s i d u e s 1 1 t o 1 7 o r 1 8 t o 2 8 o f L 2 5 , t h e S V 4 0 l a r g eT - a n t i g e n N L S o r a m i n o a c i d r e s i d u e s , 2 t o 1 3 o r 2 t o 8 o ft h e y e a s t M A T u 2 p r o t e i n , w e r e g e n e r a t e d b y i n s e r t i n g t h ea p p r o p r i a t e s e q u e n c e i n t o t h e B u m H I s i t e o f p L G S D 5u s i n g s y n t h e t i c d e o x y ol i go n u c l e ot i d e s . D e p e n d i n g u p o nt h e o r i en t a t i on , i n s e r t i o n r e c r e a t e d a B u m H I s i t e e i t h e ru p s t r e a m o r d o w n s t r e a m ( co r r e ct o r i e n t a t i o n ) f r om t h e


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2 28 P. J . S chaap e t a l .

Table 1N - t er m i n a l s e q u e n ce s o f i n t a c t L 2 5 , t h e A L 2 5 p o l y p ep t i d e a n d i t s d e r i v a t i v es

carry ing he tero logous N L8 sequen cest o l y p e p t i d e A m i n o a c i d s e q u e n c e

1 4 0L 2 5 MAPSAKATASKKAVVKGTN(;KKALKVRTSATFRLPKTLKLARAI'A J , 2 5 MTCSRAP...S A L 2 5 MTGsPKKKRKVEWRAPS S A I 2 5. MT~sPKKKRKVE~SPKKKRKVEIXRAIS X A L 2 5 MTGsPKKKRKVEGSTNGKKALKVRT~~RAI'

T h e t o p l i n e d e p i ct s t h e s e q u e n c e o f t h e 4 4 N - t e r m i n a l a m i n o a c i d r e s i d u e s o f L 2 5 . T h e s e q u e n c em o t i f s h a r e d b y t h e 2 N L S s e q u e n c e s i s u n d e r l i n e d . T h e n e x t l i n e s d i s p l a y t h e N - t e r m i n a l s e q u e n c e s o ft h e p r o t e i n t r u n c a t e d a t a m i n o a c i d r e s i d u e 42 (A L 2 6 ) a n d i t s d e r i va t i v e s c a r r y i n g 1 ( S AL 2 5 ) o r 2( S S A L 2 5 ) c o p i e s o f t h e S V 4 0 l a r g e T - a n t i g e n N L S . o r 1 c o p y o f t h i s N L S f o ll o w e d b y a m i n o a c i dr e s i d u e s 1 8 t o 2 8 of L 2 . i i t s e lf ( R N A 1 95 ) . T h e a m i n o a r i d r e s i d u e s a r e g iv e n i n t h e s t a n d a r d I Iettrrcdr. N L R s e q u e n c e s o f t h e f u s i on c o n s t r u c t s a r e m a r k e d w i t h b o l d fa c e . R e s i d u e s o r ig i n a t i n g f r o mn u c ~ l e o t i d e s e q u e n c e s u s e d i n t h e c lo n i n g p r o c e d u r e a r r i n s m a l l t y p e .

i n s e r t e d o l ig o n u c l e o t i d e . a l l o w i n g u s t o e s t a b l i s h i t s o r i e n -t a t i o n b . y r e s t r i c t i on a n a l y s i s .

(d ) (onstr uct ion of th e A L 2 5 q p n e a n d d r r i v a t i v r sT h e s e c o n n t r u & a r e d e p i c t e d s c h e m a t i c a l l y i n

F i g . I ( d ) . T h e H i n d H I - E c o R I f r a g m e n t o f p B M C Y 1 3 8( F i g . l ( b )) w a s c l on e d i n t o t h e p o l yl i n k e r o f t h e s i n g l e -c o p y y e a s t s h u t t l e v e c t o r Y C p L a c 2 2 ( G i e t z & S u g i n o ,1 9 8 8 ) t o g i v e Y Q L 2 5 . N e x t . a s y n t h e t i c d e o x y o l i g o n u c -l e ot i d e w a s c lo n e d i n t o t h e B a r n H I s i t e o f p L G S D 5 t oc r e a t e a S a c I s i t e i m m e d i a t e l y d o w n s t r e a m f r o m t h e( r e s t o r e d ) B a l r L H T s i t e ( F i g . l ( a )) . T h e r e s u l t i n g p L G S a cp l a s m i d w a s l in e a r i z e d w i t h X h o I a n d t h e 5 . ov e r h a n g i n ge n d w a s m a d e b l u n t w i t h t h e a i d o f T 4 D N A p o l ym e r a s e .T h e l in e a r p l a s m i d w a s c le a v e d w i t h & X I a n d t h e l ib e r -a t e d X h o I -S a c 1 f r a g m e n t w a s t h e n u s e d t o r e p l a c e t ,h rA \ r T s i I -A S a c Tr a g m e n t o f t h e T J 2 5 i n s e r t o f Y (l p T ,2 5 i n t h ef o ll o w i n g w a y . Y (p L 2 5 w a s l i n e a r i z e d w i t h N s i I a n d t h er e s u l t i n g e n d w a s m a d e b lu n t u s i n g T 4 D N A p o l ym e r a s r .T h e l in e a r p l a s m i d w a s t h e n c l e a v e d w i t h S a c I t o r e m o v et h e 5 . e n d o f t h e 1 ~ 2 5 c o d i n g r e g i o n ( F i g . l ( b )) . T h er e s u l t i n g p l a s m i d w a s l i ga t e d w i t h t h e X h o T - - f J a e I f r a g -m e n t f r o m p L G S a c . T h e r e s u l t w a s p l a s m i d Y C p L 2 5e n c o d i n g a m i n o a c i d r e s i d u e s 4 2 t o 1 4 2 o f L 2 5 p r e c e d e db y t h e I s t , 4 a m i n o a c i d r e s i d u e s o f / I -g a l a c t o s i d a s e(F ig . 1 (d) . Table I ) .

T h e f o ll o w i n g d e r i v a t i v e s o f Y C p A L 2 . 5 w e r ec o n s t r u r t ) e d b y i n s e r t i n g s y n t h e t i c d e o x y o l i g o n u c l e o t i d e si n t o t h e B a m H T s i t e cl os e t o t h e 5 t e r m i n u s o f t h e c o d in gr e g i o n : Y C p S A L 2 . i a n d Y C p S S A L 2 5 e n c o d e fu s i o n p r o -t e i n s o f I a n d 2 ( t a n d e m ) c o p ie s . r e s p e c t i v e l y . o f t h e S V 4 0l a r g e T - a n t i g e n N L S ( a m i n o a c i d r e s i d u e s 1 2 6 t o 1 3 2 ) t ot h e t r u n c a t e d L b 5 p o l yp e p t i d e ; Y C p S N A L 2 5 , e n c o d e s as i m i l a r f u s i o n p r o t e i n o f t h e S V 4 0 N L S f o l l ow e d b y a m i n oa c i d r e s i d u e s I 8 t o 2 8 o f L 2 5 . T n s e r t i o n o f t h e s e o l i g o -n u c l e ot i d e s r e c s r e a t e d a s i n g l e B a r n H I s i t e e i t h e r u p s t r e a mo r d o w n s t r e a m ( c> o r r e ct o r i en t a t i o n ) f r o m t h e i n s e r t .a l l o w i n g u s t o c h e c k i t s o r i e n t a t i o n b y r e s t r i c t i on a n a l y -s i s . T h e N - t e r m i n a l a m i n o a c i d s e q u e n c e s o f t h e A L 2 5d e r i v a t i v e s a r e g i v e n i n T a b l e 1 .

( e) ~ V ubc e l lu la r l oca l i za t i on o f f i - ga lac tos idas rfu s ion p ro t e in s

C e l ls t r a n s f or m e d w i t h t h e a p p r o p r i a t e l a & f u s i o ng e n e s w e r e g r o w n o n Y N B c on t a i n i n g l a ct a t e a s a c a r b o ns o u r c e ( 1 6 0 m M - l a ct i c a c i d , 1 0 0 m w - s o d i u m - l a c t a t e .a d j u s t e d w i t h 5 M - K O H t o p H 4 5 ) . 5 0 p g e a c h o f a d e n i n e .

h i s t i d i n r a n d l e u c i n e p & r l i t e r ) t o a d e n s i t ? - o f6 x I O 6 c e l ls / m l . ( :a l a c t o s e w a s t h e n a d d e d t o a f i n a l c ogc e n t , r a t , i on o f 2 % ( w / v ) a n d g r o w t h w a s co n t i n u e d f o r 4 h .( e l l s w e r e fi x e d h y s lo w l y a d d i n g t , o t h e cu l t u r e 0 . 1 v o l . o fa 3 7 ; , ( v /v ) a q u e o u s s o l u t i o n o f f o r m a l d e h y d e f o ll o w e db > s h a k i n g a t r o o m t e m p e r a t u r e f or 2 h .T r n r n u n o f l u o r e s ce n c e s t a i n i n g o f t h e y e a s t c e l ls w a s p r r -f o r m e d e s s e n t i a l l y a s d e s c r i b e d ( A d a m s & P r i n g l e , 1 9 8 4 ) .u s i n g m o u s e m o n o c lo n a l a n t i - f i- g a l a c t o s i d a s e ( P r o m r g a )a s t h r 1 s t a n t i b o d y a n d a f lu o r e s c r in - i s ot h i o cy a n a t e -c on j u g a t e d ( F I T (I ) a n t i - m o u s e -I g G ( S ig m a ) a s t h e 2 n da n t , i b o d y . ( J e l l s w e r e m o u n t e d o n p o l y l y s i n e - co a t e d s l i d e si n m o u n t i n g m e d i u m c o m p o s e d o f 0 . 1 O , ; , ( w / v ) p - p h e n y l r -m & a m i n e / m l . 2 0 n g 4 . 6 - d i a m i d i n o - % - p h e n y l i n d o l e( I )A l l ) /m l i n 9 0 9 : ) ( v /v ) g ly c e r o l a n d e x a m i n e d w i t h aZ e i s s m i c r o s r o p e e q u i p p e d f o r e p i f l u o r e s c r n c e m i c r o s c o p y .S t a n d a r d Z e i s s f il t , e r c o m h i n a t i o n s f o r d e t e c t i o n o f D A P Ta n d F t T ( f l u o r e s c e n c e w e r e u s e d .

