bur
J
Biochem
109,
217-229
(1980)
(c:
by
FEBS
1980
Transport
of
Proteins into Mitochondria
Posttranslational Transfer of
ADP/ATP
Carrier into Mitochondria
in
vitro
Richard
ZIMMERMANN
and Walter
NEUPERT
Physiologisch-Chemisches
Instilut der Universitit Gottingen
(Received March
10,
1980)
The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner mem-
brane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is trans-
ferred into mitochondria in a posttranslational manner. The following results provide further
evidence for such
a
mechanism and provide information on its details.
1.
In homologous and heterologous translation systems the newly synthesized ADPjATP carrier
protein is present in the postribosomal supernatant.
2.
Analysis by density gradient centrifugation and gel filtration shows, that the ADPjATP
carrier molecules in the postribosomal fraction are present as soluble complexes with apparent
molecular weights of about I20000 and
500000
or larger. The carrier binds detergents such as
Triton
X-I
00
and deoxycholate forming mixed micelles with molecular weights of about
200000-
400
000.
3.
Incubation
of
a postribosomal supernatant
of
a reticulocyte lysate containing newly syn-
thesized ADP/ATP carrier with mitochondria isolated from
N~zrrosporu
spheroplasts results
in
efficient transfer of the carrier into mitochondria. About
20-30",
of
the transferred carrier are
resistant
to
proteinase in whole mitochondria. The authentic mature protein is also largely resistant
to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent.
Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position.
4.
The transfer
in
vitro
into
a
proteinase-resistant form is inhibited by the uncoupler carbonyl-
cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding.
These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via
the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by
intact mitochondria into an integral position in the membrane. These findings are considered
to
be
of general importance for the intracellular transfer of insoluble membrane proteins. They support
the view that such proteins can exist in a water-soluble form
its
precursors and upon integration
into the membrane undergo a conformational change. Uptake into the membrane may involve
the cleavage of an additional sequence in some proteins, but this appears not to be a pre-
requisite as demonstrated by the ADP/ATP carrier protein.
A
large body of evidence has been presented that
transfer of cytoplasmically synthesized proteins into
mitochondria occurs by a posttranslational mecha-
nism. The first support for such a mechanism came
from kinetic experiments
in
vivo
and in homologous
cell-free systems
[l
-
41.
More recently, mitochondrial
proteins synthesized in heterologous cell-free systems
were found to be translocated in reconstitution experi-
ments employingisolated mitochondria
[5-71.
Through
Ahhrevratron.
CIPhzC(CN)2,
carbonylcyanide
m-chlorophenyl-
hydrazone.
these findings, attention is centered on the structure
and properties
of
the extramitochondrial precursor
forms. There appears to be no general rule for the
structure of such precursors, since some are made as
larger molecules with additional sequences, which are
cleaved during or after transfer to the mitochondria
[6-
161,
whereas others are not synthesized as larger
molecules
[4,5,17
-
201.
A
route of the precursors
through the cytosolic compartment
is
suggested by
the experimental data in several cases
[3-5,7].
Special
attention should be given to proteins which are integral
membrane components in their functional form.
If
218
Biogenesis
of
Mitochondria1
ADP/ATP
Carrier
they pass through the cytosolic compartment the
properties of the precursors can be anticipated to
differ strongly from those of the membrane-bound
form.
In this study, we have investigated the transfer of
the ADP/ATP carrier, an integral traiismembrane
protein of the inner mitochondrial membrane [21-241.
We have already shown that ADP/ATP carrier syn-
thesized in homologous or heterologous cell-free
systems has the same apparent molecular weight as
the functional protein and is made on free ribosomes
[20]. We report here that the precursor form is present
soluble in the cytosol fraction of
Neurosporu
cell-free
systems and of reticulocyte lysates programmed with
Neurospora
messenger RNA. It is not found as a
monomer but as an oligomeric complex. The pre-
cursor binds detergents such
a
TritonX-100 and
deoxycholate. It is transferred to isolated mitochon-
dria
in
such a form that it becomes resistant
to
added
proteinase. Proteinase resistance is a characteristic
property of the mature functional protein
in
intact
mitochondria. Integration into the membrane can be
blocked by the uncoupler CIPhzC(CN)Z. Thc hypo-
thesis is put forward, that the water-soluble precursor
makes contact with the mitochondrial membrane
through a specific binding site and in a second step
is integrated into the membrane thereby undergoing
a change in its conformation.
