Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria and inserted into the membrane from the matrix side (Tzagoloff, A., and Meagher, P. (1972) J. Biol. Chem. 247, 594- 603). The transfer of the protein was dependent on an energized mitochondrial inner membrane. It was accompanied by proteolytic processing of the precursor to the mature protein with the correct NH2 terminus as determined by Edman degradation of the transferred protein. The possibility is discussed that there are common features in the uptake machinery neither specific for one species nor specific for individual precursor proteins in the same species

Hennig, Bernd

Köhler, Helmut

Neupert, Walter

Schmidt, Bernd

English

Universität München: Elektronischen Publikationen

Communication Vol. 258, No. 8, Issue of April 25, pp. 4687-4689,  1983 THE JOURNAI. OF BIOLOGICAL CHEMISTRY Prrnted in U.S.A.  
Transport of the  Precursor  to 
Neurospora ATPase  Subunit 9 into 
Yeast  Mitochondria 
IMPLICATIONS ON THE DIVERSITY OF THE 
TRANSPORT MECHANISM* 
(Received for publication, December 21, 1982) 
Bernd  Schmidt,  Bernd  Hennig,  Helmut  Kohler, and 
Walter NeupertS 
From the Institut fur Biochemie, Uniuersitat  Gottingen, 
0-3400 Gottingen,  Federal  Republic of Germany 
Isolated yeast mitochondria were able to take up 
Neurospora ATPase subunit 9 in vitro although the 
homologous yeast protein is synthesized within the 
mitochondria  and  inserted into the  membrane  from the 
matrix side (Tzagoloff, A., and Meagher, P. (1972) J. 
Biol. C h m .  247, 694-603). The transfer  of  the  protein 
was dependent on an energized mitochondrial inner 
membrane.  It was accompanied  by  proteolytic  process- 
ing of the precursor to the mature protein with the 
correct NH2 terminus as determined  by Edman degra- 
dation of the transferred protein. The possibility is 
discussed  that  there  are common features in the  uptake 
machinery  neither  specific  for  one species nor specific 
for  individual  precursor  proteins in the  same  species. 
Import of proteins of mitochondria occurs by a  post-trans- 
lational mechanism (1-3). Precursor proteins are synthesized 
on free cytoplasmic polysomes and released into the cytosol 
(3-5). They  are  then imported into the mitochondrion in a 
step which is, with most but not all proteins, dependent on an 
electrical potential across the inner mitochondrial membrane 
(6, 7). With the majority of precursor proteins, this transfer is 
accompanied by proteolytic processing (3). 
Several hundred different proteins are transported into  the 
mitochondria by this mechanism. Studies with a number of 
mitochondrial proteins have clearly indicated that more than 
one pathway exists. Cytochrome c  appears  to use a receptor 
and  a mechanism for transmembrane transfer which is not 
shared with any other protein tested so far (6, 8). Mitochon- 
drial porin, a protein of the outer mitochondrial membrane, 
seems also to have an assembly pathway different from other 
mitochondrial proteins (5). On the other  hand, we consider it 
unlikely that every precursor protein has  a specific receptor 
and uptake mechanism; rather, we suggest that groups of 
different proteins may be transported by a common pathway. 
A number of different precursor proteins may then have a 
common structure required for the interaction with the com- 
* This work W ~ S  supported by Deutsche Forschungsgemeinschaft 
Project 101/17-3 and by a grant from the Fonds der Chemkchen 
Industrie. The costs of publication of this article were defrayed in 
part by the payment of page charges. This article  must  therefore be 
hereby  marked “aduertisement” in accordance with 18  U.S.C. Section 
1734 solely to indicate this fact. 
$ T o  whom correspondence should be sent at, Institut fur Bio- 
chemie der Universitit Gottingen, HumboldtaUee 7, D-3400 Gottin- 
gen, Federal Republic of Germany. 
ponents of that pathway. One indication that this is actually 
true is the observation that precursor proteins of Neurospora 
can be imported in vitro into mitochondria from organisms 
phylogenetically as distant  as  rat and yeast (7, 9). 
Here, we address the question of common  recognition struc- 
tures on different precursors and a limited number of receptor 
structures on mitochondria by investigating the special case 
of intracellular transport of subunit 9 of the mitochondrial 
ATPase. 
In yeast, subunit 9 of the mitochondrial ATPase is coded 
for by mitochondrial DNA and is translated on mitochondrial 
ribosomes (10, 11). It is inserted into the inner membrane 