F o r c o n f oc a l s c a n n i n g l a s e r m i c r o s c o p y ( (S L M) . y e a s t ,( a e ll s w e r e f ix e d . t r e a t e d w i t h t h e 1 s t a n d 2 n d a n t i b o d i esa n d p l a c e d b r t ~ w r r n 2 c ov e r s l ip s . K x c s it a t i o n o f t h e R u o r o -s t e i n c o n j u g a t e w a s p e r fo r m e d b y u s i n g t h e 4 8 2 n m l in eo f a k r y p t o n l a s e r i n c om b i n a t i on w i t h a 5 1 0 n m d i oh r o i c.m i r r o r a n d a 5 2 0 n m b l oc k i n g f il t ,r r . T h e y e a s t c e ll s w e r rs c a n n e d i n I O t o 2 0 s e c t i o n s w i t h a c o n f oc a l m i c r o s c o p r( B r a k e n h o f f s f a l . . 1 9 7 9 ) . C o n d i t i o n s w e r e c h o s e n t o e n s u r ea l i n e a r r e l a t i on s h i p b e t w e e n f l u o r e s ce n c e i n t e n s i t y a n dt h e n u m b e r o f f lu o r e s c en t g r o u p s ( Va n O o s t v e l d t r t ~ 1 . .1 9 7 8 ). E a c h s e c t i o n w a s s cs a n n e d 3 t i m e s t o i m p r o v e t h es i g n a l - t o - n o i s e r a t i o . T h e d i s t a n c e i & w r e n c x jn s e c n t i v ro p t i ca l s r c %i on s w a s 3 0 0 t ,o 5 0 0 n m . F l u o r r s c ~ r n v r i n t e n s i t yd a t , a o f i n d i v i d u a l y e a s t , ce l l s w e r e s t o r e d a s i n t e g e r v a l u e sb e t w e e n 0 a n d 2 5 5 a s a f u n c t i on o f t h e p o s i t i on i n t h ep r e p a r a t i o n , T h r e e - d i m e n s i o n a l i m a g e p r o ce s s i n g w a s p e r -f or m e d u s i n g a V M E - b u s - b a s e d s y s t e m e q u i p p e d w i t h aMo t o r o l a 6 8 0 2 0 p r o c es s o r . A n A p o l l o D X 4 0 0 0 w o r k -s t a t i o n w a s u s e d f or g r a p h i c s r e p r e s e n t a t i on . T h e s o ft w a r eu s e d i s d e s c a r i b r d e l s e w h e r e ( H o u t , x m u l l r r e f r r l . . 1 9 9 0 ) .

(f ) M i c m i n j ec t if m r x p e ri m r n t s[ 3 5 S ] m e t ~ h i o n i n e - l a b e l e d p r o t e i n s ( I ,% . A L 2 5 ) a - e r r

s y n t , h e s i z e d b y i n v i t r o t r a n s c r i p t i o n a n d t r a n s l a t i o n a sd e s c r i b e d ( R u t g e r s r t al . . 1991) . T h e m i r r o i n j e ct i o n t e r h -n i q u r s a n d o oc y t e c .u l t u r e c xo n d i t i on s w e r e a s d e s c r ib e d b yC o l m a n (1 9 8 4 ) . X e nopus 1ae ~ ~ i . s o o y t , r s w e r e i n j e c t e d w i t h7 5 n l o f t h e i n vitro t r a n s l a t i o n m i x t u r e c on t a i n i n g t h e3 5 S - l a b e l e d p r o t e i n . A f t e r i n j e ct i o n , t , h e o o c. y t e s w e r ei n c u b a t e d i n m o d i f ie d B a r t h s a l i n e f or 5 h . N u c l e i w e r ee x t r a c t e d m a n u a l l y b , v m a k i n g a s m a l l in c i s io n i n t h e


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Nuclear I m p o r t ?f L25 229

a n i m a l p o l e o f t h e o o cy t e . T o t a l n u c le a r a n d c y t o p l a s m i cf r a c t i on s f r o m 1 0 t o 1 5 oo cy t e s w e r e h o m o g e n i z e d a t 4 Ci n 4 0 m l S D S s a m p l e b u f f e r ( 6 M - W e a , 1 0 m M - T r i s - H C l(pH 7 .4) ) 2 y . (w/v) S D S , 2 0 m r & m e r c a p t oe t h a n o l, 2 0 y .(v/v) glycerol , O-01% (w/v) b r o m o p h e n o l b l u e ) a n d t h es o lu t i o n w a s c l a r i fi e d b y c e n t r i f u g a t i o n f or 5 m i n a t1 2 , 0 0 0 g i n a n E p p e n d o r f c e n t r i f u g e . T h e s a m p l e s w e r eb o i le d f o r 3 m i n a n d a n a l y z ed b y S D S / p o ly a c r y l a m i d e g e le l e ct r o p h o r e s i s ( L a e m m l i , 1 9 7 0 ) . T h e g e l s w e r e f ix e d ,t r e a t e d w i t h 1 M - s o d iu m s a l i cy l a t e ( C h a m b e r l a i n , 1 9 7 9 ) ,d r i e d u n d e r v a c u u m a n d f lu o r o g r a p h e d u s i n g p r e- fl a s h e dX om a t S X -r a y f il m ( K o d a k C h e m i ca l s L t d , U K ) a t-70C .

(g) In v ivo a na lys i s o f h y b r i d L 2 5 p r o t e i n sY C R 6 1 c e l l s ( R u t g e r s e t aZ . , 1 9 9 0 ) , t r a n s f o r m e d w i t hv a r i o u s d e r i v a t i v e s o f t h e s i n g l e -c op y y e a s t s h u t t l e v e c t o rY C p L a c 2 2 c a r r y i n g t h e co m p l e t e o r 5 - t e r m i n a l l y t r u n -c a t e d f or m s o f t h e L 2 5 g e n e ( s e e s e c t i on ( d ), a b o v e ), w e r e

g r o w n o n Y E B c on t a i n i n g 2 % ( w /v ) ga l a c t o s e a n d 5 0 p ge a c h o f a d e n i n e a n d h i s t i d i n e p e r l i t e r u n t i l m i d -l o g p h a s e( lo 7 c e ll s p e r m l ) . T h e c e ll s w e r e h a r v e s t e d b y b r i e fc e n t r i f u g a t i o n , a n d r e s u s p e n d e d i n 1 0 x t h e or i g i n a l v o l.of YPD m e d ium (1% (w/v) ye a s t e x t r a c t (D i fc o) , 2%(w/v) pe ptone (D i fc o) , 2% (w/v) g luc ose ) . Growth wa sf ol l ow e d b y m e a s u r i n g t h e A ,, , o f t h e c u l t u r e f or a t le a s t5 d o u b l i n g s . D u r i n g t h i s p e r i o d s a m p l e s w e r e a l s oi n s p e c t e d p e r i o d i ca l l y b y p h a s e co n t r a s t m i cr o s c op y .

3. Results(a ) Del inea t ion o f the nu c lear loca l i za t ion

sequences o f L25Y e a s t r - p r o t e i n L 2 5 c on t a i n s 1 4 2 a m i n o a c id

r e s i d u e s a n d h a s a m o l e cu l a r m a s s o f 1 5 . 7 k D a ( s e eR a & et al . , 1 9 9 0 ). W e h a v e p r e v i o u s l y d e s c r i b e d a na n a l y s is o f t h e s u b c e l lu l a r d i s t r i b u t i o n o f t w oL 2 5 / b -g a l a c t o s id a s e f u n c t i o n s t h a t i n d i c a t e d t h a tt h i s r - p r o t e i n d o e s i n d e e d c on t a i n n u c le a r t a r g e t i n gi n fo r m a t i o n t h a t i s c on t a i n e d w i t h i n i t s N - t e r m i n a l6 1 r e s i d u e s ( R u t g e r s et al . , 1 9 9 0 ) . I n o r d e r t o d e -l i n e a t e t h e N L S s e q u e n c e s o f L 2 5 m o r e p r e c i s el y ,s e v e r a l a d d i t i o n a l f u s i on p r o t e i n s c on t a i n i n g v a r i o u s

p a r t s o f t h i s N - t e r m i n a l r e g i o n w e r e s t u d i e d . T h er e s u l t s f o r t w o o f t h e s e f u s i on p r o t e i n s , c on t a i n i n ga m i n o a c i d r e s i d u e s 1 t o 1 7 a n d 1 t o 1 0 , r e s p e c -t i v e l y, o f L 2 5 a r e p r e s e n t e d i n F i g u r e 2 , w h e r e (a )a n d ( b ) s h o w t h a t t h e f ir s t of t h e s e fu s i o n p r o t e i n s i sl oc a l iz e d e x cl u s i v e l y t o t h e y e a s t n u c le u s . T h es e c on d f u s i o n p r o t e i n , h o w e v e r , s h o w s u n e q u i v o ca lc y topla sm ic loca l i z a t ion (F ig . 2 (c) a n d (d) ). F r omt h e s e d a t a w e c on c l u d e t h a t a m i n o a c id r e s i d u e s 1 t o1 7 o f L 2 5 c on t a i n a s t r o n g N L S . B e c a u s e r e m o v a l o ft h e C -t e r m i n a l s e v e n r e s id u e s o f t h i s s eg m e n ta b o l is h e s i t s c a p a c i t y t o p r o m o t e n u c le a r l oc a l iz a -t i o n , r e s i d u e s 1 1 t o 1 7 c on s t i t u t e a n e s s e n t i a l p a r t o ft h i s N L S .

