RAPID COMMUNICATION

ZIPGRAM

INITIATION FACTOR DISTRIBUTION I N XENOPUS LAEVIS

EGGS

N. E. GARRISON AND M. S. KAULENAS Department of Zoology, U n i v e r s i t y o f Massachusetts, Amherst, Massachusetts 01 002 ABSTRACT The d i s t r i b u t i o n o f a c t i v i t y s i m i l a r t o t h a t o f one o f the i n i t i a t i o n f a c t o r s ( I F 2 ) was determined as a f u n c t i o n o f developmental stage i n Xenopus laeviseggs and embryos. I n v i v o exposure o f mature oocytes t o hormone increases t h e t o t a l amount o f d e t e c t a b l e f a c t o r a c t i v i t y and f a c i l i t a t e s t h e b i n d i n g o f I F E - l i k e f a c t o r t o ribosomes. The q u a l i t y o f f a c t o r bound i s n o t c o r r e l a t e d w i t h polysome content. The development o f amphibian oocytes i s c h a r a c t e r i z e d by an extended f i r s t m e i o t i c prophase, d u r i n g which t h e b u l k o f t h e growth takes place.

The e a r l y

stages of prophase a r e devoted t o uptake o f l a r g e q u a n t i t i e s o f p r o t e i n as w e l l as t h e synthesis and accumulation o f massive amounts o f ribosomal, t r a n s f e r and informational RNAs (Davidson, '68).

The major p a r t o f t h e accumulated n u c l e i c

a c i d and p r o t e i n products i s n o t f o r immediate use, b u t i s s t o r e d t o p r o v i d e i n formation and s t r u c t u r a l precursors f o r embryonic development.

Eventually,

growth o f t h e oocyteceases, and metabolic a c t i v i t y i s reduced.

The a c t u a l l e v e l

o f p r o t e i n synthesis i n such oocytes appears t o vary f o r d i f f e r e n t species (Smith, '72; Gurdon, '73), a1 though f o r a1 1 amphibians an a p p r o p r i a t e hormonal stimulus i s needed t o t r i g g e r f u r t h e r development (Schorderet-Slatkine and Drury,

' 73).

The c h a r a c t e r i s t i c s o f i n v i v o p r o t e i n synthesis i n mature and hormones t i m u l a t e d oocytes have been described (Smith,

' 72).

Less a t t e n t i o n has been

p a i d t o t h e i n v i t r o c a p a c i t y o f t h e components which p l a y a r o l e i n t h e p r o t e i n synthetic a c t i v i t y .

Such a n a l y s i s c o u l d be u s e f u l i n a f u l l d e s c r i p t i o n o f t h e 295

As a f i r s t

mechanisms i n v o l v e d i n t h e i n i t i a t i o n o f t h e developmental program.

step, we have determined t h e t o t a l q u a n t i t y and 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 one o f t h e i n i t i a t i o n f a c t o r s as a f u n c t i o n o f developmental stage i n Xenopus eggs and embryos. MATERIALS AND METHODS

Ripe unovulated oocytes were d i s s e c t e d from o v a r i e s .

O v u l a t i o n and amplexus were induced (Landesman,

'72).

Numbers o f eggs were de-

termined by comparing t h e p r o t e i n c o n t e n t o f an unknown number o f eggs t o t h a t Eggs were d e j e l l i e d , washed 4 times w i t h 0.05 DeBoers s o l u t i o n

o f standards.

(Gusseck and Hedrick, ' 7 1 ) , t w i c e w i t h b u f f e r A (250 mM sucrose, 25 mM Tris-HC1, pH 7.5 a t 25O, 100 mM KC1, 10 mM MgSOg, 0.1 mM EDTA, 3 mM 2-mercaptoethanol) and homogenized a t 4' homogenizer.

i n 2.5 v o l o f b u f f e r A w i t h 2 s t r o k e s o f a Potter-Elvehjem

The homogenate was c e n t r i f u g e d a t 26,000 g f o r 10 min a t 4';

post-mitochondria1 supernate was c e n t r i f u g e d f o r 90 min a t 105,000 g. speed supernate was d i a l y s e d (see below) and assayed f o r f a c t o r .

the

The h i g h -

The microsomal

p e l l e t was suspended i n b u f f e r A c o n t a i n i n g 1.0% T r i t o n X- 00, c l e a r e d by cent r i f u g a t i o n and again p e l l e t e d as described.