from the matrix side.  No  cleavage of a presequence is  involved 
in this process; in fact, the initiating formylmethionine is 
retained in the  mature protein (12,  13). In  contrast, in Neu- 
rospora and in higher organisms, subunit 9 is coded  for  by a 
nuclear gene (14), is synthesized by cytoplasmic polysomes as 
a larger precursor (15), and is post-translationally transferred 
into  the inner membrane with concomitant proteolytic proc- 
essing (8). Yeast and Neurospora subunit 9 are closely related 
in their amino acid sequences with some 40% homology (16). 
The question then is: can the Neurospora precursor be  im- 
ported into yeast mitochondria and processed, although this 
is clearly not the assembly pathway in uiuo? We show here 
that indeed the heterologous precursor is imported by yeast 
mitochondria and correctly processed to  the mature size. 
MATERIALS AND METHODS 
Growth of Neurospora  crassa wild type 74A, radioactive labeling 
of cells, and isolation of mitochondria was carried out  as described (4, 
17). Saccharomyces cereuisiae diploid wild type strain 211-lb was 
grown according to Maccecchini et al. (18). Mitochondria were iso- 
lated after converting cells to spheroplasts according to  the following 
procedure. After washing in distilled water, cells were incubated in 
0.1 M Tris, 10 mM dithioerythritol, pH 9.2, for 5 min at 30 “C. Then, 
cells were harvested again and resuspended in 1.3 M sorbitol, 10 mM 
MOPS,’ pH 7.2, at 300  mg (wet  weight)/ml  and  incubated with 3 mg/ 
ml of Zymolase 5000 (Seikagaku Kogyo  Co., Ltd., Tokyo, Japan) for 
20 min at 30 “C. Cells were washed once in sorbitol/MOPS buffer and 
resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2, 
at a  concentration of 1 g of spheroplasts  (wet  weight)/lO ml. After 
addition of PMSF (final concentration 1 mM), they were homogenized 
in a Dounce homogenizer by 10 strokes. After a low speed spin at 
1,000 X g for 5 min, mitochondria were pelleted at 17,300 X g for 10 
min. 
Synthesis of Neurospora precursor proteins in rabbit reticulocyte 
lysates  and  transfer  into isolated mitochondria was performed accord- 
ing to published procedures (7, 17). 
Isolation of Neurospora ATPase subunit 9 and preparation of 
antibodies was carried out as described (7). Immunoprecipitation 
from mitochondria was performed under the following conditions. 
Mitochondria (250 pg of protein) were dissolved in 50 p1 of 2% SDS, 
5% 2-mercaptoethanol, 60 mM Tris-HC1, pH 6.8, and boiled for 3 min. 
1 ml of Triton buffer (1% Triton X-100,0.3 M NaCI, 5 mM EDTA, 10 
mM Tris-HCI, pH 7.5) and 10 pl of a 0.1 M solution of PMSF and o- 
phenanthroline in ethanol was added. The solution was freed of 
insoluble material by centrifugation for 15 min at 27,000 X g .  Protein 
A-agarose (Sigma) with bound immunoglobulins against subunit 9 
was added to  the  supernatant and the mixture was shaken for 30 min 
at 4 “C. The protein A-agarose immune complex was washed three 
times in Triton buffer and once in 10 mM Tris-HCI, pH 7.5. The 
I The abbreviations used are: MOPS, 4-morpholinepropanesulfonic 
acid PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl 
sulfate. 
4687 
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4688 Transfer of Proteins  into  Mitoch ndria 
immunoprecipitates were dissolved in 100 pl of 2% SDS. 5% 2-mer- 
captoethanol, IO<+ glycerol, 60 mM Tris-HCI, pH 6.8, by shaking for 
30 min a t  4 "C and  then  heating  to 95 "C for 2 min. 
Precipitation of precursor to subunit 9 from reticulocyte lysate 
supernatants was carried  out  as follows. The  supernatant was adjusted 
to 1% Triton, 0.3 M NaCI, 5 mM EDTA  and centrifuged for 15 rnin a t  
27,000 X g. Antiserum was added  to  the  supernatant,  and  the  mixture 
was incubated for 16 h at  4 "C. Then,  the  sample was mixed with 
preswollen protein A-agarose for 30 min. Washing and dilution of 
immunoprecipitates was carried  out as described  above. 
For microscale sequencing  experiments,  the  precursor  was  synthe- 
sized in a reticulocyte  lysate in the presence of 0.08 mCi/ml of ["HI 
tyrosine (76 Ci/mmol, New England Nuclear). The postribosomal 
supernatant was subjected  to  immunoprecipitation. The  mature  pro- 
tein  was obtained by labeling intact cells  with  [,"H]tyrosine (100 pCi/ 
100 ml of culture) for 15 min. Mitochondria were  isolated and  subunit 
9 was immunoprecipitated. In citro transferred  subunit 9  was obtained 
by carrying out a transfer experiment with yeast mitochondria as 
described in the legend to Fig. 1. Subunit 9 was immunoprecipitated 
