MATTHEW A. HARMEY*, EVA-MARIA NEHER, RICHARD ZIMMERMANN and WALTER NEUPERT. Physiologisch-Chemisches Institut der Universitdt G6ttingen ...
BioSystems, 12 (1980) 283-287 © Elsevier/North-Holland Scientific Publishers Ltd.
283
ASSEMBLY OF MrroCHONDRIA: SYNTHESIS AND INTRACEIJ.ULAR TRANSFER OF MITOCHONDRIAL PROTEINS
MATTHEW A. HARMEY*, EVA-MARIA NEHER, RICHARD ZIMMERMANN and WALTER NEUPERT Physiologisch-Chemisches Institut der Universitdt G6ttingen, Humboldtallee 7, 3400 Gottingen, F.R.G.
(Received August 20th, 1979) The majority of mitochondrial proteins are synthesized on cytoplasmic ribosomes and transferred to the mitochondria where they are assembled to supramolecular structures. The intracellular transfer of these proteins appears to occur by a post-translational mechanism, i.e., it involves extramitochondrial precursor forms which are translocated in a step independent from translation. The synthesis and transfer of individual proteins was investigated in vivo, or in vitro employing homologous and heterologous cell free systems for protein synthesis. Cytochrome c was initially made as the apoprotein. This precursor protein was converted to the holoprotein on uptake by mitochondria in reconstituted systems. Integrity of mitechondria was essential for the ape to holo conversion. In the case of the ADP/ATP carrier protein, an integral transmembrane protein of the inner mitochondrial membrane, the initial translation product had the same apparent molecular weight as the mature protein. It was found in soluble form in the post-ribosomal supernatant. Citrate synthase, a matrix protein, was synthesized as a precursor with an apparent molecular weight of 47 000. Transfer to the mitochondria was accompanied by cleavage to yield a molecular weight of 45000. The significance of these results in relation to the mechanisms of intracellular transfer and of assembly of the individual proteins is discussed.
Introduc~on T h e biogenesis of m i t o c h o n d r i a r e p r e s e n t s a c o o r d i n a t e d s y n e r g i s m b e t w e e n two segregated translation systems. Translation
products of the nuclear coded cytoplasmic s y s t e m s are t r a n s p o r t e d to t h e o r g a n e l l e w h e r e t h e y are i n t e g r a t e d w i t h t h e t r a n s l a t i o n p r o d u c t s of t h e organelle, to g e n e r a t e a functional mitochondrion. In mitochondria the i m p o r t e d p r o t e i n s h a v e v a r i o u s destinations. Some few are d e s t i n e d for t h e o u t e r m e m b r a n e , o t h e r s for a location b e t w e e n t h e i n n e r a n d o u t e r m e m b r a n e , o t h e r s still for t h e i n n e r m e m b r a n e , while a f u r t h e r g r o u p of l a r g e l y h y d r o p h i l i c p r o t e i n s are destined for t h e m a t r i x . A close, look a t t h e s t r u c t u r e of t h e m i t o c h o n d r i o n a n d t h e n a t u r e of t h e p r o t e i n s i m p o r t e d shows t h a t some p r o t e i n s m a y h a v e to pass t h r o u g h one or two m e m b r a n e s while some p r o t e i n s m a y become a n i n t e g r a l p a r t of
* Present address: Department of Botany, University College Dublin, Ireland.