The ribosoma

salt-wash was pre-

pared by suspending t h e ribosomal p e l l e t i n TNM b u f f e r (50 mM Tris-HC1, pH 7.5,

500 mM NH4C1, 10 mM MgSOq), and p e l l e t i n g t h e ribosomes as d e s c r i b e d above

The

c e l l sap and salt-wash f r a c t i o n were d i a l y s e d f o r 4 h a t 4O, w i t h s t i r r i n g a g a i n s t 200 v o l

of

b u f f e r A w i t h o u t sucrose, b u t w i t h g l y c e r o l added t o

0%.

B i n d i n g a c t i v i t y was determined by t h e procedure o f Z a s l o f f and Ochoa ( ' 7 1 Developmental stages:

as i n Nieuwkoop and Faber ( ' 6 7 ) .

The a c t i v i t y measured

was l i k e t h a t o f i n i t i a t i o n f a c t o r 2 (IF2), which, a l o n g w i t h i n i t i a t i o n f a c t o r 1, i s r e q u i r e d f o r b i n d i n g o f n a t u r a l mRNA t o ribosomes, and f o r e f f i c i e n t p o l y p h e n y l a l a n i n e s y n t h e s i s w i t h p o l y U as template ( S h a f r i t z e t a l . RESULTS AND DISCUSSION

The r e s u l t s a r e presented i n t a b l e 1.

,

'70).

T o t a l IF2-

l i k e a c t i v i t y i s d e f i n e d as t h a t found i n t h e c e l l sap p l u s t h a t which i s removed

296

TABLE 1 I F 2 - l i k e a c t i v i t y present i n Xenopus c e l l sap and bound t o ribosomes Devel opmenta 1 Stage

Units o f I F 2 - l i k e a c t i v i t y / 1 0 0 0 eggs* Unbound i n c e l l sap

Mature oocytes U n f e r t i 1i z e d eggs Stage 8 Stage 12

80 289 253 273

(235) (763) (780) (747)

Ribosomebound

Total bound t unbound

2.3 ( 85) 212 (1,239) 55 (1,156) 40 ( 641)

82.3 501 308 313

-

Bound Unbound

0.029 0.73 0.22 0.15

*1 u n i t of a c t i v i t y = t h e amount o f a c t o r r e q u i r e d t o c a t a l y s e t h e AUG-dependent b i n d i n g o f 1 pmol o f fMet-tRNA t o Artemia 40s ribosomal subunits i n 30 min ( Z a s l o f f and Ochoa, '71). Numbers i n parentheses a r e "raw" counts/min, b e f o r e c o r r e c t i o n f o r non-enzymatic b i n d i n g and number o f eggs used t o e x t r a c t IF2-like activity.

from ribosomes by a h i g h

s a l t wash.

A substantial quantity o f IF2-like a c t i v i t y

i s present i n oocytes p r i o r t o i n v i v o hormone s t i m u l a t i o n .

The t o t a l amount o f

d e t e c t a b l e f a c t o r , however, increases approximately s i x - f o l d i n hormone-stimulated

u n f e r t i l i z e d eggs.

This h i g h l e v e l i s n o t maintained i n developing

f e r t i l i z e d eggs, b u t decreases by about 40% i n morulae and gastrulae. The IF2-dependent b i n d i n g a c t i v i t y can be subdivided i n t o unbound and r i b o some-bound categories.

Unbound IF2-1 i k e a c t i v i t y increases 3 . 6 - f o l d a f t e r hor-

mone treatment and then remains e s s e n t i a l l y a t t h e same l e v e l i n developing f e r t i l i z e d eggs.