after reisolating the  mitochondria. 
The immunoprecipitates were  dissolved and  subjected  to  electro- 
phoresis. The gels were fluorographed (19). The radioactive bands 
were cut  out;  the gel slices  were washed in water,  and  extracted  with 
80% (v/v) formic  acid. After removal of the formic  acid, the  proteins 
were subjected  to  manual  Edman  degradation  (20). 
Electrophoresis on 16% polyacrylamide  gels  was  performed accord- 
ing to Laemmli (21). After  fluorography (19) dried  gels  were analyzed 
as described (17). 
RESULTS 
Neurospora Subunit 9 Is Transferred into Yeast Mito- 
chondria-The  precursor  to  subunit 9 was synthesized in a 
reticulocyte  lysate  programmed with Neurospora  RNA in the 
presence of [:"S]methionine. It could be immunoprecipitated 
from the  postribosomal  supernatant (Fig. 1, track 1 ) with the 
antibody against the mature subunit 9. The postribosomal 
supernatant was incubated  either  with  mitochondria isolated 
from Neurospora  or  yeast.  In  both cases, the  mitochondria, 
which were reisolated after  incubation,  contained processed 
subunit 9 (Fig. 1, tracks 2 and 4 ) .  Subunit 9 processed in  vitro 
had  the  same  apparent molecular  weight as  mature  subunit 9 
immunoprecipitated  from  Neurospora  mitochondria labeled 
in uiuo (Fig. 1, track 3 ) .  Neurospora subunit 9, transferred 
into  yeast  mitochondria  in vitro, is largely resistant  to  Pro- 
teinase K (Fig. 1, track 5 ) .  Breakdown of the membrane 
potential of the  yeast  mitochondria by addition of valinomycin 
1 2 3 4 5 6 7  
- ( p )  
- (m) 
FIG. 1. Transfer of the Neumspom precursor to subunit 9 
into yeast mitochondria. I ,  Precursor immunoprecipitated from 
reticulocyte lysate supernatant; 2, transfer into Neurospora mito- 
chondria; 3, mature  subunit 9 immunoprecipitated  from ["'S]sulfate 
labeled mitochondria; 4-7, transfer  into  yeast  mitochondria; 4, control 
transfer; 5, after transfer mitochondria were suspended in isotonic 
medium and  then  treated  with 300 pg/ml of Proteinase K for 1  h a t  
0 "C; 6, transfer in the  presence of 2 pM valinomycin; 7, transfer in the 
presence of 10 p~ oligomycin, 4 p~ antimycin A, 1 mM KCN. ( p ) ,  
precursor; (m), mature  subunit 9. 
Tronslcr ~n 
n l o  yeast mltochondrlo 
Moturc  protcln 
from Weurosporo 
Precursor  f rom 
ret lculocytc lysotc 
FIG. 2. Edman degradation of different forms of subunit 9 
labeled with ["H]tyrosine. The total activities employed were: 
subunit 9 transferred into yeast mitochondria, 1160 cpm; mature 
subunit 9 from Neurospora  mitochondria, 1640 cpm;  and  precursor 
protein, 2800 cpm.  Total  radioactivities in each  case were set  to 100% 
(dotted  bars)  and  the  radioactivities released in the first three  Edman 
cycles are expressed as % of total  radioactivities  (hatched  bars). 
(Fig. 1, track 6) or  carbonyl cyanidep-chlorophenylhydrazone 
(not  shown)  leads  to a complete elimination of import  and 
processing. The  same  has been found for the  import of the 
Neurospora precursor into Neurospora and rat liver mito- 
chondria (7). Transfer into Neurospora mitochondria was 
completely inhibited by a combination of oligomycin and 
antimycin A plus KCN which inhibit the formation of a 
membrane  potential  either  through  electron  transport  or 
through  the oligomycin-sensitive ATPase (7). The  import of 
Neurospora  subunit 9 into  yeast  mitochondria was also largely 
inhibited by these  agents (Fig. 1, track 7). 
The  Precursor  to  Neurospora  Subunit 9 Is Correctly Proc- 
essed by Yeast Mitochondria-Neurospora subunit 9 was 
isolated from  yeast  mitochondria  subsequent  to  transfer  and 
processing in vitro. The precursor in this experiment was 
synthesized in the  presence of ["Hltyrosine, which is the  NHr- 
terminal amino acid in the mature protein. A total of two 
tyrosine residues are  present in the  Neurospora  protein (16). 
Edman degradation was  performed, and the radioactivity 
released during the first three steps was compared to that 
obtained when the precursor was analyzed. Also, mature 
subunit 9, labeled in vivo by adding  ["Hltyrosine  to  the  culture 
medium, was subjected  to  Edman  degradation. As shown in 
Fig. 2, about 30% of total  radioactivity  appeared in the first 
Edman step when both mature subunit 9 and subunit 9 
transferred  into  yeast  mitochondria were sequenced.  In  con- 
trast,  no significant radioactivity  was released in the first three 
steps when the  precursor  protein was  analyzed  (Fig. 2). These 
