The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs- porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding")

Ito, Masaki

Kleene, Ralf

Neupert, Walter

Pfaller, Rupert

Pfanner, Nikolaus

Tropschug, Maximilian

English

Universität München: Elektronischen Publikationen

Communication Vol. 263, No. 9, Issue of March 25, pp. 4049-4051, 1988 THE  JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. 
Printed in U. S. A. 
Role of ATP in Mitochondrial 
Protein Import 
CONFORMATIONAL ALTERATION OF A 
PRECURSOR PROTEIN CAN SUBSTITUTE FOR 
ATP REQUIREMENT* 
(Received for publication, November 23,  1987) 
Nikolaus Pfanner, Rupert Pfaller, Ralf Kleene, 
Masaki ItoS, Maximilian  Tropschug,  and 
Walter  NeupertP 
From the Znstitut fur Physwlogische  Chemie,  Universitcit 
Munchen,  Goethestrasse 33, 8000 Miinchen 2, Federal 
Republic of Germany 
The role of nucleoside  triphosphates (NTPs) in  the 
import of porin  into  the  mitochondrial  outer  membrane 
was investigated with two forms  of  the  porin  precur- 
sor:  the in vitro synthesized  biosynthetic  precursor  (bs- 
porin) and a water-soluble form of porin (ws-porin) 
obtained  by  subjecting  the  membrane-derived  porin  to 
an acid-base treatment (exposure to trichloroacetic 
acid, followed by alkali and  rapid neutralization). The 
import  of ws-porin into  mitochondria  did  not  require 
NTPs, whereas the  import  of  bs-porin  required  NTPs. 
In other characteristics, such as binding  to a specific 
receptor protein on the mitochondrial surface, two- 
step insertion  into the outer  membrane,  and  formation 
of specific membrane  channels, ws-porin was indistin- 
guishable  from  bs-porin.  Thus,  the  acid-base  treatment 
applied in the  preparation of ws-porin can  substitute 
for  the NTP-requiring step in mitochondrial protein 
import. We conclude  that NTPs are required  for  un- 
folding  mitochondrial  precursor  proteins  (“transloca- 
tion  competent folding”). 
The  transport of cytoplasmically synthesized precursor pro- 
teins into mitochondria requires NTPs,’ e.g. ATP  or  GTP 
(Pfanner  and Neupert, 1986; Chen and Douglas, 1987; Eilers 
et QL, 1987; Hart1 et al., 1987; Kleene et at, 1987; Pfanner et 
al., 1987). Several observations suggested that  the  NTPs  are 
needed for the import competence of the precursor proteins 
rather  than solely interacting with mitochondrial elements. 
Precursor proteins displayed different degrees of protease 
sensitivity at  different levels of NTPs, suggesting that  NTPs 
are affecting their folding. Chimeric precursor  proteins with 
identical  targeting  signals required different  concentrations 
of NTPs for import (Pfanner et al., 1987). Finally, import of 
* This work  was supported by the Sonderforschungsbereich 184, 
the Genzentrum Miinchen, and  the Fonds der Chemischen 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 “advertisement” in accordance with 18 U.S.C. Section 1734 
solely to indicate this fact. 
$ Present address: Institute of Comprehensive Medical Science, 
Fujita Gakuen Health University, Toyoake, Aichi 470-11, Japan. 
3 To whom correspondence should be addressed. 
The abbreviations used are: NTPs, nucleoside triphosphates; bs- 
porin, biosynthetic porin precursor; ws-porin, water-soluble porin; 
MOPS, 3-(N-morpholino)propanesulfonic acid. 
incompletely synthesized precursor chains showed a decreased 
requirement for NTPs (Verner and Schatz, 1987). It  has been 
proposed that  NTPs were required to keep precursor proteins 
in an import-competent (“unfolded”) conformation (Pfanner 
and Neupert, 1986; Rothman and Kornberg, 1986; Chen and 