On t h e o t h e r hand, l i t t l e f a c t o r a c t i v i t y appears t o be r i b o -

some-bound i n ovarian eggs.

Hormone treatment b r i n g s about a spectacular i n -

crease (x 93) i n t h e q u a n t i t y of bound f a c t o r .

L a t e r developmental stages show

a progressive d e c l i n e i n t h e amount o f detectable ribosome-associated f a c t o r . I n some o t h e r systems, ribosome-associated i n i t i a t i o n f a c t o r s appear t o be predominantly l o c a t e d on polysomes (Kaulenas,

' 71).

The p r o p o r t i o n o f ribosomes

i n polysomes f o r t h e various developmental stages o f Xenopus i s given i n t a b l e 2.

I t i s c l e a r t h a t the amount o f bound

297

I F 2 - l i k e factor i s not correlated

TABLE 2 Polysome c o n t e n t o f Xenopus ribosome p r e p a r a t i o n Ribosome source

1. 2. 3.

% o f ribosomes as polysomes*

Mature oocytes U n f e r t i l i z e d eggs Gastrulae

4 9 14

*Estimated as p r e v i o u s l y d e s c r i b e d (Kaulenas, '72). The v a l i d i t y o f t h e assay was checked by d e t e r m i n i n g t h e polysorne c o n t e n t o f a number o f Xenopus t i s s u e s , which were found t o be: l i v e r , 62%, lung, 50%, kidney, 58%, muscle, 14% and t o t a l ovary, 18%. A l l values a r e means from f o u r experiments.

w i t h t h e polysome c o n t e n t o f t h e stages.

The d i f f e r e n t i a l between o v a r i a n and

u n f e r t i l i z e d eggs i s g r e a t e r than would be expected f o r an approximate d o u b l i n g

i n polysome c o n t e n t .

W h i l e i t i s l i k e l y t h a t a t l e a s t some o f t h i s f a c t o r ac-

t i v i t y i n Xenopus i s a s s o c i a t e d w i t h polysomal ribosomes, i t appears u n l i k e l y t h a t i t c o u l d be a l l o f t h e bound I F 2 - l i k e f a c t o r , since, as development progresses t h e amount o f polysomes increases, b u t t h e amount o f bound f a c t o r act i v i t y declines.

I t appears, t h e r e f o r e , t h a t a t l e a s t some o f t h e bound IF2-

l i k e a c t i v i t y i n hormone-stimulated u n f e r t i l i z e d eggs i s n o t a s s o c i a t e d w i t h polysomes, n o r i s i t a v a i l a b l e t o promote more e f f i c i e n t i n i t i a t i o n o f p o l y U a t low Mg2+ on f r e e ribosomes ( G a r r i s o n e t a l . ,

'72).

A possible interpretation

o f t h e hormone-stimulated r e d i s t r i b u t i o n o f I F 2 - l i k e f a c t o r , from t h e s o l u b l e t o t h e ribosomal compartments,

i s that, i n part, the f a c t o r i s required f o r the

f o r m a t i o n o f i n i t i a t i o n complexes w i t h maternal mRNA.

The bound f a c t o r used i n

such a way would n o t be a v a i l a b l e t o f a c i l i t a t e p o l y U t r a n s l a t i o n (Loeb, '70). Such complexes need n o t n e c e s s a r i l y a l l p a r t i c i p a t e immediately i n polysome f o r mation.

As maternal messengers a r e m o b i l i z e d d u r i n g t h e e a r l y development, t h e

t o t a l q u a n t i t y o f ribosome-bound f a c t o r would a l s o decrease. 298

An a s s o c i a t i o n o f

i n a c t i v e mRNA w i t h ribosomal monomers has been d e s c r i b e d f o r o t h e r systems (Schul t z e t a l .