data  show  that  Neurospora  subunit 9 is correctly  processed 
when transferred  into  yeast  mitochondria in vitro. 
DISCUSSION 
We have  shown  that  yeast  mitochondria  have  the  ability  to 
import  and  correctly process a protein  that in yeast cells is 
synthesized within mitochondria  and  inserted  from  the  matrix 
side. The most likely explanation of this  phenomenon is that 
the  import  mechanism is not  constructed in such a way that 
each precursor protein has i t s  own uptake device. Rather, 
recognition on  the  mitochondrial  surface  must involve struc- 
tures  not specific for  individual mitochondrial precursor  pro- 
teins. Also, further steps in the transfer process such as 
translocation  across  the  outer  membrane  and proteol-ytic proc- 
essing appear  to be  common  for  different  mitochondrial pro- 
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Transfer  of Proteins into Mitochondria 4689 
teins. The observation that  the yeast mitochondrial processing 
enzyme(s) correctly process a precursor protein from a differ- 
ent organism,  which  in yeast is assembled via a quite different 
pathway, suggests that there must be signals common to at 
least classes of mitochondrial precursors. On the  other  hand, 
it is clear that there is not one unique pathway. Subunit 9 of 
ATPase belongs to  that class of proteins which requires an 
energized inner membrane for transfer (7).  Within this class 
it falls into  the group of precursors which are proteolytically 
processed during or after membrane translocation. This group 
containi the majority of proteins studied so far. Precursor 
proteins which belong to a second class, not requiring a 
membrane potential, are cytochrome c and the porin of the 
outer mitochondrial membrane (6, 5). These two proteins do 
not have to be inserted into or translocated across the inner 
membrane. Both of these proteins are not proteolytically 
cleaved during assembly. However, they do use different 
pathways, as demonstrated by the observation that excess 
unlabeled apocytochrome c does not compete with porin for 
assembly.’ Furthermore, cytochrome bz (22) in yeast and 
cytochrome c1(9,22) in yeast and Neurospora are transported 
and assembled in a two-step processing event which seems to 
involve a  “detour” of the precursors to  the matrix before they 
reach their functional site in the intermembrane space or at 
the  outer face of the inner membrane (22). 
REFERENCES 
1. Chua, N.-H., and  Schmidt, G. W. (1979) J. Cell Biol. 81,461-483 
2. Sabatini, D. D., Kreibich, G., Morimoto, T., and Adesnik, M. 
(1982) J. Cell Biol. 92, 1-22 
H. Freitag, B. Hennig, and W. Neupert, unpublished data. 
3. Neupert, W., and  Schatz, G. (1981) Trends Biochern. Sci. 6, l -4  
4. Korb, H.,  and  Neupert, W. (1978) Eur. J. Biochern. 91,609-620 
5. Freitag, H., Janes, M., and  Neupert, W. (1982) Eur. J. Biochern. 
6. Hennig, B., and  Neupert, W. (1981) Eur. J. Biochern. 121, 203- 
7. Schleyer, M., Schmidt, B., and  Neupert, W. (1982) Eur. J. Bio- 
8. Zimmermann, R., Hennig, B., and Neupert, W. (1981) Eur. J. 
9. Teintze, M., Slaughter, M., Weiss, H., and  Neupert, W. (1982) J. 
10. Tzagoloff, A,, and Meagher, P. (1972) J. Biol. Chern. 247, 594- 
11. Sierra, M. F., and Tzagoloff, A. (1973) Proc. Natl. Acad. Sci. 
12. Macino, G., and Tzagoloff, A. (1979) J.  Biol. Chern. 254, 4617- 
13. Hensgens, L. A. M., Grivell, L. A., Borst, P.,  and Bos, J. L. (1979) 
14. Viebrock, A., Perz, A., and  Sebald, W. (1982) EMBO  Journal 1, 
15. Michel, R., Wachter, E., and  Sebald, W. (1979) FEBS Lett. 101, 
373-376 
16. Sebald, W., and Wachter, E. (1978) in 29th Moosbach Collo- 
quium: Energy  Conservation in Biological  Membranes. 
(Schafer, G., and Klingenberg, M., eds) pp. 228-263, Springer 
Verlag, Berlin 
17. Zimmermann,  R., and  Neupert, W. (1980) Eur. J. Biochern. 109, 
18. Maccecchini, M.-L., Rudin, Y., and Schatz, G.  (1979) J. Biol. 
19. Chamberlin, J. P. (1979) Anal. Biochern. 98, 132-135 
20. Tarr, G. E. (1975) Anal. Biochern. 63,361-370 
21. Laemmli, U. K. (1970) Nature  (Lond.) 277,680-685 
22. Gasser, S. M., Ohashi, A., Dann, G., Bohni, P. C., Gibson, J., Reid, 
G.  A., Yonetani, T., and Schatz, G.  (1982) Proc. Natl.  Acad. 
Sci. U. S. A. 79, 267-271 
126, 197-202 
212 
chern. 125,109-116 
Biochern. 116,455-460 
Biol. Chern. 257, 10364-10371 
603 
U. S. A. 70,3155-3159 
4623 
Proc. Natl.  Acad. Sci. U. S. A. 76, 1663-1667 
565-571 
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Transport of the precursor to neurospora ATPase subunit 9 into yeast mitochondria