Douglas, 1987; Pfanner et al., 1987; Verner and Schatz, 1987). 
A demonstration that unfolding a mitochondrial precursor 
protein, whose import requires NTPs, can bypass the require- 
ment for NTPs would constitute direct proof for this hypoth- 
esis. 
Recently, we showed that  the mitochondrial import of the 
biosynthetic precursor form of the outer membrane protein 
porin (bs-porin) required NTPs (Kleene et al., 1987). A dif- 
ferent form of porin (ws-porin) was obtained by treatment of 
the purified membrane protein with 5% trichloroacetic acid, 
followed by treatment with 100 mM NaOH and by rapid 
neutralization with NaH2P04 (Pfaller et d., 1985). This ws- 
porin will also specifically bind to a receptor protein on the 
mitochondrial surface, compete with bs-porin for specific 
binding, insert into  the outer membrane in  a two-step reac- 
tion, and form specific membrane channels (Pfaller et al., 
1985; Pfaller and Neupert, 1987). We now report that the 
import of ws-porin does not require NTPs. 
EXPERIMENTAL PROCEDURES 
Published procedures were  used for the import of porin into isolated 
Neurospora crussa mitochondria (Pfaller et aZ., 1985; Kleene et al., 
1987; Pfaller and Neupert, 1987). Either bs-porin was  employed  which 
was synthesized in rabbit reticulocyte lysates and labeled with [%I 
methionine, or ws-porin was  employed  which  was prepared from the 
membrane form and labeled with 14C by reductive methylation. The 
import reactions included mitochondria (200 pg of protein), 10-30 p1 
of a 100 mM phosphate buffer (pH 7),  25 pl of rabbit reticulocyte 
lysate, antimycin A (8 p~ final concentration), oligomycin (20 pM 
final concentration),  and  a buffer consisting of 250 mM sucrose, 80 
mM KCI, 5 mM MgCl,, 10 mM MOPS, and 3% (w/v) bovine serum 
albumin, adjusted to  pH 7.2 with KOH (Pfanner  and Neupert, 1985). 
The final  volume was 200 pl. 
RESULTS AND DISCUSSION 
An in vitro import reaction containing Neurospora mito- 
chondria and rabbit reticulocyte lysate was depleted of endog- 
enous ATP (and ADP) by preincubation with apyrase (an 
ATPase and  an ADPase from potato) (Fig. 1, lanes 1 and 3 ) .  
Control samples received an apyrase preparation  heated to 
95 “C prior to use (Fig. 1, lanes 2 and 4 ) .  Samples 1 and 2 
contained W-labeled ws-porin, whereas samples 3 and 4 
contained 35S-labeled bs-porin synthesized in  rabbit reticulo- 
cyte lysates. Oligomycin was included to  prevent formation 
of ATP by the FoF1-ATPase. After incubation for 15 min at 
25 “C,  the mitochondria were treated with proteinase K at  a 
high concentration which completely degraded precursors 
which were not imported (Kleene et al., 1987; Pfaller and 
Neupert, 1987). Mitochondria were then reisolated and  ana- 
lyzed by sodium dodecyl sulfate-polyacrylamide gel electro- 
phoresis. Fluorographs of the dried gels are shown in Fig. 1. 
The import of bs-porin was inhibited by the pretreatment 
with apyrase (lane 3 )  as expected. Readdition of ATP or of 
GTP was shown to restore import  demonstrating the  NTP 
requirement of import of bs-porin into mitochondria (Kleene 
et al., 1987). The import of ws-porin, however,  was not affected 
by the pretreatment with apyrase (lane 1 )  suggesting that 
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4050 Role of ATP  in Mitochondrial  Protein Import 
ws-porin bs-porin 
1 2  3 4  
Apyrase + - + -  
FIG. 1. Depletion of  the in vitro import system from ATP 
does not inhibit the import of ws-porin into mitochondria. 
Isolated mitochondria (10 mg/ml) and reticulocyte lysate were prein- 
cubated with apyrase (8 units/ml; lanes I and 3 )  or with an  apyrase 
