Synthesis of mitochondrial proteins. The synthesis of cytochrome cin vitro

PRELIMINARY NOTES

651

aqueous phenol at t e m p e r a t u r e s b e t w e e n 6o ° a n d 8o ° a n d p r o b a b l y corresponds to t h e D N A - l i k e R N A r e p o r t e d b y GEORG]EV AND MANTIEVA5. The o t h e r t y p e cannot be released w i t h aqueous phenol a t a n y t e m p e r a t u r e , b u t o n l y b y the use of phenol a n d s o d i u m d o d e c y l sulfate at 95 °. E x p e r i m e n t s are now in progress to characterize t h e n a t u r e a n d biological role of these two t y p e s of r a p i d l y - l a b e l e d , D N A - l i k e R N A . This work was s u p p o r t e d b y N a t i o n a l I n s t i t u t e s of H e a l t h G r a n t N B o 6615-Ol a n d A m e r i c a n Cancer S o c i e t y G r a n t I N - I 6 - G .

Departments o/ Psychiatry and Physiological Chemistry, The Ohio State University, Columbus, Ohio (U.S.A.) I 2 3 4 5

W. W. MORRISON R. H. McCLUER

G. P. GEORGIEVAND M. [. LERMAN,Biochim. Biophys. Acla, 91 (1964) 678. \'V. \¥. MORRISON AND W. J. FRAJOLA, Biochem. Biophys. Res. Commun., 17 (1964) 597. S. KATZAND D. G. COMB,J. Biol. Chem., 238 (1963) 3o65 . R. J. MANS .aND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 3 (196o) 54o. G. P. GEOR~IEV AND V. L. MANTIEVA, Biochim. Biophys. Acta, 61 (1962) I53.

Received F e b r u a r y 27th, 1967

Biochim. Biophys. Acta, 138 (1967) 649-65 1

BBA 91165 Synthesis of mitochondriol proteins. The synthesis of cytochrome c in vitro E a r l i e r claims concerning t h e d e m o n s t r a t i o n of the synthesis of c y t o c h r o m e c in isolated r a t liver 1 a n d calf h e a r t m i t o c h o n d r i a 2 were l a t e r w i t h d r a w n a. In fact, several workers have now e s t a b l i s h e d t h a t isolated m i t o c h o n d r i a are unable to synthesize their soluble p r o t e i n s 4-G. O n l y the insoluble fraction ~,8, m a i n l y the struct u r a l p r o t e i n ~,8,9, is s y n t h e s i z e d in vitro. Therefore it was assumed, a n d also i n d i r e c t l y shown, t h a t m o s t of t h e m i t o c h o n d r i a l enzymes are s y n t h e s i z e d at the microsomes 9-11. Direct evidence i n d i c a t i n g t h a t the microsomes represent the site of synthesis of c y t o c h r o m e c has been o b t a i n e d r e c e n t l y b y CAMPBELL AND CADAVID12. However, t h e m e c h a n i s m b y which these proteins enter the m i t o c h o n d r i a was a m a t t e r of speculation. In a previous p a p e r we d e m o n s t r a t e d the t r a n s f e r of labelled proteins from microsomes into m i t o c h o n d r i a in vitro 6. This p a p e r describes the labelling of c y t o c h r o m e c in m i t o c h o n d r i a which h a v e been i n c u b a t e d with labelled microsomes. A special c h r o m a t o g r a p h i c t e c h n i q u e was d e v e l o p e d for the purification of v e r y small a m o u n t s of c y t o c h r o m e c. The c o m b i n a t i o n of an a n i o n - e x c h a n g e ( T E A E cellulose) a n d a c a t i o n - e x c h a n g e column (CM-cellulose) in tandem (i.e. so t h a t t h e effluent from the first enters t h e second) allows t h e one-step exclusion of 95 % of t h e p h o s p h a t e - s o l u b l e m i t o c h o n d r i a l protein. The a n i o n - e x c h a n g e colunm retains 83 o0, whereas 12 % is e x c l u d e d from t h e c a t i o n - e x c h a n g e c o l u m n b y elution w i t h T r i s HC1 (pH 7-5). C y t o c h r o m e c a p p e a r s at t h e t o p of t h e CM-cellulose c o l u m n as a n a r r o w red b a n d . The elution of the l a t t e r column with a linear g r a d i e n t results in an almost pure fraction of c y t o c h r o m e c (Fig. I). R e c h r o m a t o g r a p h y of t h e c y t o chrome c on CM-cellulose d i d not change t h e specific a c t i v i t y .