,

' 7 2 ) ; t h e proposed hormone-mediated f o r m a t i o n o f such complexes

would be pecul i a r t o Xenopus. LITERATURE CITED Davidson, E. H. 1968 Gene A c t i v i t y i n E a r l y Development. Academic Press, New York. Garrison, N. E., R. A. Bosselman and M. S. Kaulenas 1972 The e f f e c t o f ribosomal p r o t e i n exchange on t h e a c t i v i t y o f Xeno us l a e v i s ribosomes. Biochem. Biophys. Res. Commun. , 49: Gurdon, J. B. 1973 I n : P r o t e i n Synthesis i n Reproductive Tissue, K a r o l i n s k a I n s t i t u t e t , Stockholm, Sweden, pp. 225-243. Gusseck, D. J . , and J. L. H e d r i c h 1971 A m o l e c u l a r approach t o f e r t i l i z a t i o n I . D i s u l f i d e bonds i n Xenopus l a e v i s j e l l y c o a t and a m o l e c u l a r h y p o t h e s i s f o r f e r t i l i z a t i o n . Develop. B i o l . , 25: 348-359. Kaul enas , M. S. 1971 Col d - i nduced " r u n - o f f " r ibosomes o f Acheta domesticus. Biochem. Biophys. Res. Comm. , 43: 1 0 8 1 - 1 0 8 r Kaulenas, M. S. 1972 The s t a b i l i t y o f s u b u n i t a s s o c i a t i o n i n Acheta ribosomes. J. I n s e c t Physiol., 18: 649-673. Landesman, R. 1972 Ribosomal RNA s y n t h e s i s i n pre- and p o s t - g a s t r u l a embryos o f Xenopus l a e v i s . C e l l D i f f e r e n , 1: 209-213. Loeb, J. N. 1970 Leucine and p h e n y l a l a n i n e i n c o r p o r a t i o n by r a t l i v e r microsomes c o n t a i n i n g endogenous template RNA: A k i n e t i c s t u d y o f t h e e f f e c t o f p o l y u r i d y l i c a c i d . Arch. B ophys. Biochem., 139: 306-310. Nieuwkoop, P. D., and J . Faber 1967 Normal Table o f Xenopus l a e v i s (Daudin). Second e d i t i o n , North-Holland P u b l i s h i n g Company, Amsterdam, p. 252. S c h o r d e r e t - S l a t k i n e , S., and K. C. Drury 1973 Progesterone induced m a t u r a t i o n i n oocytes o f Xenopus l a e v i s . Appearance o f a " m a t u r a t i o n promoting f a c t o r " i n enucleated oocytes. C e l l D i f f e r e n . , 2: 247-254. Schultz, G. A., D. Chen and E. K a t c h a l s k i 1972 L o c a l i z a t i o n o f a messenger RNA i n a ribosomal f r a c t i o n from ungerminated wheat embryos. J. Mol. B i o l . , 66: 379-390. S h a f r i t z , D. A., P. M. P r i c h a r d , J. M. G i l b e r t and W. F. Anderson 1970 S e p a r a t i o n o f two f a c t o r s , M1 and Mz, r e q u i r e d f o r p o l y U dependent p o l y p e p t i d e s y n t h e s i s by r a b b i t r e t i c u l o c y t e ribosomes a t l o w magnesium i o n c o n c e n t r a t i o n . Biochem. Biophys. Res. Commun., 38: 721-727. Smith, L. D. 1972 I n : Oogenesis. J. D. Biggers, A. W. Schuetz, eds., U n i v e r s i t y Park Press, B a l t i m o r e , pp. 227-239. Z a s l o f f , M., and S. Ochoa 1971 A supernatant f a c t o r i n v o l v e d i n i n i t i a t i o n complex f o r m a t i o n w i t h e u k a r y o t i c ribosomes. Proc. N a t l . Acad. Sci., 68: 3059-3063.

&.

299

Initiation factor distribution in Xenopus laevis eggs.

The distribution of activity similar to that of one of the initiation factors (IF2) was determined as a function of developmental stage in Xenopus lae...
237KB Sizes 0 Downloads 0 Views