preparation heated to 95 "C prior to use (corresponding to 8 units/ 
ml; lanes 2 and 4 )  as described (Pfanner  and Neupert, 1986). The 
import reactions were performed as described under "Experimental 
Procedures." Samples 1 and 2 contained "C-labeled ws-porin, and 
samples 3 and 4 contained  93-labeled bs-porin. After incubation for 
15 min at  25 "C, the samples were treated with proteinase K (250 pg/ 
ml) as described (Kleene et al., 1987). Mitochondria were reisolated 
and resolved by sodium dodecyl sulfate-polyacrylamide gel electro- 
phoresis. Fluorographs of the dried gels are shown. The protein band 
corresponding to imported porin is marked by an arrowhead. 
0 io 80 
Apyrase (Ulrnl) 
FIG. 2. Import of ADP/ATP carrier and of bs-porin, but not 
of ws-porin, is inhibited by pretreatment with different con- 
centrations of apyrase. The experiment was performed as de- 
scribed in the legend of  Fig. l with the following modifications. The 
import reactions contained ascorbate and  N,N,N',N'-tetramethyl- 
phenylenediamine as described (Pfanner  and Neupert, 1987b). The 
reactions contained "C-labeled ws-porin or ?!+labeled bs-porin or 
3sSS-labeled precursor of the ADP/ADP carrier (AAC) together with 
unlabeled ws-porin. The concentrations of apyrase were as indicated. 
Results were quantified by densitometry. 
\+ apyrase 
0 JJ. C 5  15 25 
llme 01 lncubatlon (minl 
FIG. 3. The kinetics  of import of ws-porin are not affected 
by pretreatment with apyrase. The experiment was performed as 
described in the legends of Figs. 1 and 2. The concentration of apyrase 
was 80  units/ml.  Time  points and temperatures of import reactions 
were as indicated. 
converted 
bs-porin 
L 
0 
a 
- E 
01 ' 
0 LO 80 
Apyrase (Ulml) 
FIG. 4. Import of a converted form of bs-porin is not inhib- 
ited by pretreatment with apyrase. The experiment was per- 
formed as described in the legend of  Fig. 2 with the following  modi- 
fications. Ascorbate and N,N,N',N'-tetramethylphenylenediamine 
were omitted. The samples contained bs-porin which  was precipitated 
out of "S-labeled reticulocyte lysate with (NH4),S04 (64% saturation) 
and then treated with trichloroacetic acid, NaOH, and NaH,PO, as 
described (Pfaller et al., 1985). 
NTPs were not required. The  same result was obtained when 
the import of ws-porin was performed in the absence of 
reticulocyte lysate. 
In  order  to be sure  that  the samples containing ws-porin 
were depleted of ATP, we performed the following experiment 
(Fig. 2). The in vitro import system was pretreated with 
different  concentrations of apyrase. The  import of bs-porin 
was strongly inhibited by pretreatment with a low concentra- 
tion of apyrase (0.8 unit/ml), whereas the  import of ws-porin 
was not affected even by a 100-fold higher concentration of 
apyrase (80 units/ml).  Import of ADP/ATP  carrier was per- 
formed in  the presence of unlabeled  ws-porin, and also  import 
of this precursor was strongly inhibited by the  pretreatment 
with  apyrase. The  total  amount of precursor proteins in these 
import reactions and  the protease  resistance of imported  porin 
and  ADP/ATP  carrier were not affected by the apyrase treat- 
ment; furthermore, the ratios between free and imported 
precursor proteins were similar  with  ws-porin,  bs-porin, and 
ADP/ATP  carrier  under  the  experimental conditions  applied 
(Pfanner  and Neupert, 1986; Kleene et al., 1987; Pfanner et 
al., 1987; and  data  not shown). This suggests that ws-porin 
can be imported into mitochondria in an ATP-depleted in 
uitro system in  contrast  to bs-porin, ADP/ATP carrier, and 