S e v e r a l k a r y o p h i l i c p r o t e i n s , y e a s t r - p r o t e i n L 2 9a m o n g t h e m , c on t a i n t w o i n d e p e n d e n t N L Ss e q u e n c e s ( R i c h a r d s o n et al . , 1986; S iom i et al . ,1 9 8 8 ; D a n g & L e e , 1 9 8 8 ; R o b e r t s , 1 9 8 9 ; H a l l et al . ,1 9 9 0 ; U n d e r w o o d & F r i e d , 1 9 9 0 ) . T h e ex p e r i m e n ts h o w n i n F i g u r e 3 m a k e s i t c le a r t h a t a s im i l a rs i t u a t i on h o l d s fo r L 2 5 . A f u s i on p r o t e i n c a r r y i n gr e s i d u e s 1 8 t o 6 1 o f L 2 5 , a n d t h u s l a c k i n g t h ea m i n o -t e r m i n a l N L S i d e n t i f ie d a b o v e , n e v e r t h e l e s si s l o ca l i z ed e x cl u s i v e l y t o t h e n u c le u s . S i n c e t h es e g m e n t o f L 2 5 e n c o m p a s s i n g r e s i d u e s 4 2 t o 1 4 0 i su n a b l e t o p r o m o t e n u c le a r i m p o r t o f P - g a l a c t o s id a s e( R u t g e r s e t a E . , 1 9 9 0 ), w e co n c l u d e t h a t a t l e a s t o n ea d d i t i on a l s t r o n g N L S is c on t a i n e d w i t h i n t h es e q u e n c e e n c om p a s s i n g a m i n o a c id r e s i d u e s 1 8 t o4 1 . T h e t w o L 25 se g m e n t s t h a t c on t a i n X L Ss e q u e n c e s d o d i s p l a y a c e r t a i n s e q u e n c e s i m i l a r i t y( s e e T a b l e 1 ) . B o t h s e g m e n t s c on t a i n a m o t i fc ons i s t in g of Lys -Lys -Ala fo llowe d by e i th e r tw o ort h r e e h y d r o p h o b i c r e s i d u e s a n d a f u r t h e r l ys i n er e s i d u e . M o r e o v e r , i n t h e N - p r o x i m a l N L S t h i ss e q u e n c e m o t i f c oi n c id e s w i t h t h e s e g m e n t f ou n d t ob e e s s e n t i a l f or i t s fu n c t i on .

A s o u t l i n e d i n I n t r o d u c t i o n , a c t i v e i m p o r t o f p r o -t e i n s i n t o t h e n u c le u s c on s i s t s o f a t le a s t t w o s t , e p s :i n i t i a l i n t e r a c t i o n o f t h e p r o t e i n w i t h a c or n -p o n e n t ( s ) o f t h e n u c le a r p o r e , fo l lo w e d b y e n e r g y -d e p e n d e n t t r a n s l o ca t i o n a c r o s s t h e n u c le a r

F i g u r e 2 . S u b c e l l u l a r l oc a l iz a t i o n o f / ?- g a la c t o s i d a s e f u s i on p r o t e i n s c a r r y i n g a m i n o a c id r e s i d u e s 1 t o 1 7 ( (a ) a n d ( b )) o r1 t o 1 0 ( (c ) a n d ( d )) o f L 2 5 . Y e a s t c e ll s e x p r e s s i n g t h e r e s p e c t i v e f u s i o n p r o t e i n s w e r e a n a l y z e d b y i n d i r e c ti m m u n o fl u o r e s c e n c e u s i n g a s t a n d a r d f lu o r e s c e n c e m i cr o s c op e . ( a ) a n d ( c) F I T C f lu o r e s c e n c e i n d i c a t i n g t h e l o ca t i o n o ft h e f u s i on p r o t e i n I n ( a ) c e ll s w e r e s im u l t a n e o u s l y p h o t o g r a p h e d u s i n g p h a s e c on t r a s t t o v is u a l i z e t h e ce l l b o u n d a r y .( b ) a n d ( d ) D A P I f lu o r e s c e n c e o f t h e s a m e ce l ls , i n d i c a t i n g t h e l oc a t i o n o f t h e n u c l e u s .


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Figure 3 . S u b c e l l u l a r l o ca l i z a t i o n o f / I -g a l a c t o s i d a s ef u s i on p r o t e i n s c a r r y i n g a m i n o a r i d r e s i d u e s 1 8 t o 6 1 o fL 2 5 . (a ) F T T C fl u o r e s c e n c e i n d i c a t i n g t h e l o ca t i o n o f t h ef u s i on p r o t e in . C e l ls w e r e s i m u l t a n e o u s l y p h o t o g r a p h e du s i n g p h a s e c o n t , r a s t t o v i s u a l iz e t h e c e ll b o u n d a r y .( b ) D A P I f l u o r e s c e n c e o f t h e s a m e c e ll s . i n d i c a t i n g t h el o ca t i o n o f t h e n u c l e u s . F o r u n k n o w n r e a s o n s t h e c on c e n -t , r a t i o n s o f b - g a l a c t o s i d a s e f u s i o n p r o t e i n i n t h e n u c l e i o fm o t , h e r a n d d a u g h t e r c el l d o n o t e a l w a y s c or r e s p o n d t , o t h es t , a i n i n g i n t e n s i t i es o f t h e 2 n u c l e i w i t h D A P T .

envelope. An intrinsic d rawback of standard two-dimensional immu nofluorescence microscopy is thatdiscrimination between th e occurrence of only thefirst, step. wh ich results in perinuclear localization,and nuclear import proper is ra,ther difficult.Therefore, we have also analyzed the subcellularlocalization of several of the fusion p roteinsdescribed above by CSLM. Since this techniqu ecombines zero depth-of-field with high resolutionin the viewing plane, it enables one to inspect indivi-dual cells in a series of very thin op tical sections.Moreover. the relative amoun ts of protein in thecytoplasm and nucleus can be determined with goodaccuracy by quan tifying the fluorescence indifferent areas of each section. Figure 1 show s anum ber of optical sections of a yeast cell expressingthe fusion p rotein that carries residues 1 to 17 of125. The FTTC fluorescence is distribu ted evenlythroughout the nucleus in all sections without anytrace of accumulation at the nuclear envelope. We

_Figure 5 . C o n f o c a l s c a n n i n g l a s e r s c a n n i n g m i c r o s c op y

o f y e a s t c e ll s e x p r e s s i n g / I -g a l a c t o s i d a s e f u s i o n p r o t e i n st h a t c a r r y ( a ) r e s i d u e s 1 1 t o I 7 o r (b ) r e s i d u e s 1 8 t , o 2 8 o fL 2 5 . T h e fu s i o n p r o t e i n w a s v i s u a l i z e d b > - i n d i r e c ti m m u n o f lu o r e s c e n c r . I n e a c h cs a s e , o n l y I s e c t i o n f r o ma b o u t t h e m i d d l e o f t h e c e ll is s h o w n .

obt)ained the same result, in t)he case of the fusionprotein carrying residues 18 to 61 (data not shown).It is clear. th erefore. that, both segm ents of 125 areindeed true NLS sequences. since each stronpl?promotes the actual import of the fusion proteininto the nucleus.

In a further set of experiments, we sought toestablish whether the sequence mot,if shared by thetw o SLS sequen ces of L25 is in itself sufficient tocause nu clear accum ulat ion. (ISLM analysis of cellsexpressing fusions of P-galactosida se with eitheramino acid residues 11 t,o 17 or 18 to 28 of 123 do esindeed reveal nuclear import of the two fusion pro-teins (Fig. 5). How ever. quan tification of thefluorescence in the cytoplasm and nucleus of indiv-dua l t,ransformants (see Materials and Methods)shows that, t,he nu clear concentrat ion of each of thetwo fusion prot,eins was only about two- or threefoldgreat,er than its (Bytopla smic concentrat ion. Bysimilar analysis of transformants expressing fusionsthat carry amino acid residues I t,o 17 or 18 to 61.UT found over 98 Cl of the fusion protein in thenu cleus (cf. Figs 2 and 3). Thus. the m otif sharedbetween the two sequences does act as an NLS butat) a relative ly low level of efficiency. It,s f+Fec~tvC-ness is enhanced by add itional amino acid residuesthat . in the case of the N-proximal NLS. a re locatedbetween positions 1 and 11. i.e. upstream from thecommon motif, whereas in the case of the N-distal

Figure 4 . C o n f o ca l s c a n n i n g l a s e r m i c r o s c o p y o f a s i n g l e y e a s t c e l l e x p r e s s i n g a b - g a l a c t o s i d a s e f u s i o n p r o t e i n t h a tc a r r i e s a m i n o a c i d r e s i d u e s 1 t o 1 7 o f L 2 5 . T h e f u s i o n p r o t e i n w a s v i s u a l i z e d b y i n d i r e c t i m m u n o fl u o r e s c e n c r . ( a ) t o ( e )O p t i c a l s e c t i o n n u m b e r s 1 , 4 . 7 . 1 0 a n d 1 4 o f t h e ce l l , r e s p e c t i v e l y . E a c h s e c t i o n w a s 2 0 0 n m t h i c k .


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N u c l e a r I m p o r t of L 2 5 2 31

N L S a n a d d i t i o n a l r e s i d u e ( s ) l oc a t e d d o w n s t r e a mf r o m t h e m o t i f i s r e q u i r e d .