Open Access LMU

Communication Vol. 258, No. 8, Issue of April 25, pp. 4687-4689,  1983 THE JOURNAI. OF BIOLOGICAL CHEMISTRY Prrnted in U.S.A.  Transport of the  Precursor  to Neurospora ATPase  Subunit 9 into Yeast  Mitochondria IMPLICATIONS ON THE DIVERSITY OF THE TRANSPORT MECHANISM* (Received for publication, December 21, 1982) Bernd  Schmidt,  Bernd  Hennig,  Helmut  Kohler, and Walter NeupertS From the Institut fur Biochemie, Uniuersitat  Gottingen, 0-3400 Gottingen,  Federal  Republic of Germany Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria  and  inserted into the  membrane  from the matrix side (Tzagoloff, A., and Meagher, P. (1972) J. Biol. C h m .  247, 694-603). The transfer  of  the  protein was dependent on an energized mitochondrial inner membrane.  It was accompanied  by  proteolytic  process- ing of the precursor to the mature protein with the correct NH2 terminus as determined  by Edman degra- dation of the transferred protein. The possibility is discussed  that  there  are common features in the  uptake machinery  neither  specific  for  one species nor specific for  individual  precursor  proteins in the  same  species. Import of proteins of mitochondria occurs by a  post-trans- lational mechanism (1-3). Precursor proteins are synthesized on free cytoplasmic polysomes and released into the cytosol (3-5). They  are  then imported into the mitochondrion in a step which is, with most but not all proteins, dependent on an electrical potential across the inner mitochondrial membrane (6, 7). With the majority of precursor proteins, this transfer is accompanied by proteolytic processing (3). Several hundred different proteins are transported into  the mitochondria by this mechanism. Studies with a number of mitochondrial proteins have clearly indicated that more than one pathway exists. Cytochrome c  appears  to use a receptor and  a mechanism for transmembrane transfer which is not shared with any other protein tested so far (6, 8). Mitochon- drial porin, a protein of the outer mitochondrial membrane, seems also to have an assembly pathway different from other mitochondrial proteins (5). On the other  hand, we consider it unlikely that every precursor protein has  a specific receptor and uptake mechanism; rather, we suggest that groups of different proteins may be transported by a common pathway. A number of different precursor proteins may then have a common structure required for the interaction with the com- * This work W ~ S  supported by Deutsche Forschungsgemeinschaft Project 101/17-3 and by a grant from the Fonds der Chemkchen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article  must  therefore be hereby  marked “aduertisement” in accordance with 18  U.S.C. Section 1734 solely to indicate this fact. $ T o  whom correspondence should be sent at, Institut fur Bio- chemie der Universitit Gottingen, HumboldtaUee 7, D-3400 Gottin- gen, Federal Republic of Germany. ponents of that pathway. One indication that this is actually true is the observation that precursor proteins of Neurospora can be imported in vitro into mitochondria from organisms phylogenetically as distant  as  rat and yeast (7, 9). Here, we address the question of common  recognition struc- tures on different precursors and a limited number of receptor structures on mitochondria by investigating the special case of intracellular transport of subunit 9 of the mitochondrial ATPase. In yeast, subunit 9 of the mitochondrial ATPase is coded for by mitochondrial DNA and is translated on mitochondrial ribosomes (10, 11). It is inserted into the inner membrane from the matrix side.  No  cleavage of a presequence is  involved in this process; in fact, the initiating formylmethionine is retained in the  mature protein (12,  13). In  contrast, in Neu- rospora and in higher organisms, subunit 9 is coded  for  by a nuclear gene (14), is synthesized by cytoplasmic polysomes as a larger precursor (15), and is post-translationally transferred into  the inner membrane with concomitant proteolytic proc- essing (8). Yeast and Neurospora subunit 9 are closely related in their amino acid sequences with some 40% homology (16). The question then is: can the Neurospora precursor be  im- ported into yeast mitochondria and processed, although this is clearly not the assembly pathway in uiuo? We