many other mitochondrial  precursor proteins (see the  Intro- 
duction). 
The  data  in Fig. 2 did not exclude that ATP depletion  might 
lead to a reduced rate of porin  import.  Therefore, we examined 
the kinetics of import a t  different  temperatures (25 and 0 "C) 
(Fig. 3). Pretreatment with apyrase (80 units/ml) did not 
affect the  rates of import of ws-porin. 
As a further control, the biosynthetic  porin  precursor was 
subjected to a procedure  similar to  that used for the  prepara- 
tion of ws-porin including precipitation with  trichloroacetic 
acid,  solubilization in 100 mM NaOH, and neutralization with 
NaH,PO,. This converted  bs-porin was tested for import  into 
mitochondria. It was efficiently  inserted into  the  outer mem- 
brane. Pretreatment of the in vitro import reaction with 
different concentrations of apyrase did not reduce import 
(Fig. 4). Thus, also the biosynthetic porin precursor could be 
converted to a form whose import was independent of NTPs. 
In conclusion, two different forms of a  mitochondrial  pre- 
cursor protein show completely different requirements for 
NTPs for the  import  into mitochondria. There  are essentially 
two possible explanations for the  nature of the  NTP-requiring 
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Role of ATP  in  Mitochondrial  Protein Import 405 1 
step which can be bypassed by the acid-base treated precursor. 
NTPs might be required for release of precursor proteins from 
cytosolic transport complexes in which the precursors might 
interact with so far uncharacterized  components  (for review 
see Pfanner and Neupert, 1987a). The import of a largely 
purified precursor protein, however, also requires NTPs (Ei- 
lers et al., 1987). It is thus unlikely that  the dissociation of 
complexes between precursor proteins and  other components 
represents the crucial function of NTPs in  protein  import. It 
is much more likely that  NTPs  are involved in modifying the 
precursor proteins  in such a manner  that they assume a less 
rigid ("unfolded") conformation. In support of such a  function, 
NTPs increase the protease  sensitivity of precursor proteins 
in reticulocyte lysate (Pfanner et al., 1987). The proposed 
unfolding reaction may  be mimicked by the acid-base treat- 
ment which is known to greatly destabilize the conformation 
of proteins. This conclusion extends recent studies which 
suggested that mitochondrial precursor proteins  must  be at  
least partially unfolded to be competent for transport into 
mitochondria (Schleyer and Neupert, 1985; Eilers and Schatz, 
1986). An NTP-dependent unfolding enzyme was proposed to 
participate in this process (Rothman and Kornberg, 1986; 
Pfanner et al., 1987). It is not known if the proposed unfolding 
enzyme is located in the cytoplasm or associated with the 
mitochondrial membranes, or both. In addition, the translo- 
cation of precursors into or across the inner membrane may 
require NTPs for further steps, e.g. for the phosphorylation 
of components of the translocation machinery. 
A  similar role of NTPs  as in unfolding precursor proteins 
upon import into mitochondria may exist in the case of 
translocation of proteins across other biological membranes 
and organelles such as the endoplasmic reticulum, chloro- 
plasts, and  the prokaryotic plasma membrane. An ATP re- 
quirement was also described for protein transport into or 
across these membranes (Grossman et al., 1980; Chen and 
Tai, 1985;  Fliigge and Hinz, 1986; Geller et al., 1986; Hansen 
et al., 1986; Mueckler and Lodish, 1986; Perara et ul., 1986; 