Biochim. Biophys. Acta, 138 (1967) 651-65¢

652

PRELIMINARY

NOTES

°I,T 0 Transmission

Absorbamce

Grad}ent

08

20

I] 280 m

i:

lJ

280 mJJ

40

0.6

Cytochrome C oxidized i

i

'~ 405 mp

j /

reduced

",

I i 637

"

75.7

04

02

41

1/---L

100

5

C

E

F

7

8 Hours

G

Fig. I. C h r o m a t o g r a i n of c a t i o n i c p r o t e i n s from r a t l i v e r n i i t o c h o n d r i a on CM cellulo~:e. Tile e f f l u e n t froni t h e c o l u m n p a s s e d a t cm flow cell a n d t h e t r a n s m i t t a n c e or a b s o r b a n c e w a s nl e a s ured c o n t i n u o u s l y a t 4o5 m/l ( ) a n d 28o m # ( - ) s i m u l t a n e o u s l y . SanIples w e re t a k e n b y h a n d . The n u m b e r s u n d e r t h e p e a k s r e p r e s e n t t h e a r e a s in cm 2 for t h e fra c t i ons . 1.25 nil cont a i n i n g 44 m g p r o t e i n of t h e c o n c e n t r a t e d p h o s p h a t e - s o l u b l e e x t r a c t from T a b l e I ( t r a n s f e r systen1 I) w a s a p p l i e d to t h e T E A E - c e l l u l o s e c o l u m n (o.8 c n l X 23 cln, e q u i l i b r a t e d w i t h IO mM Tris-HC1, p H 7.5) w h i c h w a s c o n n e c t e d w i t h a CM ce l l ul os e c o l u m n (o. 5 c m :4 23 cm, equilib r a t e d w i t h 20 mM p h o s p h a t e , p H 6.5). E l u t i o n w a s w i t t l lO inM Tris-HC1, p H 7.5 (4 .16 nil/h). A f t e r a b o u t 2 tl t h e CM-cellulose c o l u m n w a s d i s c o n n e c t e d a n d c l u t c d w i t h a l i n e a r g r a d i e n t of p h o s p h a t e b u f f e r from 2o to 3o0 mM (3o ml, p H 6.5). F l o w s pe e d: 3.46 ml/h. TABLE 1 L A B E L L I N G OF P R O T E I N F R A C T I O N S IN S L I C E S A N D I N T H E T R A N S F E R S Y S T E M

All i n c u b a t i o n s w e r e p e r f o r m e d for 2o rain a t 37 °. Slices: 8.8 g r a t l i v e r slices w e re i n c u b a t e d in 6o inl final v o l u m e as d e s c r i b e d before 6 e x c e p t t h a t t h e a m i n o a c i d c o n c e n t r a t i o n w a s t w i c e t h a t in vivo '3 a n d l y s i n e w a s a d d e d as l a b e l (12 #C L-[14C]lysine, 247 m C / m m o l e ) . A f t e r i n c u b a t i o n m i c r o s o m e s a n d n l i t o c h o n d r i a w e r e i s o l a t e d in m e d i u m B; m i t o c h o n d r i a w e re w a s h e d 3 t i n l e s in m e d i u m A n. Trans/er system I: M i t o c h o n d r i a (148 ing p r o t e i n ) , cell s a p (lO8 nag p r o t e i n ) a n d l a b e l l e d microsonaes (75 nag p r o t e i n , 1274 c o u n t s / m i n p e r rag) f r o m t h e a b o v e slices w e r e i nc ub a t e d in t h e t r a n s f e r m e d i u m 6 in a final v o l u n m of 25 ml. The p u r i t y of t h e m i c r o s o m a l f r a c t i o n was c h e c k e d b y t h e a m o u n t of c y t o c h r o m e c r e c o v e r e d u n d e r t h e s a m e c o n d i t i o n s of c h r o m a t o g r a p h y (Fig. 1 ) w h i c h w a s o.o 3 }~g c o m p a r e d w i t h 1. 3 / ~ g / m g p r o t e i n from n i i t o c h o n d r i a . Trans/er system II: M i t o c h o n d r i a (23o nag p r o t e i n ) , cell sap (2IO m g p r o t e i n ) a n d l a b e l l e d m i c r o s o m e s (124 m g p r o t e i n , 888 c o u n t s / r a i n p e r nlg) w e r e i n c u b a t e d in t h e t r a n s f e r m e d i u m 6 in a final v o l u m e of 4 ° nil. T h e n l i c r o s o m e s w e r e l a b e l l e d in vitro, as d e s c r i b e d be fore 6 w i t h 3 o F C L-[a4C,lysine in a final v o l u m e of 3 ° ml.