show here that indeed the heterologous precursor is imported by yeast mitochondria and correctly processed to  the mature size. MATERIALS AND METHODS Growth of Neurospora  crassa wild type 74A, radioactive labeling of cells, and isolation of mitochondria was carried out  as described (4, 17). Saccharomyces cereuisiae diploid wild type strain 211-lb was grown according to Maccecchini et al. (18). Mitochondria were iso- lated after converting cells to spheroplasts according to  the following procedure. After washing in distilled water, cells were incubated in 0.1 M Tris, 10 mM dithioerythritol, pH 9.2, for 5 min at 30 “C. Then, cells were harvested again and resuspended in 1.3 M sorbitol, 10 mM MOPS,’ pH 7.2, at 300  mg (wet  weight)/ml  and  incubated with 3 mg/ ml of Zymolase 5000 (Seikagaku Kogyo  Co., Ltd., Tokyo, Japan) for 20 min at 30 “C. Cells were washed once in sorbitol/MOPS buffer and resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2, at a  concentration of 1 g of spheroplasts  (wet  weight)/lO ml. After addition of PMSF (final concentration 1 mM), they were homogenized in a Dounce homogenizer by 10 strokes. After a low speed spin at 1,000 X g for 5 min, mitochondria were pelleted at 17,300 X g for 10 min. Synthesis of Neurospora precursor proteins in rabbit reticulocyte lysates  and  transfer  into isolated mitochondria was performed accord- ing to published procedures (7, 17). Isolation of Neurospora ATPase subunit 9 and preparation of antibodies was carried out as described (7). Immunoprecipitation from mitochondria was performed under the following conditions. Mitochondria (250 pg of protein) were dissolved in 50 p1 of 2% SDS, 5% 2-mercaptoethanol, 60 mM Tris-HC1, pH 6.8, and boiled for 3 min. 1 ml of Triton buffer (1% Triton X-100,0.3 M NaCI, 5 mM EDTA, 10 mM Tris-HCI, pH 7.5) and 10 pl of a 0.1 M solution of PMSF and o- phenanthroline in ethanol was added. The solution was freed of insoluble material by centrifugation for 15 min at 27,000 X g .  Protein A-agarose (Sigma) with bound immunoglobulins against subunit 9 was added to  the  supernatant and the mixture was shaken for 30 min at 4 “C. The protein A-agarose immune complex was washed three times in Triton buffer and once in 10 mM Tris-HCI, pH 7.5. The I The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate. 4687  by on November 12, 2008 www.jbc.orgDownloaded from 4688 Transfer of Proteins  into  Mitoch ndria immunoprecipitates were dissolved in 100 pl of 2% SDS. 5% 2-mer- captoethanol, IO<+ glycerol, 60 mM Tris-HCI, pH 6.8, by shaking for 30 min a t  4 "C and  then  heating  to 95 "C for 2 min. Precipitation of precursor to subunit 9 from reticulocyte lysate supernatants was carried  out  as follows. The  supernatant was adjusted to 1% Triton, 0.3 M NaCI, 5 mM EDTA  and centrifuged for 15 rnin a t  27,000 X g. Antiserum was added  to  the  supernatant,  and  the  mixture was incubated for 16 h at  4 "C. Then,  the  sample was mixed with preswollen protein A-agarose for 30 min. Washing and dilution of immunoprecipitates was carried  out as described  above. For microscale sequencing  experiments,  the  precursor  was  synthe- sized in a reticulocyte  lysate in the presence of 0.08 mCi/ml of ["HI tyrosine (76 Ci/mmol, New England Nuclear). The postribosomal supernatant was subjected  to  immunoprecipitation. The  mature  pro- tein  was obtained by labeling intact cells  with  [,"H]tyrosine (100 pCi/ 100 ml of culture) for 15 min. Mitochondria were  isolated and  subunit 9 was immunoprecipitated. In citro transferred  subunit 9  was obtained by carrying out a transfer experiment with yeast mitochondria as described in the legend to Fig. 1. Subunit 9 was immunoprecipitated after reisolating the  mitochondria. The immunoprecipitates were  dissolved and  subjected  to  electro- phoresis. The gels were fluorographed (19). The radioactive bands were cut  out;  the gel slices  were washed in water,  and  extracted  with 80% (v/v) formic  acid. After removal of the formic  acid, the  proteins were subjected  to  manual  Edman  degradation  (20). Electrophoresis on 16% polyacrylamide  gels  was  performed accord- ing to Laemmli (21). After  fluorography (19) dried  gels  were analyzed as described (17). RESULTS Neurospora Subunit 9 Is Transferred into Yeast Mito- chondria-The  precursor  to  subunit 9 was synthesized in a reticulocyte  lysate  programmed with Neurospora  RNA in the presence of [:"S]methionine. It could be immunoprecipitated from the  postribosomal  supernatant (Fig. 1, track 1 ) with the antibody against the mature subunit 9. The postribosomal supernatant was incubated  either  with  mitochondria isolated from Neurospora  or  yeast.  