e i t h e r of t h e two m e m b r a n e s . A f u n d a m e n t a l p r o b l e m in e l u c i d a t i n g t h e n a t u r e of t h e prot e i n t r a n s p o r t arises in t r y i n g to decide w h e t h e r t h e r e is a unified t r a n s p o r t s y s t e m for all t h e p r o t e i n s or a v a r i a b l e m e c h a n i s m of t r a n s p o r t , d e p e n d i n g , e.g., on t h e n u m b e r of membranes a protein must traverse. K i n e t i c studies w i t h i n t a c t cells a n d w i t h h o m o l o g o u s cell free s y s t e m s f r o m N e u r o s p o r a were c a r r i e d o u t to follow t h e i n t r a c e l l u l a r t r a n s f e r of m i t o c h o n d r i a l proteins. A p p l y i n g a n u m b e r of c r i t e r i a it w a s concluded t h a t t r a n s f e r of p r o t e i n s is by a post t r a n s l a t i o n a l m e c h a n i s m ( H a l l e r m a y e r et al., 1977; H a r m e y et al., 1977): (a) t h e r e is a t i m e lag b e t w e e n t h e s y n t h e s i s of p r o t e i n s in t h e cytosol a n d t h e i r a p p e a r a n c e in t h e m i t o c h o n d r i a ; (b) cycloheximide inhibits p r o t e i n s y n t h e s i s on c y t o p l a s m i c ribosomes c o m p l e t e l y b u t allows c o n t i n u e d t r a n s p o r t of p r o t e i n s into t h e mitochondria; (c) different p r o t e i n s h a v e different kinetics of a p p e a r a n c e in t h e m i t o c h o n d r i a . T h e s e findings s u g g e s t e d t h e existence of e x t r a m i t o c h o n d r i a l p r e c u r s o r pools of mitoc h o n d r i a l proteins.
284 Results The experiments described here were aimed at elucidating the nature of the extramitochondrial precursors, and their transport to their functional locations in the mitochondria. Three proteins from different topographical locations within the mitochondrion were studied viz. cytochrome c, ADP/ATP carrier protein and citrate synthase. Cytochrome c is a small protein (mol. wt 13000) consisting of an apoprotein and a covalently attached heme group (Heller and Smith, 1966). It is generally described as a peripheral protein of the inner membrane (De Pierre and Ernster, 1977). The ape and holo forms of cytochrome c were distinguished from each other by monospecific antibodies (Korb and Neupert, 1978). A cytosolic pool of apocytochrome c could be demonstrated in vivo and in vitro. In vivo pulse labelling experiments showed a correlation between the appearance and disappearance of apocytochrome c in the cytosol and the appearance of labelled holocytochrome c in the mitochondria. In vitro reconstitution experiments have shown that apocytochrome c is first released into the postribosomal supernatant (Korb and Neupert, 1978). Import into the mitochondria is a time dependent process accompanied by attachment of heine and binding to the mitochondrial membrane (Fig. 1). The uptake and binding is dependent on the amount of mitochondria and on their integrity. Disruption of the mitochondria by agents such as Triton X-100, treatment with deoxycholate or hypotonic media destroyed the capacity of the mitochondria to import and convert apocytochrome c to the holo form. Heterologous translation systems (Pelham and Jackson, 1976; Roberts and Paterson, 1973) were programmed with messenger RNA from Neurospora and apocytochrome c immunoprecipitated from the translation products. When the dissociated immunoprecipitates were subjected to SDS electrophoresis the original translation product does not have an aminoterminal extension, nor does it have a carboxyterminal extension
3H cpm 2OOO
1000
A
~
/ 1'0 time [min]
3H cpm 3000.
o
5'0
9()
B
2000.
1000"
0.1
0.5 1.0 mitochondnol protein [mg J
Fig. 1. Conversion of alx)cytochrome c to holocytochrome c in a reconstituted system. [3I-I]leucine was incorporated into a postmitechondrial fraction of Neurospora a n d the postribosomal s u p e r n a t a n t prepared (Harmey et ell., 1977). The s u p e r n a t a n t was incubated with mitochondria. Following incubation, lysis of mitochondria was carried out by addition of Triton X-100 a n d holocytochrome c was immunoprecipitated. The immunoprecipitate was subjected to SDS gel electrophoresis a n d the radioactivity in cytochrome c determined. A: Time dependence of the formation of holocytochrome c. B: Dependence of the formation of holocytochrome c on the a m o u n t of mitochondria in the reconstituted system (30 rain incubation).