(b ) Fun c t iona l an a lys is o f L25 nu c lear loca l i za t ions i g n a l s i n X e n o p u s l a e v i s oocytes

A lt h o u g h t h e r e s u l t s o f t h e e x p e r im e n t s d e s cr i b e da b o v e a r e i n a g r e e m e n t w i t h t h e n o t i on t h a t t h er e g i o n o f L 2 5 e n c o m p a s s i n g a m i n o a c id r e s id u e s 1 t c4 1 c on t a i n s s e q u e n c e s r e q u i r e d f or e f fi ci e n t n u c le a ri m p o r t o f t h e p r o t e i n , t h e y d o n o t co n s t i t u t e d i r e c tp r o o f o f s u c h a r o l e . T h e r e f or e , w e h a v e s t u d i e d t h es u b c e l lu l a r d i s t r i b u t i o n o f w i l d -t y p e L 2 5 a s w e l l a s at r u n c a t e d f or m l a c k i n g a m i n o a c i d r e s i d u e s 2 t o 4 1( AL 2 5 ) a f t e r i n j e ct i o n i n t o t h e c y t op l a s m o fX . l a e v i s o o cy t e s . B o t h p r o t e i n s w e r e p r e p a r e d b y i nv i t r o t r a n s c r i p t i o n o f t h e r e s p e c t i v e c od i n g r e g i on sc lo n e d i n t h e v e ct o r p G E M 4 u n d e r c o n t r o l o f t h ep h a g e T 7 p r o m o t e r , f ol l ow e d b y i n v i t r o t r a n s l a t i o no f t h e t r a n s c r ip t i n a w h e a t g e r m s y s t e m i n t h ep r e s e n c e o f [ 3 5 S ]m e t h i on i n e ( R u t g e r s et al., 991) .S a m p l es o f t h e in v i t r o t r a n s l a t i on m i x t u r e w e r ei n j e ct e d i n t o o o cy t e s w i t h o u t f u r t h e r p u r i f ic a t i o n .A f t e r i n c u b a t i o n , c yt o p la s m a n d n u c l eu s w e r es e p a r a t e d a n d t h e i r p r o t e in c on t e n t a n a l y z e d b yS D S / p o l y a c r y la m i d e g e l e l e ct r o p h o r e s i s . T h e r e s u l t so f a t y p i c a l e x p e r i m e n t a r e s h o w n i n F i g u r e 6 .C l e a r l y , a l m o s t a l l o f t h e A L 2 5 p o l y p e p t i d e i sr e t a i n e d i n t h e c yt o p la s m , w h e r e a s a b o u t 9 0 % o ft h e w i l d -t y p e p r o t e i n i s f ou n d i n a s s o c ia t i o n w i t ht h e n u c le u s a f t e r t h e f iv e h o u r i n c u b a t i o n p e r i o d .S i n c e w e d i d n o t o b s e r v e a n y s i g n i fi ca n t d i m i n u t i o ni n t h e t o t a l a m o u n t o f t h e l a b e l e d p r o t e i n o v e r ap e r i o d o f 2 4 h ( P . J . S c h a a p , u n p u b l i s h e d r e s u l t s ) ,t h i s d i f fe r e n c e c a n n o t b e d u e t o s e l e ct i v e d e g r a d a -

(a) (b)

F i g u r e 6 . S u b c e l l u l a r d i s t r i b u t i o n o f i n . v i t ro p r e p a r e d( a ) i n t a c t L 2 5 a n d ( b ) L 2 5 l a c k i n g a m i n o a c i d r e s i d u e s 2t o 4 1 a f t e r i n j e c t i o n o f t h e 35 S - l a b e l e d p r o t e i n i n t oX . l a e v i s o o cy t e s a n d i n c u b a t i o n f o r 5 h . T o t a l p r o t e i nf r o m t h e n u c l e i ( l a n e s 1 ) a n d c y t o p l a s m ( l a n e s 2 ) o f 1 5o o c y te s w a s a n a l y z e d b y S D S / p o l y a c ry l a m i d e g e l e l e ct r o -p h o r e s i s . T h e r e l a t i v e a m o u n t s o f r a d i o a c t i v i t y i n t h eb a n d s w e r e q u a n t i fi ed b y s c a n n i n g t h e a u t o r a d i og r a m s .

t i o n . T h e s e r e s u l t s s t r o n g l y s u p p o r t t h e c on c l u s i o nt h a t t h e r e g i o n o f L 2 5 e n c o m p a s s i n g a m i n o a c i dr e s i d u e s 1 t o 4 1 c on t a i n s o n e o r m o r e N L S s e q u e n c e sr e q u i r e d f or n u c le a r i m p o r t o f t h e r - p r o t e i n a n d ,fu r t h e r m o r e , t h a t t h e s e y e a s t N L S s e q u e n ce s a r ea l s o f u n c t i on a l i n v e r t e b r a t e c e ll s .

(c ) An alys is o f the ro le o f am ino ac id res iduesI to 41 in v ivo

C o m p u t e r - a i d e d s t r u c t u r a l c o m p a r i s on h a s c le a r l ys h o w n t h a t y e a s t r -p r o t e i n L 2 5 c on t a i n s a nN - t e r m i n a l e x t e n s i o n o f a b o u t 5 0 a m i n o a c idr e s i d u e s t h a t h a s n o e q u i v a le n t i n i t s b a c t er i a l a n dc h l o r o p l a s t h o m o l o g s ( R a u e et al . , 1989) . In vitrob i n d in g s t u d i e s p e r f or m e d i n o u r l a b or a t o r y d e m o n -s t r a t e d t h a t t h i s e x t e n s i o n i s d i s p e n s a b l e f or s p e c if ica n d f u l ly e f fi ci e n t b i n d i n g o f L 2 5 t o 2 6 S r R N A( R u t g e r s et al . , 1 9 9 0 , 1 9 9 1 ), w h i l e t h e d a t a r e p o r t e da b o v e l o ca t e t h e s e q u e n c e s o f L 2 5 in v o l v e d i n i t sn u c l e a r i m p o r t w i t h i n t h e N - t e r m i n a l e x t e n s io n .T h e s e f in d i n g s s u p p o r t a v i e w o f L 2 5 a s c on s i s t i n g o ft w o i n d e p e n d e n t f u n c t i on a l , a n d p r o b a b l y s t r u c -t u r a l , d o m a i n s .

I n o r d e r t o d e t e r m i n e w h e t h e r t h e N - t e r m i n a le x te n s ion of L25 is r e q ui r e d e xc lus ive ly for e f fi c ie n tt r a n s l o ca t i o n o f t h e p r o t e i n i n t o t h e y e a s t n u c le u s ,w e r e p l a c e d a m i n o a c i d r e s i d u e s 2 t o 4 1 b y v a r i o u sh e t e r o l og o u s N L S s e q u e n ce s . W e t h e n a s s e ss e d t h ea b i l it y o f e a c h o f t h e m u t a n t p r o t e i n s t o s u b s t i t u t efor w i ld- type L25 i n v i v o b y i n t r o d u c in g t h e e n g i n -e e r e d g e n e s i n t o ce l ls o f t h e m u t a n t y e a s t s t r a i nY C R 6 1 . T h i s s t r a i n ( fo r m e r l y c a l le d B W G l -7 A /4 2 )c on t a i n s a w i l d -t y p e c h r o m o s o m a l L 2 5 g e n e u n d e rc on t r o l o f t h e G A L l -1 0 U A S ( U A S , ) a n d , t h e r e f o r e ,i s c om p l e t e l y d e p e n d e n t u p o n p l a s m i d - en c o d e d L 2 5w h e n g r ow n i n g l u co s e- ba s e d m e d i u m ( R u t g e r s etal. , 1990) .

Y C R 6 1 c e ll s w e r e t r a n s f o r m e d w i t h g e n e se n c o d i n g w i l d -t y p e L 2 5 , A L 2 5 (w h i c h l a ck s a m i n oa c i d r e s i d u e s 2 t o 4 1 ) , or A L 2 5 f it t e d w i t h v a r i o u ss e g m e n t s c a r r y in g o n e o r m o r e h e t e r o lo g ou s N L Ss e q u e n c e s ( T a b l e 1 ) . E a c h o f t h e s e g e n e s w a s c lo n e di n t h e s i n g l e -c op y s h u t t l e v e c t o r Y C p l a c 2 2 ( G i e t z &S u g i n o , 1 9 8 8 ) u n d e r c on t r o l o f t h e w i l d -t y p e I ~ 2 5p r o m o t e r , i n o r d e r t o e x c lu d e a n y s p u r i ou s e f fe c t sd u e t o o v e r p r o d u c t i on o f t h e p r o t e i n .

T h e d a t a i n T a b l e 2 s h o w t h a t Y C R 6 1 ce l ls t r a n s -f or m e d w i t h t h e w i l d -t y p e L 2 5 g e n e c a r r i e d o np l a s m i d Y C p L 2 5 , h a v e a d o u b l i n g t i m e of a b o u tt w o h o u r s a f t e r t h e i r t r a n s f e r f r o m g a l a c t o s e- b a s e dm i n i m a l m e d i u m ( Y N B - G a l ) t o g l u c os e - b a s e d Y P Dm e d i u m . T h i s g r o w t h r a t e is i d e n t ic a l w i t h t h a t o ft h e u n t r a n s f o r m e d B W G l -7 A p a r e n t s t r a i n o n Y P Dm e d i u m , c on f i r m i n g o u r e a r l i e r c on c l u s i o n ( R u t g e r set al . , 1 9 9 0 ) t h a t t h e p l a s m i d - en c o d e d w i l d -t y p e L 2 5g e n e fu l l y co m p e n s a t e s f or t h e a b s e n c e o f e x p r e s s i o no f t h e c h r o m o s o m a l g e n e i n Y P D m e d i u m . I t s h o u l db e n o t e d t h a t u n t r a n s f or m e d Y C R G I c el ls g r o ws o m e w h a t s l ow e r o n Y N B -G a l c om p a r e d t o e i t h e rc e ll s c on t a i n i n g t h e Y C p L 2 5 p l a s m i d o r c e ll s o f t h eB W G l - 7 A p a r e n t s t r a i n ( T a b l e 2 ) . A p p a r e n t l y , t h er a t e o f e x p r e s s i o n o f t h e L 2 5 g e n e c on t r o l l ed b y t h e


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2 3 2 P . J . S c h a a p e t a l .