In  both cases, the  mitochondria, which were reisolated after  incubation,  contained processed subunit 9 (Fig. 1, tracks 2 and 4 ) .  Subunit 9 processed in  vitro had  the  same  apparent molecular  weight as  mature  subunit 9 immunoprecipitated  from  Neurospora  mitochondria labeled in uiuo (Fig. 1, track 3 ) .  Neurospora subunit 9, transferred into  yeast  mitochondria  in vitro, is largely resistant  to  Pro- teinase K (Fig. 1, track 5 ) .  Breakdown of the membrane potential of the  yeast  mitochondria by addition of valinomycin 1 2 3 4 5 6 7  - ( p )  - (m) FIG. 1. Transfer of the Neumspom precursor to subunit 9 into yeast mitochondria. I ,  Precursor immunoprecipitated from reticulocyte lysate supernatant; 2, transfer into Neurospora mito- chondria; 3, mature  subunit 9 immunoprecipitated  from ["'S]sulfate labeled mitochondria; 4-7, transfer  into  yeast  mitochondria; 4, control transfer; 5, after transfer mitochondria were suspended in isotonic medium and  then  treated  with 300 pg/ml of Proteinase K for 1  h a t  0 "C; 6, transfer in the  presence of 2 pM valinomycin; 7, transfer in the presence of 10 p~ oligomycin, 4 p~ antimycin A, 1 mM KCN. ( p ) ,  precursor; (m), mature  subunit 9. Tronslcr ~n n l o  yeast mltochondrlo Moturc  protcln from Weurosporo Precursor  f rom ret lculocytc lysotc FIG. 2. Edman degradation of different forms of subunit 9 labeled with ["H]tyrosine. The total activities employed were: subunit 9 transferred into yeast mitochondria, 1160 cpm; mature subunit 9 from Neurospora  mitochondria, 1640 cpm;  and  precursor protein, 2800 cpm.  Total  radioactivities in each  case were set  to 100% (dotted  bars)  and  the  radioactivities released in the first three  Edman cycles are expressed as % of total  radioactivities  (hatched  bars). (Fig. 1, track 6) or  carbonyl cyanidep-chlorophenylhydrazone (not  shown)  leads  to a complete elimination of import  and processing. The  same  has been found for the  import of the Neurospora precursor into Neurospora and rat liver mito- chondria (7). Transfer into Neurospora mitochondria was completely inhibited by a combination of oligomycin and antimycin A plus KCN which inhibit the formation of a membrane  potential  either  through  electron  transport  or through  the oligomycin-sensitive ATPase (7). The  import of Neurospora  subunit 9 into  yeast  mitochondria was also largely inhibited by these  agents (Fig. 1, track 7). The  Precursor  to  Neurospora  Subunit 9 Is Correctly Proc- essed by Yeast Mitochondria-Neurospora subunit 9 was isolated from  yeast  mitochondria  subsequent  to  transfer  and processing in vitro. The precursor in this experiment was synthesized in the  presence of ["Hltyrosine, which is the  NHr- terminal amino acid in the mature protein. A total of two tyrosine residues are  present in the  Neurospora  protein (16). Edman degradation was  performed, and the radioactivity released during the first three steps was compared to that obtained when the precursor was analyzed. Also, mature subunit 9, labeled in vivo by adding  ["Hltyrosine  to  the  culture medium, was subjected  to  Edman  degradation. As shown in Fig. 2, about 30% of total  radioactivity  appeared in the first Edman step when both mature subunit 9 and subunit 9 transferred  into  yeast  mitochondria were sequenced.  