Fractions

Slices (counts/rain per ~ng protein)

Trans/er system (counts/rnin per mg protein) I I [--

Mitochondria, phosphate-soluble Mitochondria, phosphate-insoluble Cell sap M icroso ines C y t o c h r o n l e c*

316 374 244 1274 12o

231 187 114 705 4[

* 18o :< lO 6 c o u n t s / n l i n = i # m o l e c y t o c h r o m e c.

Biochim. Biophys. Acta, i38 (i967) 651-654

lO4 204 96 487 o

653

PRELIMINARY NOTES TABLE II SPECIFIC

RADIOACTIVITY

OF FRACTIONS

FROM FIG.

I.

Fraction

Total counts/rain

Protein (rag)

Counts~rain per mg protein

A B C+D+E F G H

1181 9° 13. 7 27 54 49

5.5 o o-35 -o.62 0.87 0.53

213 255 -44 63 91

J K L

28 13.8 13.5

o.o37" o. Io5"

-4 I**

* Mg of c y t o c h r o m e c c a l c u l a t e d f r o m tile a r e a of a b s o r b a n c e a t 4o5 m # ~4, a s s u m i n g t h a t t h e r e w a s no o t h e r m a t e r i a l a b s o r b i n g a t t h i s w a v e l e n g t h . "* Specific a c t i v i t y of c y t o c h r o m e c. T h e r a t i o A 405 m~:A 280 my for p u r e c y t o c h r o m e c is 4.03 (ref. 14). I n f r a c t i o n L t h i s r a t i o is 3.33 w h i c h c o r r e s p o n d s to 17 % i m p u r i t y ( p a r t l y r e d u c e d c y t o c h r o m e c, w h i c h h a s a l o w e r r a t i o ) . The a c t i v i t y of c y t o c h r o r n e c w a s c a l c u l a t e d b y subt r a c t i o n of t h e r a d i o a c t i v i t y of i m p u r i t y , a s s u m i n g t h e s a m e a c t i v i t y p e r c m 2 a r e a as for F r a c t i o n J.

Low amounts of radioactivity were estimated accurately by using filter discs to which the protein sample was transferred after precipitation with IO o~ ,o trichloroacetic acid and solubilization in 0.5 M KOH. The discs were washed and counted as described before 6. Table I compares the specific activity of several protein fractions labelled either directly with il~Cllysine in slices, or by transfer of proteins from labelled microsomes. The transfer reaction was mediated with microsomes labelled either within slices (I) or after isolation (II). Radioactive proteins were recovered in all fractions. Cytochrome c was isolated from the phosphate-soluble s mitochondrial fractions as described in Fig. I and Table II. As can be seen, the specific radioactivity of cytochrome c labelled by transfer of proteins from microsomes (derived from slices, I) is 1/3 of that obtained by labelling the enzyme within the intact cell (slices). In contrast, no radioactivity could be detected in the cytochrome c isolated from mitochondria which were labelled by transfer system II. This system differs from transfer system I only in the way in which the microsomes had been labelled. This seems to indicate that the most labile step for the synthesis of mitochondrial proteins in vitro is not the transfer reaction, but rather the synthesis of the polypeptide chains on the microsomes. After disruption of the cell, the organized synthesis of definite proteins m a y be lost even though an active protein synthesis remains. These results do not indicate whether cytochrome c is synthesized on the microsomes in toto or whether only the apoenzyme is synthesized on the microsomal unit (see ref. 12). They m a y be summarized as follows: I. The polypeptide chain of cytochrome c is synthesized on the microsomes and, in a second step, transferred (with or without a haemin group) into the mitochondria. 2. The previously described transfer of proteins from microsomes into mitochondria 6 represents a physiological mechanism by which most of the mitochondrial enzymes enter the organelle. The author is indebted to Professor KLINGENBERGfor his interest and valuable criticism. This work was supported by the Deutsche Forschungsgemeinsehaft.