T a b l e 2G r o w t h r a t es a n d m o r p h o l o g y o f Y C R G I c el l s t r a n s f o r m e d w i t h v a r i o u s m u t a n t

f o r m s o f L 2 5

M u t a n t L 2 5 g e n ei n t r o d u c e d

D o u b l i n g t i m e ( h )Y N B - g a l Y P I )

M o r p h o l o g yY N U - g a l Y P I )

N o n e ( s t r a i n B W G I - 7 A ) 2.5 1 .9 N NN o n e ( s t r a i n Y C R G I ) 4 5 NL 2 5 2 . 5 1 . 9 N NA L 1 5 4 . 8 2 4 N AS A L 2 5 4 . 8 lo .5 N AS S A L 2 5 7 .2 1 2 N ATSXAL25 6 .5 9 .8 s A

Y (J R 6 1 c el l s w e r e t r a n s f o r m e d w i t h t h e s in g l e -c op y p l a s m i d Y C p la c 2 2 c a r r y i n g a g e n e e n c od i n gw i l d - t y p e L 2 5 , L 2 5 l a c k i n g a m i n o a c i d r e s i d u e s 2 t o 4 1 (A L 2 5 ) o r d e r i v a t i v e s o f t h e la t t e r c a r r y i n gf or e i g n N L S s e q u e n c e s l a b e le d a s in T a b le 1 . T r a n s f o r m a n t s w e r e g r o w n o n m i n i m a l m e d i u mc on t a i n i n g g a l a ct o s e ( YN B - G a l ) a n d t h e n d i l u t e d i n t o Y P D m e d i u m c on t a i n i n g g l u c o s e . a f t e r w h i c ht h e A , , , o f t h e c u l t u r e w a s d e t e r m i n e d a t r e g u l a r in t e r v a l s . T h e d o u b l in g t i m e s g iv e n w e r e d e r i v e da f t e r s t a b i li z a t io n o f t h e g r o w t h r a t e . T h e m o r p h o l o gy o f t h e c el ls w a s in s p e c t e d b y p h a s e co n t r a s tm i c r o s c o p y , s c o r e d a s : A , a b n o r m a l m o r p h o l o g y ( s e e F i g . 7 ) ; N , n o r m a l m o r p h o l o g y .

U A S G i s l o w e r t h a n t h a t o f a w i l d -t y p e L 2 5 g e n e ,t h u s l im i t i n g t h e g r o w t h r a t e o f u n t r a n s fo r m e dYCR61 c e l l s .

I n co n t r a s t t o YC p L 2 5 -t r a n s fo r m a n t s ! a c u l t u r eo f Y C R 6 1 c e ll s c on t a i n i n g t h e Y C p A L 2 5 p l a s m i ds h o w s o n l y a v e r y s l o w , a l b e i t c o n t i n u a l , i n c r e a s e i na b s o r b a n c e a f t e r t r a n s fe r t o Y P D m e d i u m( T a b l e 2 ) . T h i s r e s u l t is i n a g r e e m e n t w i t h t h e s e v e r er e d u c t i on i n n u c l ea r u p t a k e o f t h e t r u n c a t e d L 2 5e x p e c t e d f r o m t h e r e m o v a l o f i t s N L S s e q u e n c e s .B e c a u s e o f i t s s m a l l s i z e, h o w e v e r , s o m e o f t h e AL 2 5p r o t e i n i s l i k e ly t o e n t e r t h e n u c le u s b y p a s s i v edi f fus ion (Robe r t , s , 1989) , g iv ing r i s e to func t ion a l6 0 S s u b u n i t s a n d t h u s s u s t a i n i n g a (ve ry) l im i t . e da m o u n t o f g r o w t h . I n d e e d . i m p r o v in g t h e n u c le a ri m p o r t o f t h e A l, 2 5 p o l y p e p t i d e b y f u s i n g i t t oe i t h e r o n e o r t w o c o p i e s o f t h e S V 4 0 l a r g e T - a n t i g e nN L S ( YC p S A L 2 5 o r Y C p S S A L 2 5 t r a n s f or m e d c e ll s )s u b s t a n t i a l l y i n c r e a s e s t h e g r o w t h r a t e o f t h e c e ll so n Y P D m e d i u m ( T a b l e 2 ). H o w e v e r , d e s p i t , e t h ef a ct t h a t t h i s h e t e r o l og o u s N L S h a s b e e n s h o w n t ob e h i g h l y e f f ic ien t , i n p r o m o t i n g i m p o r t o f p r o t e i n si n t o t h e y e a s t n u c l e u s ( K a l i n i ch & D o u g l a s , 1 9 8 9 ;N e l s o n $ S i lv e r , 1 9 8 9 ) , n e i t h e r t h e S A L 2 F i n o r t h eS S A L 2 5 p r o t e in s u p p o r t s t h e s a m e g r o w t h r a t e a si n t a c t p l a s m i d - en c o d e d 1 ~ 2 . 5 . A s i m i l a r r e s u l t w a so b t a i n e d f or c el l s e x p r e s s i n g A L 2 5 f it t e d w i t h ac om b i n a t i o n o f t h e S V 4 0 S L S a n d t h e r e g i o ne n c o d i n g a m i n o a c i d r e s i d u e s 1 8 t o 2 8 f r o m i t s o w nN - d i s t a l N L S ( YC p S N A L 2 5 ; T a b l e 2 ). T h e s eo b s e r v a t i o n s s u g g e s t a r o l e f or a m i n o a c i d r e s i d u e s 2t ,o 41 of I ,25 in r i b o s o m e a s s e m b l y a n d / or fu n c t i ono v e r a n d a b o ve t h e i r i n v ol v em e n t i n n u c l e a r i m p o r t .

Mic rosc opic insp e c t ion of c e l ls e xpr e s s in g th ev a r i o u s f or m s o f N - t e r m i n a l l y t r u n c a t e d L 2 5 a f t e rt h e i r t r a n s fe r t , o Y P D m e d i u m s h o w e d t h a t t h ef u n c t i o n a l d e f e ct i n L 2 5 d u e t o r e m o v a l o f a m i n oa c i d r e s i d u e s 2 t , o 4 1 o r t h e i r r e p l a c e m e n t b y ah e t e r o l o go u s N J , S i s m a n i f e s t n o t on l y a s s l o w e rg r o w t h b u t a l s o a s a h i g h l y a b n o r m a l c e ll m o r p h o -logy (F ig . 7 ) . Ce l l s e xc lus ive ly e xpr e s s in g a n y one of

t h e N - t e r m i n a l ly t r u n c a t e d L P 5 p o ly p e p t i d e sd e v e l o p m u l t i p l e , a b n o r m a l ly l a r g e b u d s . A s s h o w nb y t h e D A P T s t a i n i n g ( F i g . 7 (b ) ), m o s t o f t h e s e c el lsc on t a i n a s i n g l e l a r g e n u c le u s , i n d i c a t i n g t h a t t , h e r ei s a d e f e ct i n n u c le a r d i v is i on . F u r t h e r m o r e , Y C R 6 1c e ll s c o- e xp r e s s i n g w i l d -t y p e L 2 5 a n d e i t h e r R S A L 2 5o r S N A L 2 5 m u t a n t , p r o t e i n g r o w m o r e s l o w l y t h a nu n t r a n s f o r m e d h o s t ce l ls , w h i l e c el l s c o- e x p r e s s i n gw i ld - t yp e L 2 5 a n d S A L 2 5 m u t a n t p r o t e i n g r o wn o r m a l l y . C e l l m o r p h o l o g y i s n o t a f fe c t e d i n a n y o fth e se c o-e xpr e s s in g c e l ls (Ta ble 2) . Appa re n t IT , int h e c o- e xp r e s s i n g c e ll s t h e r a t i o o f m u t a n t D W S U . Sw i l d -t y p e L 2 5 i n t h e n u c le u s , w h i c h m a y b ei n f l u e n c e d b y t h e n u m b e r o f N L S s e q u e n c e s c a r r i e db y t h e m u t a n t p o l y p e p t i d e ( fo r a r e v i e w . s e eG a r c i a - B u s t o s et al . , 1 9 9 1 ), h a s t o e x c e e d a c a r r t a i nv a l u e b e f or e a n e E e c + o n g r o w t , h b e c o m e s m a n i f e s t .E:ven i n t h e S S A L 2 5 o r K N A L 2 5 t r a n s f or m a n t s c o-e x p r e s s i n g t h e w i ld - t yp e a n d m u t a n t , p r o t ,e i n s .

F i g u r e 7 . A b n o r m a l c e l l m o r p h o l o g y o f y e a s t Y C R 6 lc el ls t h a t e x p r e s s o n l y L 2 5 la c k i n g a m i n o a c i d r e s i d u e s 2t o 4 1 . Y C R G I c e ll s t r a n s f o r m e d w i t h p l a s m i d Y C p A L 2 5w e r e t r a n s f e r r e d f r o m g a l a c t o s e - b a s e d t o g l u c o s e -b a s em e d i u m . S a m p l e s w e r e t a k e n a t r e g u l a r i n t e r v a l s , s t a i n e dw i t h D A P I a n d i n s p e ct e d b y ( a ) p h a s e c o n t r a s t a n d ( b )i m m u n o f lu o r e s c e n c e m i c r o s co p y . T h e F i g u r e s h o w s ar e p r e s e n t a t i v e c e ll f r om a s a m p l e t a k e n a t 2 4 h a f t e rt r a n s f e r .


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N u c l ea r I m p o r t o f L 2 5 2 3 3

however, this rat io appear s to be insufficient toinduce developmen t of th e abnorm al morphology.

4. DiscussionEfficient a ssem bly of euk ar yotic r ibosomes

depends u pon the rapid upta ke of newly synthesizedribosomal proteins into the nucleus. In order to gainmore insight int o the factors cont rolling nu clearimport of r-proteins, we have initiat ed a sear ch forsegments of yeast r-proteins tha t actively pr omoteaccum ulat ion of th ese proteins in th e nu cleus. H erewe report the resu lts of such an ana lysis on th e 60 Ssubunit protein L25, one of the two yeast r-proteinsso far shown to be able to bind specifically an dindependent ly to high molecular weight rRNA (El-Baradi e t a l . , 1985) an d thu s likely to play a centr alrole in assembly of th e large ribosomal subu nit.