In  con- trast,  no significant radioactivity  was released in the first three steps when the  precursor  protein was  analyzed  (Fig. 2). These data  show  that  Neurospora  subunit 9 is correctly  processed when transferred  into  yeast  mitochondria in vitro. DISCUSSION We have  shown  that  yeast  mitochondria  have  the  ability  to import  and  correctly process a protein  that in yeast cells is synthesized within mitochondria  and  inserted  from  the  matrix side. The most likely explanation of this  phenomenon is that the  import  mechanism is not  constructed in such a way that each precursor protein has i t s  own uptake device. Rather, recognition on  the  mitochondrial  surface  must involve struc- tures  not specific for  individual mitochondrial precursor  pro- teins. Also, further steps in the transfer process such as translocation  across  the  outer  membrane  and proteol-ytic proc- essing appear  to be  common  for  different  mitochondrial pro-  by on November 12, 2008 www.jbc.orgDownloaded from Transfer  of Proteins into Mitochondria 4689 teins. The observation that  the yeast mitochondrial processing enzyme(s) correctly process a precursor protein from a differ- ent organism,  which  in yeast is assembled via a quite different pathway, suggests that there must be signals common to at least classes of mitochondrial precursors. On the  other  hand, it is clear that there is not one unique pathway. Subunit 9 of ATPase belongs to  that class of proteins which requires an energized inner membrane for transfer (7).  Within this class it falls into  the group of precursors which are proteolytically processed during or after membrane translocation. This group containi the majority of proteins studied so far. Precursor proteins which belong to a second class, not requiring a membrane potential, are cytochrome c and the porin of the outer mitochondrial membrane (6, 5). These two proteins do not have to be inserted into or translocated across the inner membrane. Both of these proteins are not proteolytically cleaved during assembly. However, they do use different pathways, as demonstrated by the observation that excess unlabeled apocytochrome c does not compete with porin for assembly.’ Furthermore, cytochrome bz (22) in yeast and cytochrome c1(9,22) in yeast and Neurospora are transported and assembled in a two-step processing event which seems to involve a  “detour” of the precursors to  the matrix before they reach their functional site in the intermembrane space or at the  outer face of the inner membrane (22). REFERENCES 1. Chua, N.-H., and  Schmidt, G. W. (1979) J. Cell Biol. 81,461-483 2. Sabatini, D. D., Kreibich, G., Morimoto, T., and Adesnik, M. (1982) J. Cell Biol. 92, 1-22 H. Freitag, B. Hennig, and W. Neupert, unpublished data. 3. Neupert, W., and  Schatz, G. (1981) Trends Biochern. Sci. 6, l -4  4. Korb, H.,  and  Neupert, W. (1978) Eur. J. Biochern. 91,609-620 5. Freitag, H., Janes, M., and  Neupert, W. (1982) Eur. J. Biochern. 6. Hennig, B., and  Neupert, W. (1981) Eur. J. Biochern. 121, 203- 7. Schleyer, M., Schmidt, B., and  Neupert, W. (1982) Eur. J. Bio- 8. Zimmermann, R., Hennig, B., and Neupert, W. (1981) Eur. J. 9. Teintze, M., Slaughter, M., Weiss, H., and  Neupert, W. (1982) J. 10. Tzagoloff, A,, and Meagher, P. (1972) J. Biol. Chern. 247, 594- 11. Sierra, M. F., and Tzagoloff, A. (1973) Proc. Natl. Acad. Sci. 12. Macino, G., and Tzagoloff, A. (1979) J.  Biol. Chern. 254, 4617- 13. Hensgens, L. A. M., Grivell, L. A., Borst, P.,  and Bos, J. L. (1979) 14. Viebrock, A., Perz, A., and  Sebald, W. (1982) EMBO  Journal 1, 15. Michel, R., Wachter, E., and  Sebald, W. (1979) FEBS Lett. 101, 373-376 16. Sebald, W., and Wachter, E. (1978) in 29th Moosbach Collo- quium: Energy  Conservation in Biological  Membranes. (Schafer, G., and Klingenberg, M., eds) pp. 228-263, Springer Verlag, Berlin 17. Zimmermann,  R., and  Neupert, W. (1980) Eur. J. Biochern. 109, 18. Maccecchini, M.-L., Rudin, Y., and Schatz, G.  (1979) J. Biol. 19. Chamberlin, J. P. (1979) Anal. Biochern. 98, 132-135 20. Tarr, G. E. (1975) Anal. Biochern. 63,361-370 21. Laemmli, U. K. (1970) Nature  (Lond.) 277,680-685 22. Gasser, S. M., Ohashi, A., Dann, G., Bohni, P. C., Gibson, J., Reid, G.  A., Yonetani, T., and Schatz, G.  (1982) Proc. Natl.  Acad. Sci. U. S. A. 79, 267-271 126, 197-202 212 chern. 125,109-116 Biochern. 116,455-460 Biol. Chern. 257, 10364-10371 603 U. S. A. 70,3155-3159 4623 Proc. Natl.  Acad. Sci. U. S. A. 76, 1663-1667 565-571 217-229 Chem. 254,4368-4372  by on November 12, 2008 www.jbc.orgDownloaded from 

b y o n N o v e m b e r , w w w . j b c . o r g D o w n l o a d e d f r o m

in 29th Moosbach Colloquium: Energy Conservation in Biological Membranes.

Trends Biochern.

https://epub.ub.uni-muenchen.de/7414/1/Neupert_Walter_7414.pdf

Transport of the precursor to neurospora ATPase subunit 9 into yeast mitochondria

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