Physiologisch-Chemisches Institut der Philipps Universitdt, Marburg/Lahn (Germany)

B. KADENBACH

Biochim. Biophys. Acta, 138 (1967) 651-654

654 I 2 3 4 5

6 7 8 9 io II I2 13 14

PRELIMINARY NOTES

]-I. 1~{. BATES, \7. 1~. CRADDOCK AND .'V[. V. SIMPSON, J. Biol. Chem., 235 (196o) 14o. H. M. BATES AND M. V. SIMPSON', Biochim. Biophys. Acta, 32 (1959) 49. M. V. SIMPSON, D. M. SKINNER AND J. M. LUCAS, J. Biol. Chem., 236 (1961) I' C 8 i . D. 13. ROODYN, J. \ ¥ . SUTTIE AND T. S. WORK, Biochem. J., 83 (1962) 29. U. BRONSERT AND W. NEUPERT, in i. M. TAGER, S. PAPA, E. QUAGLIARIELLO AND E. C. SLATER, Regulation o/2VIetabolic Processes in Mitochondria, B B A L i b r a r y Vol. 7, E l s e v i e r , A m s t e r d a m , 1966, p. 426. B. lSZADENBACH,Biochim. Biophys. ,4eta, 134 (1967) 43 o. D. E. S. TRUMAN, Biochem. ,[., 9t (1964) 59. D. B. ROODYN, Biochem. J., 85 (I962) 177. D. HALDAR, K. FREEMAN AND T. S. WORK, Nature, 211 (1966) 9. T. HIGASHI AND TH. PETERS, J. Biol. Chem., 238 (1963) 3952. D. BEATTIE, [{. JBASFORD AND S. KORITZ, Biochemistry, 5 (1966) 926. P. N. CAMPBELL AND G. CADAVID, in t h e press. H. SCHIMASSEK AND \V. GERO~, IHochem. Z., 343 (I965) 4°7 E. MAR(~OLIASH AXD N. FROHWlRT, Biochem. J., 71 (I959) 57 o.

Received December 27th, 1966 Revised manuscript received March I4th , 1967 Biochim. Biophys..4eta, I38 (1967) 6 5 1 - 6 5 4

BBA 91166

Incorporation of radioactive amino acids into 7-globulin in a cell-free system from rat spleen Recently a number of papers on the attempted synthesis ot immunoglobulins in cell-free systems from lymphoid tissue have been published 1-5. Direct evidence for such a synthesis would consist in incorporation of radioactive amino acids into immunoglobulins. Although spleen microsomes are able to incorporate amino acids into proteins 2 a it is difficult to identify the radioactive proteins 6. For this purpose, STENZEL AND RVBIN" have made use of radioimmunoelectrophoresis but this approach does not afford quantitative results. It was therefore considered much more advantageous to identify and determine radioactive proteins quantitatively by means of immunoadsorbents. However, the first attempts to use this method for the detection of antibodies or y-globulins possibly synthesized in vitro by microsomes of rabbit or pig lymphoid tissues met with failure~, 5. On the other hand, experiments with microsomes of rat spleen proved successful and using antibody immunoadsorbents we were able to detect and quantitatively characterize the synthesis of y-globulin effected in vifro by these particles. The experiments were carried out with the spleen and liver of rats following two injections on successive days with 2 ml of a IO o~ ,o suspension of sheep erythrocytes; the animals were killed on the third day after the second injection. In order to obtain microsomes, minced tissues were placed in a glass homogenizer and disrupted by four strokes of a loosely fitting spherically ended Teflon pestle. The homogenization medium had the following composition: o.o35 M Tris buffer (pH 7.8), o.15 M sucrose, o.o 7 M KC1, o.oi M MgC12 and o.oo6 M mercaptoethanol. To isolate the microsomes, the homogenates were centrifuged at 15 o o o × g for 2o min and then for 6o min at lO5 ooo ×g. The cell sap used to isolate the pH-5 fraction was obtained by centrifugation of the homogenates at IO5 ooo ×g for 9° min. Biochi~*~. Biophys. Acta, 138 (1967) 654-656