Using conditionally expressed fusions of variousportions of L25 to E. co l i fi-galactosida se, we ha veidentified t wo distinct regions of th is r-protein th atact as highly efficient n uclear localization signa ls i nv iv o , i.e. th ey cau se accumu lat ion of virtu ally all ofth e fusion pr otein inside the yeast n ucleus (Figs 2an d 3). Tran slocation across the nu clear envelopewas observed by mean s of confocal scanning lasermicroscopy (Fig. 4), a techn ique allowing opticalsectioning of th e insp ected cell. The first of th e NLSsequ ences of L25 is locat ed in th e region en com-passing amin o acid residues 1 to 17 (Fig. 2). Thesecond is positioned immediately adjacent to th efirst , but its borders have been defined less precisely(Fig. 3). However, it is likely to extend no fur th erth an amino acid residue 41, since a C-terminal L25fragment star ting at amino acid residue 42 does notstimulate nuclear import of b-galactosidase(Rutgers e t a l . , 1990). Moreover, th e sameC-termina l fragm ent fails to associate preferentiallywith th e nucleus when injected into X. laevisoocytes (Fig. 6). Becaus e fragm ent s of L25 lackingup to 61 N-termina l amin o acid residues do notshow any significant redu ction in affinity for th eL25 binding site on 26 S rRNA (Rutgers e t a l . , 1991)and, thus, appear to fold into their native conforma-tion, th ese data can be tak en a s firm evidence forthe absence of any further NLS sequences C-distalof residue 41.

A more detailed an alysis revealed t hat amino acidresidues 11 to 17 and 18 to 28 of L25, when linked to/I-galactosidase, also show NLS activity. Thesesegments , however, ar e less potent th an th e longerNLS sequ ences defined a bove of which th ey are par t(Fig. 5). The respective tr an sform ant s reach anu cleocytoplasmic concent ra tion ra tio for th e fusionprotein of only 2 to 3. The two su b-segment s show acert ain sequence similarity in th at th ey contain themotif Lys-Lys-(H),_,-Lys in which H rep res ent s ahydr ophobic res idue (Table 1). Clear ly, th e form ofthis motif presen t in th e N-proximal NLS (Lysl2-Lys-Ala-Val-Val-Lys17) const itu tes a genu ine NLS(Fig. 5(a)). However, for full activity, it requ iresamin o acid residu es 1 to 10, a region tha t does not.by itself, act as an NLS. This composite nat ur e is

remin iscent of the situa tion described for th e SV40large T-antigen where nuclear u pta ke, whichdepends absolutely upon am ino acid residues 126 to132, is accelera ted consid era bly by am ino acidresidues 111 to 125, again a region th at, by itself,shows no NLS activity (Rhis & Peter s, 1990).Although L25 contains a serine residue a t position4, it seems un likely tha t ph osphorylation of theNLS enha ncer plays a role in its nu clear import,as has been suggested for t he SV40 large T-antigen(Rhis & Peter s, 1990). Experimen ts in our labora -tory (Kruiswijk e t a l . , 1978) have not provided anyevidence for phosphorylation of L25. It should benoted th at, even with out its enha ncer, the SV40NLS is considera bly more efficient th an eitheram ino acids 11 to 17 or 18 to 28 of L25, since, un derthe sa me conditions as used in the experimentsdescribed above, it cau ses a virtua lly completetr an slocation of /I-galactosidase to th e yeast nu cleus(P. J . Schaap, un published resu lts).

Whether the slightly different form of th ecomm on motif presen t in th e N-distal NLS of L25(Lys21-Lys-Ala-Leu-Lys25) can, by itself, pr omotenu clear im port is not quit e cert ain, s ince the fra g-ment tested (amino a cid r esidues 18 to 28) encom-pass es several additional residues (including t hebas ic Arg27; Ta ble 1) th at ma y be crit ical for NLSactivity. Like tha t of its N-proximal coun terp ar t,however, th e effectiveness of th is second N LS isincreased by a dditional, in th is case C-distallylocated, residues. Our dat a do not allow us toconclude wheth er th is potentiat ing effect is due toan enha ncer as defined above, or is caused by thepresen ce of still anoth er NLS between positions 28and 42. Since basic amino acid residues are bothscarce an d scatt ered between positions 28 an d 42(Table l), we favor the form er explanat ion.

With one exception, the motif shared by the twoNLS sequences of L25 shows no significant simi-larity to any of the limited number of nuclearlocalization signals identified in other yeast proteinsso far (Garcia-Bustos e t a l . , 1991). The exception isth e sequence Lys-Ile-Pro-Ile-Lys (residues 3 to 7)identified by Hall and co-worker s as an importa ntpar t of th e relatively weak , N-term inal NLS of th eyeast MATa protein, report ed to encompa ss aminoacid residu es 1 to 13 (Hall e t a l . , 1984, 1990).However, we failed to observe an y NLS activity ofeither am ino acid residues 2 to 8 or 1 to 13 ofMATtx2 when we tested these segments in the sametype of fusion with /?-galactosidase th at we used toidentify th e NLS sequen ces of L25. This result is inaccordance with th e failur e of peptides consisting ofMATa amino a cid residues 1 to 13 to stimulatenu clear accum ulat ion of seru m albumin (Chelsky eta l . , 1989) or mouse IgG (Lanford et al., 990) invertebra te cells when crosslinked to either of th esepr oteins. Amino acid residu es 1 to 13 of MATcr2,including th e Lys-Ile-Pro-Ile-Lys motif, th erefore,appear to be essential but not sufficient for nucleartar geting activity of th e N-termina l NLS of theMATa protein. A motif similar to th at in theMATa protein wa s shown to be necessary but not


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P. J. Sc ha ap e t a l .

s u f f ic ie n t f or n u c le a r t a r g e t i n g a c t i v it y o f t h e N L Sof X. Zaewis uc le opla sm in (D ingwa l l e t a l . , 1988) .

I n o r d e r t o a s c er t a i n w h e t h e r t h e r e gi on s o f L 2 5i d e n t i f ie d a b o v e a s N L S s e q u e n c e s a r e i n d e e di n v o l v e d i n n u c le a r a c cu m u l a t i o n o f t h e r - p r o t e i ni t s e l f , we s tu die d th e sub c e l lu la r loca l i z a t ion of invitro p r e p a r e d i n t a c t a n d N - t e r m i n a l ly t r u n c a t e dL 2 5 a f t e r i n j ec t i on i n t o X . l aev i s o o c y t e s . T h er e s u l t s o f t h e s e e x p e r i m e n t s ( F i g . 6 ) u n e q u i v o ca l l ys u p p o r t t h e i n v ol v em e n t o f t h e N L S s e q u e n c e s i nn u c le a r i m p o r t o f L 2 5 . V i r t u a l ly a l l o f t h e i n t a c tl a b e l e d L B 5 i s f ou n d t o a s s o c ia t e w i t h t h e o o cy t en u c le u s . W h e n a m i n o a c i d r e s i d u e s 2 t ) o 4 1 a r er e m o v e d , h o w e v e r , n o m o r e t h a n 1 8 % o f t h e A L 2 5p o l y p e p t i d e i s f ou n d t o b e a s s o c ia t e d w i t h t h en u c le u s , a s s h o w n b y s c a n n i n g o f t h e p r o t , e i n b a n d si n F i g u r e 6 . T h i s i s v e r y c lo s e t o t h e 1 4 o /; e x p e c t e di f t h e A L 2 5 p ol y p e p t i d e w e r e d i s t r i b u t e d e v e n l yb e t w e e n n u c le u s a n d c y t o s ol , t h e v o lu m e s o f w h i c ha r e in a p r o p o r t i o n o f 1 : 7.3 in a X . l a e v i s ooc yte(Bonn e r , 1975) . M ore ove r , th e va lue of 18 o /o i s l ike lyt o b e a n o v e r e s t i m a t e , s i n c e a s i g n i fi ca n t a m o u n t o fr a d i oa c t i v it , y r e m a i n e d a t t h e t o p of t h e g e l w h e nth e c y t , oso li c fr a c t ion of ooc yte s in je c te d w i th th et r u n c a t e d L 2 5 w a s a n a l y z e d ( F i g . 6 ( b ). l a n e 2 ) .T h u s , t h e A L 2 5 r e c o v e r e d i n t h e n u c le a r f r a c t i onm o s t l i k e l y r e s u l t s fr o m p a s s i v e d i ff u s i on o f t h i ss m a l l p o l v p e p t i d e i n t o t h e n u c le u s ( R ob e r t I s . 1 9 8 9 ) .T h e r e q m r e m e n t o f a m i n o a c i d r e s i d u e s 2 t o 4 1 f ora c t i v e n u c le a r i m p o r t o f I , 2 5 i s f u r t h e r s u p p o r t e d b yo u r f in d i n g t h a t t ) h e A L 2 5 p o l y p e p t i d e c om p e n s a t e se x t r e m e l y p o o r l y f or t h e a b s e n c e o f i n t a c t L 2 5 i nye a s t YCR61 c e l ls g rowin g on g luc ose . S inc e . inc on t r a s t t o u n t r a n s f o r m e d Y C R A I h o s t c e ll s( R u t g e r s Pt al., 1 9 9 0 ). c e ll s t r a n s f o r m e d w i t h t h eA L 2 5 g e n e d o s u r v i v e a t r a n s f e r t o g l u c os e - b a s e dm e d i u m , a s m a l l a m o u n t o f t ~ h e A L 2 5 p r o t e i n m u s ts t i l l r e a c h t h e n u c le u s , h o w e v e r , t o b e a s s e m b l e di n t o a t l e a s t p a r t i a l ly a c t i v e 6 0 S s u b u n i t s .A s s e m b l y s h o u l d b e p o s s i b le s i n c e . a s a l r e a d ym e n t i o n e d , t h e N - t e r m i n a l 4 1 a m i n o a ci d r e s i d u e s o fL 2 5 d o n o t p l a y a n y r o l e i n r R N A b i n d i n g o f L 2 5( R u t g e r s et aZ.. 1 9 9 0 , 1 9 9 1 ). T n c r e a s i n g t h e r a t e o fn u c le a r i m p o r t o f A L 2 5 b y fi t t i n g i t ) w i t h o n e o rm o r e h e t e r o l og o u s N L S s eq u e n c e s s i g n i fi ca n t l yi m p r o v e s t h e a b i l it y o f t h e r e s u l t i n g p r o t e i n t os u b s t i t u t e f or i t s i n t a c t c ou n t e r p a r t i n g l u c os e -g r o w n Y C R 6 1 t r a n s f o r m a n t s ( T a b l e 2 ) . T h u s ,p a s s i v e d i f fu s i o n a n d r e t e n t i o n b y b i n d i n g t o an u c le a r c om p o n e n t o f e v e n a p o l y p e p t i d e a , s s m a l l a s1 2 k D a , ( t h e m o l ec u l a r m a s s o f A L 2 5 ) c le a r l y i si n s u f f ic ie n t t o s u p p o r t a n y t h i n g l i k e a n o r m a l r a t eo f r i b o s o m e a s s e m b l y . B e c a u s e i n n o r m a l c el l s t h er a t e o f r i b o s o m e b i o g en e s i s i s n o t l im i t e d b y t h ee f fi c ie nc y of nu c le a r im por t o f r -pro t e ins (Ra u C: 8 :P l a n t a , 1 9 9 1 ), w e c on c l u d e t h a t e a c h o f t h e r - p r o -t e i n s a s s e m b l ed i n t h e n u c le u s m u s t r e a c h t h i sc om p a r t m e n t b y m e a n s o f a c t iv e t r a n s p o r t a c r os st h e n u c le a r e n v e l op e , e i t h e r b e c a u s e i t c on t a i n s i t so w n N L 6 s e q u e n c e (s ) o r b e c a u s e i t a s s o c ia t e s w i t ha n o t h e r a c t i ve l y t r a n s p o r t e d ( r -) p r o t e i n . T h e f ea s i -b i l i ty of th e l a t t e r t y p e o f a s s i s t e d i m p o r t ( p i g g y -b a c k in g ) h a s b e e n d e m o n s t r a t e d i n b o t h y e a s t