since the apparent molecular weight of the in vivo and in vitro products are identical (Fig. 2). As an inner membrane protein we chose the ADP/ATP carrier protein, an integral membrane protein (Klingenberg, 1976). This protein has a monomeric molecular weight of 32000 and has a hydrophobic character. Kinetic experiments indicated the existence of an extramitochondrial pool (Hallermayer et
285 e----e
35S cpm
o-
-
-o
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A
3000
1500
2000
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1000
soo
B 3000
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al., 1977). Synthesis in a homologous Neurospora system (Harmey et al., 1977) yielded a protein identical in apparent molecular weight to the authentic protein (Fig. 3). Also, when Neurospora total or poIy(A)-RNA was translated in a rabbit reticulocyte lysate (Pelham and Jackson, 1976) or wheat germ translation system (Roberts and Paterson, 1973) the immunoprecipitable ADP/ATP cartier protein was indistinguishable from the isolated protein (Fig. 4). Furthermore when translation was carried out in the presence of 35S-labelled formylmethionyl transfer RNA (Zimmermann et al., 1979) the immunoprecipitated protein had the same molecular weight as the authentic protein (Fig. 4). The retention of the formylmethionine and the coincident molecular weight clearly indicate the absence of an aminoterminal extension. In beth homologous and heterologous cell free systems and newly formed ADP/ATP carrier was detected mainly in the postribosomal supernatant. Citrate synthase is a soluble matrix protein (Ernster and Kuylenstierna, 1970). The mature mitochondria] protein is a dimer composed of identical subunits with a o---o
e-------e
400
3H cpm
35s cpm
1500
300
1000
200
500
100
200
20
40 Fraction no
60
Fig. 2, Synthesis of apecytochrome c in heterologous cell free systems programmed with Neurospora poly(A)-RNA. Apocytochrome c was immunoprecipitated from the cell free incubation mixture with a specific antibody and protein-A-sepharose. The immunoprecipitate was subjected to SDS electrophor~.~is. The gel was sliced and radioactivity in gel fractions determined. A: Wheat germ system; incubation with [3SS]methionine. Coelectrophoresis of holocytochrome c labelled with [~rI]leucine. B: Reticulocyte lysate; incubation with [~S]methionine. C: Reticulocyte lysate; incubation with [~sS]formylmethionyl-transfer-RNA.
-_-
-
.W'.a
20
~:~-
40 Fraction no.
60
Fig. 3. Synthesis of ADP/ATP carrier in a cell free homogenate. A cell free homogenate from Neurospora cells grown in the presence of [asS]sulfate was incubated with [aI-I]leucine for 10rain (Harmey et al., 1977). ADP/ATP carrier was immunoprecipitated after lysis with Triton X-100. The immunoprecipitate was analysed by SDS polyacrylamide gel electrophoresis.
286 @-
~,-
•
-,
600
3% cpm 1500
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I000
200
500
3H cpm
=35S-" cpm 3000
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Discussion
1000
300
monomeric molecular weight of 45000. In vivo pulse labelling studies at low temperature (8°C) showed the appearance of a larger molecular weight monomer having a molecular weight of about 47 000, which was rapidly converted to the m a t u r e 45 000 form. At room temperature the conversion was so rapid t h a t the precursor form was undetectable. Homologous and heterologous translation systems also showed t h a t citrate synthase was initially synthesized as a larger precursor protein (Fig. 5). The precursor protein is quite unstable compared to the authentic protein. Translation in the presence of [3sS]formylmethionyl-RNA showed t h a t the 47 000 protein retained its initiator methionine.
C
200
100
20
40 Fraction no
60
Fig. 4. Synthesis of ADP/ATPcarrier in heterologouscell free systems programmedwith Neurospora poly(A)-RNA. ADP/ATP carrier was immunoprecipitatedfrom the cell free systems with a specific antibedy and the immunoprecipitates were analysed by SDS-gel electrophoresis (Zimmermann et al., 1979). A: Wheat germ system; incubation with [~]leucine. Coelectrophoresis of ~S labelled carrier immunoprecipitatedfrom mitochondria. B: Reticulocyte lysate; incubation with [~S]methionine. C: Reticulocyte lysate; incubation with [ssS]formylmethionyl-transfer-RNA.