Rothblatt and Meyer, 1986; Waters and Blobel, 1986; Pain 
and Blobel, 1987; Schlenstedt and Zimmermann, 1987; Ya- 
mane et al., 1987). Protein export in Escherichia coli was 
shown to require at  least partial unfolding of the precursor 
protein (Randall  and Hardy, 1986). Indeed, the  ATP require- 
ment for protein transport into the endoplasmic reticulum 
appears  to be related to preservation of transport competence 
of the precursor protein (Wiech et al., 1987). 
Acknowledgments-We are  grateful  to  Christine  Forster  and  Bri- 
gitte Stelzle for expert  technical assistance. We  thank  Heinrich  Steger 
for supplying us with cDNA for the ADP/ATP carrier and Dr. 
William Wickner for critical  comments on the  manuscript. 
Note Added in Proof-The converted  bs-porin  shows a 3-&fold 
higher  sensitivity  toward digestion by low concentrations of protein- 
ase  K than  the  authentic  bs-porin.  This  supports  the conclusion that 
the  converted  precursor  exists  in a more loosely folded ("unfolded") 
conformation  than  the  authentic one. 
REFERENCES 
Chen, L., and Tai, P. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 
Chen, W.-J., and Douglas, M. G. (1987) Cell 49, 651-658 
Eilers, M., and  Schatz, G. (1986) Nature 322, 228-232 
Eilers, M., Oppliger, W., and Schatz, G. (1987) EMBO  J. 6, 1073- 
Flugge, U. I., and  Hinz, G. (1986) Eur. J.  Biochem. 160, 563-570 
Geller, B. L., Mowa, N. R., and Wickner, W. (1986) Proc.  Natl. Acad. 
Grossman, A., Bartlett, S., and Chua, N. H. (1980) Nature 285,625- 
Hansen, W., Garcia,  P. D., and  Walter, P. (1986) Cell 45,397-406 
Hartl, F.-U., Ostermann, J., Pfanner, N., Tropschug, M., Guiard, B., 
and  Neupert, W. (1987) in Cytochrome Systems: Molecular Biology 
and Energetics (S. Papa  et al., eds),  Plenum  Publishing Corp., New 
York, in  press 
Kleene, R., Pfanner, N., Pfaller, R., Link, T. A., Sebald, W., Neupert, 
W., and  Tropschug, M. (1987) EMBO J. 6, 2627-2633 
Mueckler, M., and Lodish, H. F. (1986) Nature 322, 549-552 
Pain, D., and Blobel, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 ,  
Perara, E., Rothman, R. E., and Lingappa, V.  R. (1986) Science 232 ,  
Pfaller, R., and  Neupert, W. (1987) EMBO J. 6,2635-2642 
Pfaller, R., Freitag, H., Harmey, M. A., Benz, R., and  Neupert, W. 
Pfanner, N., and  Neupert,  W. (1985) EMBO J. 4, 2819-2825 
Pfanner, N., and  Neupert,  W. (1986) FEBS Lett. 209 ,  152-156 
Pfanner, N., and  Neupert,  W. (1987a) Curr. Top. Bioenerg. 16,  177- 
Pfanner, N., and  Neupert, W. (1987b) J.  Biol. Chem. 262,7528-7536 
Pfanner, N., Tropschug, M., and  Neupert, W. (1987) Cell 49,  815- 
Randall, L. L., and  Hardy, S. J. S. (1986) Cell 46, 921-928 
Rothblatt, J. A., and Meyer, D. I. (1986) EMBO J. 5, 1031-1036 
Rothman, J. E., and Kornberg, R. D. (1986) Nature 322, 209-210 
Schlenstedt, G.,  and  Zimmermann, R. (1987) EMBO J. 6,699-703 
Schleyer, M., and  Neupert, W. (1985) Cell 43, 339-350 
Verner, K., and  Schatz, G .  (1987) EMBO J.  6,2449-2456 
Waters, M. G., and Blobel, G. (1986) J. Cell Biol. 102,  1543-1550 
Wiech, H., Sagstetter, M., Muller, G., and Zimmermann, R. (1987) 
Yamane, K., Ichihara, S., and Mizushima, S. (1987) J.  Biol. Chem. 
4384-4388 
1077 
Sci. U. S. A. 83,4219-4222 
628 
3288-3292 
348-352 
(1985) J.  Biol. Chem. 260,8188-8193 
219 
823 
EMBO J.  6, 1011-1016 
262,2358-2362 
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Role of ATP in mitochondrial protein import

Cell 45,397-406

Cell 49, 815-Randall,

http://epub.ub.uni-muenchen.de/7485/1/Neupert_Walter_7485.pdf

Role of ATP in mitochondrial protein import

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