( M o r e l a n d et a l . , 1 9 8 7 : B o o h e r et a l . , 1989) a ndv e r t e b r a t e c e ll s (D i n g w a l l e t a E . , 1 9 8 2 ; Z h a o &P a d m a n a b h a n . 1 9 8 8 : S h a u l s k y rt al., 1990). T h rr e s u l t s o f t h e o o cy b e i n j e c t io n e x p e r i m e n t s a l s os e r v e t o u n d e r l i n e t h e a b s e n c e o f s p e c ie s - s p e ci fi ci t yo f N L S s e q u e n c e s a l r e a d y i n d i ca t e d b y t h e a b i l it y o ft h e R V 4 0 l a r g e T - a n t i g e n N L S t o f u n c t i o n e f l-c ie n t l y i n y e a s t ( N e l s on & S i l v e r , 1 9 8 9 : K a l i n i ch &D o u g l a s . 1 9 8 9 ) a n d a v a r i e t y o f v e r t e b r a t , e c e ll s( R h i s & , P e t e r s . 1 9 9 0 ) .

E & h e r o f t h e t w o N L S s e q u e n c e s o f L 2 5 b y i t s e l fa p p e a r s t o b e s u f f ic ie n t t o e n s u r e v i r t u a l ly c om p l e t > en u c le a r l oc a l iz a t i o n o f a r e p o r t e r p r o t e i n o f w h i c h i ti s p a r t . O n e m a y . t h e r e f or e , a s k w h y t h e r e a r e t , w os u c h N L S s e q u e n c es i n L 2 5 . I t , s h o u l d b e n o t e d t h a t .f or m a l l y . o u r d a t a d o n o t e xc lu d e t h e p o s s i b il it yt h a t o n l y o n e o f t h e s e s e q u e n c e s i s i n v o l v e d i nn u c le a r i m p o r t o f L 2 5 . H o w e v e r , t h e p r e s e n c e o fm u l t i p l e N L S s e q u e n c e s i s n o t w it h o u t p r e c e d e n te v e n a m o n g t , h e v e r y f e w y e a s t r - p r o t e i n s s o f a ra n a l y ze d . T w o a l m o s t i d e n t i c a l ? 1 L S s e q u e n c e ss e v e n a m i n o a c id r e s i d u e s l o n g h a v e b e e n l o ca t e dc lo s e t o t h e N t e r m i n u s o f L 2 9 (I l n d e r w o o d & F r i e d .1 9 9 0 ) . I n a c t i va t i o n o f f ir s t t h e N - p r o x i m a l a n d t h e nt h e N - d is t a l N L S r e s u l t e d i n p r o g r e s s iv e g r o w t hi m p a i r m e n t o f t h e c e ll s e x p r e s s i n g t h e m u t a n t p r o -t e i n s . w h i ch i n d i c a t e s , t h o u g h i t d o e s n o t p r o v e , ar o l e f or h o t h s e q u e n c e s i n n u c le a r i m p o r t o f t h en a t i v e r - p r o t e i n ( I T n d e r w o o d 8 : F r i e d , 1 9 9 0 ).T n a c t i va t i o n o f o n l y t h e S -d i s t a l N L S , o n t , h e o t h e rh a n d , d i d n o t c a u s e a g r o w t h d e f e ct . i n d i c a t i n g t h a tn u c le a r u p t a k e o f I , 2 9 c on t a i n i n g o n l y t h eN - p r o x i m a l N L S i s n o t r a t e - li m i t i n g f or r i b o s om ra s s e m b l y . a t ) l e a s t a t t h e r e l a t i v e l y s l o w g r o w t h r a t eo b t a i n i n g i n t h i s e x p e r i m e n t ( 0 .3 d o u b l i n g s /h :I n d e r w o o d B F r i e d , 1 9 9 0 ) . T h i s d o e s n o t e x cl u d et h e p o s s ib i l it y . h o w e v e r , t h a t m u l t i p l e N L Ss e q u e n c e s a r e r e q u i r e d t o a ll o w c e l ls t o g r o w a t ) t h e i ?m a x i m u m r a t , e u n d e r op t i m a l c on d i t i on s .R e i t e r a t i o n o f a n N L S s e q u e n c e h a s b e e n cl e a r I !-s h o w n t o i m p r o v e t h e r a t e o f n u c l e a r u p t a k e ( fo r ar e v i e w . s e e R o b e r t s . 1 9 8 9 ). A p a r t , f r o m L 2 9 w e k n o uo f n o o t h e r y e a s t r - p r ot e i n t h a t h a s b e e n a n a l y z ed i ns u f fi ci e n t , d e t a i l t o e s t a b l i s h t h e p r e s e n c e o r a b s e n c & ro f m u l t i p l e N L S s e q u e n c e s . W o r k i n o u r o w n l a b or a -t o r y . h o w e v e r , i n d i c a t e s t h e p r e s e n c e o f t w o N L Ss e y u e n c cs i n t h e s m a l l s u b u n i t r - p r o t e i n S 3 1 (1 . , I .S c h a a p . J . v a n - t R i e t , o t H . A . R a & . u n p u b l i s h e dr e s u l t s ) .

T h e t ) w o N L S s e q u e n c e s o f I, 2 5 a r e fu l l )c on t a i n e d w i t h i n t h e N - t e r m i n a l e x t e n s i o n o f t , h ep r o t e in t h a t d o e s n o t h a v e a n e q u i va l e n t i n e i t ,h e ri t s b a c t e r i a l o r c h l o r op l a s t c ou n t e r p a r t , ( R a m ? r t a l . .1 9 8 9 ) a n d i s d i s p e n s a b l e f or b i n d i n g o f t h e r - p r o t e i nt o 2 6 S r R N A ( R u t g e r s i t a l . , 1 9 9 0 . 1 9 9 1 ) . T h ee x t e n s i o n . h o w e v e r . a p p e a r s t o s e r v e s t , il l a n o t h e rp u r p o s e a p a r t f r om p r o m o t i n g i m p o r t o f t h e p r o t e ini n t o t h e n u c l e u s . T h i s i s i n f e r r e d f r o m t h e p h e n o -t y p e o f c el l s t h a t e x cl u s i v e l y e x p r e s s t h e A L 2 5 p o l y-p e p t i d e ( T a b l e 2 ). N o t o n l y d o t h e s e ce l ls s h o w t h re x t r e m e l y s l ow g r ow t h r a t e t o b e e x p e ct e d f r om t h er e m o v a l o f t h e N L S s e q u e n c e s , b u t o v e r 9 O ?b oft h e m a l s o d e v e lo p a h i g h l y a b e r r a n t m o r p h o l og >


11/13

N u c l ea r I mp o r t o f L2 5 23 5

s u g g e s t i v e o f a d e f e ct i n n u c le a r d i v i s io n . I m p r o v i n gt h e n u c le a r i m p o r t o f A L 2 5 b y m e a n s o f a h e t e r o -l og o u s N L S s e q u e n c e t h a t h a s p r o v e d t o b e h i g h l ye f fi c ie n t in ye a s t (Ne l son & Si lve r , 1 989; Ka l in ic h &D o u g l a s , 1 9 8 9 ) i n c r e a s e s t h e g r o w t h r a t e b u t d o e sn o t r e s t o r e i t t o i t s n o r m a l v a l u e (c om p a r e t h eg r o w t h r a t e o n Y P D m e d i u m o f Y C p L 2 5t r a n s f o r m e d c e ll s t o t h a t o f Y C p S A L 2 5 ,Y C p S S A L 25 o r Y C p S N A L 2 5 t r a n s f o r m a n t s i nT a b l e 2 ) . A l s o t h e a b n o r m a l c e ll m o r p h o l o g y i s n o tc or r e c t e d b y t h e i m p r o v e d n u c l e a r u p t a k e .