A comparison of the results obtained with the three proteins shows t h a t only in the case of the matrix protein is there an extension of the polypeptide chain in the. precursor, whereas the other two precursors are synthesized at their final length. Our results with cytochrome c suggest t h a t the apoprotein is synthesized on cytoribosomes and released into the cytosol. The uptake of apocytochrome c into the mitochondria is accompanied by conversion to the holo form and requires intact mitochondria which suggests a role for the outer membrane in protein uptake. The ADP/ATP carrier protein despite its highly hydrophobic character is synthesized on cytoribosomes and is probably released into the cytosol. The cytosolic precursor is t a k e n up by mitochondria and integrated into the hydrophobic domain of the inner membrane. Citrate synthase on the other h a n d is released into the cytosol as a larger molecular weight precursor which is cleaved on or after transport to form the m a t u r e protein of the matrix. The behaviour of citrate synthase resembles t h a t reported for the ATPase subunits of yeast mitochondria and of the cyt bcl complex (Maccechini et al., 1979; Cot~ et al., 1979). It appears from our results and those of
287 e ~
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3H 800
35S cpm 200
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~50
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cpm
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or in the interior of the protein structure. We propose that the precursor proteins undergo changes in conformation which lead to their correct assembly. These changes may be triggered by different events, heme attachment for cytochrome c, membrane integration for ADP/ATP translocator, proleolytic cleavage in the case of citrate synthase. At this point in time sufficient data is not available to draw generalisecl conclusions regarding the role of additional or internal sequences in the transport of mitochondrial proteins. Furthermore the exact mode of transport and the role of outer and inner membranes in this transport remains to be elucidated.
o---o
35S cpm
3H cpm
300
6O0
OOt
200
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40 Fraction no.
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O0
60
Fig. 5. Synthesis of citrate synthase in homologous and heterologous cell free systems. Immunoprecipitated citrate synthase was analysed by SDS electrophoresis. A: Neurospora cell free homogenate; [3H]leucine was incorporated into the cell free homogenate which was obtained from cells prelabelled with [~S]methionine. B: Reticulocyte lysate programmed with Neurospora-poly(A)RNA; incubation w~th [~S]methionine. Coelectrophoresis of ~H-labelled citrate synthase immunoprecipitated from mitochondria.
others (Maccechini et al., 1979; Cot~ et al., 1979) that signals for specific translocation may be located either in additional sequences
References Cot~, C., M. Solioz and G. Schatz, 1979, J. Biol. Chem. 254, 1437-1439. De Pierre, J.W. and L. Ernster, 1977, Annu. Rev. Biochem. 46, 201-262. Ernster, L. and B. Kuylenstierna, 1970, in: E. Racker (ed.), Membranes of Mitochondria and Chloroplasts (Van-Ncetrand Reinhold Co., London) p. 195. Hallermayer, G., R. Zimmermann and W. Neupert, 1977, Eur. J. Biochem. 81,523-532. Harmey, M.A., G. Hallermayer, H. Korb and W. Neupert, 1977, Eur. J. Biochem. 81,533-544. Heller, J. and E.L. Smith, 1966, J. Biol. Chem. 31653180. Klingenberg, M., 1976, in: Martenosi, A.N. (ed.), Vol. 3, The Enzymes of Biological Membranes: Membrane Transport (Plenum Publ. Co., New York) pp. 383-438. Korb, H. and W. Neupert, 1978, Eur. J. Biochem. 91, 609-620. Maccechini, M.L., Y. Rudin, G. Blobel and G. Schatz, 1979, Proc. Natl. Acad. Sci. USA 76, 343-347. Pelham, H.R.B. and R.J. Jackson, 1976, Eur. J. Biochem. 67, 247-256. Roberts, B.E. and B.M. Paterson, 1973, Proc. Natl. Acad. Sci. USA 70, 2330-2334. Zimmermann, R., N. Palnch, M. Sprinz! and W. Neupert, 1979, Eur. J. Biochem. 99, 247-252.