T h e m o s t p l a u s i b l e e x p l a n a t i o n f or t h e s e o b s e r v a -t i o n s , i n o u r v i e w , i s t h a t p r e s e n c e o f L 2 5 l a c k i n g i t sN - t e r m i n a l 4 1 a m i n o a c i d r e s id u e s i n 6 0 S s u b u n i t sc a u s e s t h e s e s u b u n i t s t o h a v e a f u n c t i on a l d e f e ctt h a t i m p a i r s t h e i r a b i li t y t o c a r r y o u t t r a n s l a t i on .T h u s , ce l ls e x p r e s s i n g o n l y t h e t r u n c a t e d p r o t e i n( e v e n i f f it t e d w i t h a n ef fi ci e n t N L S ) s u f f e r a no v e r a l l r e d u c t i on i n t r a n s l a t i o n r a t e , r e s u l t i n g i ns l o w e r g r o w t h . T h e d o m i n a n c e o f ( s om e o f ) t h e L 2 5g e n e s l a c k i n g c od o n s 2 t o 4 1 o v e r t h e i r w i l d - t y p ec ou n t e r p a r t i n t r a n s f o r m a n t s s i m u l t a n e o u s lye x p r e s s i n g b o t h t y p e s o f g e n e ( T a b l e 2 ) i s a l s o ina c c or d a n c e w i t h i m p a i r e d r i b o s om a l f u n c t i o n d u e t ot h e a b s e n c e o f t h e N - t e r m i n a l 4 1 a m i n o a c i dr e s i d u e s ( fo r a m o r e d e t a i l e d d i s cu s s i o n o f t h i s p oi n t ,s e e U n d e r w o o d & F r i e d , 1 9 9 0 ) , a l t h o u g h a n a d d i -t i o n a l e f fe c t d u e t o t h e p r e s e n c e o f t h e f or e i g n N L S s e q u e n c e s c a n n o t b e e x c lu d e d .

W e a s s u m e t h e a b n o r m a l c e ll m o r p h o l o g y t o b e as e c on d a r y c on s e q u e n c e o f t h e r i b o s om a l d e f e ct . T h ed e f e ct c ou l d c a u s e a d i ff e r e n t i a l r e d u c t i on i n t r a n s -la t ion of ind iv idu a l m R NAs (e .g . be c a u se of d i f fe r -e n c e s i n c od o n b i a s ) le a d i n g t o i m b a l a n c e dp r o d u c t i on o f s o m e f a c t or c r i t ic a l f o r n u c le a r d i v i-s i on , t h e i m p a i r m e n t o f w h i c h s e e m s t o b e p r i m a r yc a u s e f or t h e a b n o r m a l a p p e a r a n c e o f t r a n s fo r m a n t se x p r e s s i n g o n l y t r u n c a t e d L 2 5 . A t l e a s t o n e o t h e rc om p l e x p h e n o t y p i ca l c h a n g e i n e u k a r y o t i c( h u m a n ) c e ll s , d e v e l o p m e n t o f m a l i gn a n c y, h a s b e e nl i n k e d t o a m u t a t i on i n a n r - p r o t e i n g e n e p os s i b lyl e a d i n g t o a l t e r e d f u n c t i o n o f t h e l a r g e r i b os o m a ls u b u n i t ( S h a r p e t aE . , 1 9 9 0 ). F u r t h e r m o r e , s e l e ct i v ei n t e r f e r e n c e w i t h t r a n s l a t i o n i n i t i a t i o n k i n e t i cs h a sb e e n s u g g es t e d a s t h e ca u s e o f m a l i g n a n t t r a n s -f or m a t i o n o f H e L a c e ll s o v e r e x p r e s s i n g i n i t i a t i onf a ct o r e I F - 4 E ( D e B e n e d e t t i & R h o a d s , 1 9 9 0 ;L a z a r i s - K a r a t z a s e t al . , 1 9 9 0 ) . I n t e r e s t i n g l y , t h em u l t i p l y b u d d e d u n i n u c le a t e m o r p h o l o g y o f t h et r a n s fo r m a n t s e x p r e s s in g o n l y m u t a n t L 2 5 i s s t r i k -i n g l y s i m i l a r t o t h a t o b s e r v e d f or y e a s t c e ll s c a r r y -i n g a m u t a t i o n i n t h e C D C 4 g e n e ( B y e r s & G o e t s ch ,1 9 7 4 ), w h i c h i s n o t a n r -p r o t e i n g e n e ( P e t e r s o n e t al . ,1984) .

D o m i n a n c e o f m u t a n t o v e r w i l d -t y p e L 2 5 i so b s e r v e d o n l y i n Y C p S S A L 2 5 a n d Y C p S N A L 2 5t r a n s f o r m a n t s i n w h i c h t h e t r u n c a t e d p r o t e inc a r r i e s t w o N L S s e q u e n c e s . C e l l s c o- e x p r e s s i n g w i l d -t y p e L 2 5 a n d S A L 2 5 , w h i c h h a s o n l y a s i n g l e N L S ,g r o w n o r m a l l y ( T a b l e 2 ) . A p p a r e n t l y , t h e r a t i o o fm u t a n t o v e r w i l d -t y p e r i b o s o m e s h a s t o e x c e e d ac e r t a i n v a l u e b e f o r e t h e f u n c t i on a l d e f e ct o f t h ef or m e r b e c o m e s m a n i f e s t i n t h e c e ll u l a r g r o w t h r a t e .

S i n c e t h e r a t e o f n u c le a r i m p o r t o f a p r o t e i ni n cr e a s e s w i t h t h e n u m b e r o f N L S s e q u e n c e s i tc a r r i e s ( fo r a r e v i e w , s e e R o b e r t s , 1 9 8 9 ) t h e r a t i o o fm u t a n t o v e r w il d - t y p e L 2 5 in t h e n u c le u s o f g a l a c -t o s e - g r o w n Y C p S S A L 2 5 o r Y C p S N A L 2 5 t r a n s f o r -m a n t s i s l ik e l y t o b e h ig h e r t h a n i n t h e i rY C p S A L 2 5 - t r a n s f o r m e d c o u n t e r p a r t s . C o n s e -q u e n t l y , t h e f or m e r s h o u l d p r o d u c e a h i g h e r p e r -c en t a g e o f m u t a n t r i b os o m e s t h a n t h e l a t t e r .

T h e m a n i fe s t a t i on o f t h e a b n o r m a l m o r p h o l o gya p p e a r s t o r e q u i r e a n e v e n h i g h e r p e r ce n t a g e o fm u t a n t r i b o s o m e s , s inc e t r a n s f o r m a n t s co -e x p r e s s in g w i ld - t y p e L 2 5 a n d a n y o f t h e t r u n c a t e df or m s o f t h i s p r o t e i n a r e c om p l e t e l y n o r m a l( T a b l e 2 ) . F u r t h e r m o r e , u p o n t r a n s fe r o f t h e s et r a n s f o r m a n t s t o g l u co s e- ba s e d m e d i u m , a b n o r m a lc e ll s s t a r t t o a p p e a r o n l y a f t e r a d e l a y o f s e v e r a lh o u r s . D e ve l op m e n t o f t h e a b e r r a n t m o r p h o l og y ,t h e r e f o r e , d o e s n o t co i n c id e w i t h t h e s w i t c h f r o mw i ld - t y p e t o m u t a n t L 2 5 e x p r e s s io n b u t r e q u i r e se xte n s ive d i lu t ion of th e popu la t ion of o ld r ibo-som e s . The a va i l a b i l i ty of v ia b le c e l l s e xc lus ive lye x p r e s s i n g t r u n c a t e d f or m s of L 2 5 s h o u l d e n a b l e u st o a s c e r t a i n t h e n a t u r e o f t h e f u n c t i on a l d e f e ct i nt h e m u t a n t r i b os o m e s u s i n g i n v i t r o t r a n s l a t i o ns y s t e m s .

W e t h a n k M S M a r l i es B u i js f or h e r e xp e r t t e ch n i c a la s s i st a n c e d u r i n g t h e C S L M e xp e r i m e n t s , t h e o t h e rm e m b e r s o f t h e C S L M g r o u p o f t h e S e c t i on f or M o l e cu l a rC y t o l o g y a t t h e U n i v e r s i t y o f A m s t e r d a m f or t h e i r h o s p i -t a l i t y a n d D r 0. D e s t r 6 e ( H u b r e ch t L a b o r a t o r y , S t a t eU n i v e r s i t y , U t r e c h t ) f o r f a ci li t i e s a n d a s s i s t a n c e d u r i n gt h e o o cy t e i n j e ct i o n e x p e r i m e n t s . W e a r e g r a t e f u l t o D rR . J . P l a n t a a n d D r J . P . G . B a l l es t a f or s t i m u l a t i n gd i s c u s s i o n s a n d t o t h e f o r m e r a l s o f o r c r i t i ca l r e a d i n g o ft h e m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d b y t h eP r o g r a m m a c o m m i s s i e I n d u s t r i t i l e B i o t e ch n o l o g ie ( P cI B )w i t h f in a n c ia l a i d f r o m t h e N e t h e r l a n d s M i n i s t r y o fE c o n o m i c A f f a i r s .

ReferencesA d a m , S . A . (1 9 8 9 ) . N u c le a r p r o t e i n i m p o r t i n p e r m e -a b i l iz e d m a m m a l ia n c e ll s r e q u i r e s s o l u b l e cy t o -

p la sm ic f a c tor s . J . Ce l l Bi o l . 111, 8 0 7 - 8 1 6 .A d a m , S . A . , L o b l , T . J . , Mi t c h e l l , M . A . & G e r a c e , L .

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