MATERIALS AND METHODS
Groiidz
of
Neurospora
Cells and Cell Fractionation
Neurosporu
crussa
(wild type, 74 A) was cultivated
in
Vogel's minimal medium [25]. Labelling of cells
with [35S]~ulfate was performed as described [5]. Cell-
free homogenates were prepared and amino acid in-
corporation was carried out according to published
procedures
[20].
Neurospora
hyphae were converted
to spheroplasts by suspending log of hyphae (wet
weight) in 50 ml of medium A containing
1
M
sor-
bitol, 4.5 mM sucrose and Vogel's minimal medium
as employed for cultivation of cells. Then 1 ml of a
solution containing [Lglucuronidase and arylsulfatase
from
Hrlixpomatia
(5.2 and 4.5
U,
respectively,
Boehringer, Mannheim) were added. The mixture was
kept at 25 "C for
30
min with gentle agitation. Then
it was cooled to
O'T
and centrifuged for 10 min at
5000
x
g.
The pellet was washed once with medium A.
The final pellet was transferred to a Dounce homog-
enizer together with
10
ml of
a
medium employed for
protein synthesis in a homologous
Neurospora
cell-
free system as described before
[5,20].
Homogenisa-
tion was carried out by
10
strokes with a loosely
fitting pestle. The homogenate was centrifuged twice
for 5 min at 4000
xg,
then mitochondria were sedi-
mented by centrifugaton at 17 300
x
g
for 12 min.
Protein S.ynthesis
in
Reticulocyte Lysutes
Preparation of reticulocyte lysates, isolation of
Neurospora
poly(A)-containing RNA and incorpora-
tion
of
[35S]niethionine in reticulocyte lysates were
carried out as described [20,26-
281.
Postribosomal
supernatants were prepared by centrifuging the cooled
lysates for
1
h at 166000
x
g
in a Beckman rotor Ti
75.
Sucrose Gradient Centrifugation
Linear sucrose gradients were prepared by mixing
6 ml of 5
%
sucrose with
6
ml45
%
sucrose (w/w). The
solvent was
0.3
M
KCI,
10
mM Tris-HCI
pH
7.5
and
when indicated contained
1
Triton X-100. Samples
to be centrifuged were first made
0.3
M
KCI, or
0.3 M
KCI and
1
%
Triton. Samples of
0.5
ml were layered
on the gradients. Centrifugation was carried out in a
Beckman ultracentrifuge (rotor Ti 75) at
166
000
x
g
and
1
"C
for 15 h or
60
h. Gradient fractions were
obtained by puncturing tubes at the bottom and
collecting
1.3-nl1
fractions. Density of fractions was
determined in a Zeiss refractometer. For electro-
phoretic analysis of total proteins,
0.
l-ml aliquots were
mixed with 0.1 ml acetone. Precipitated proteins were
dissolved in
dodecylsulfate-containing
buffer as used
for the solubilisation of immunoprecipitates (see be-
low). The remaining 1.2 ml were subjected
to
immuno-
precipitation.
Gel Filtration
Columns containing Sephadex
G-1
00
(Pharmacia,
Uppsala, Sweden) or Ultrogel AcA
34
(LKB, Bromma,
Sweden) (55
x
0.9
cm) were equilibrated with 0.3
M
KCI,
10
mM Tris-HC1
pH
7.5 and, when indicated,
1
Triton. For calibration, the following components
were applied and eluted in the same buffer: dextran
blue, thyroglobulin, ferritin, catalase, lactate dehy-
drogenase, serum albumin, cytochrome
c
and
t3H]-
Triton
X-100
(NEN Corp., Boston, MA). Super-
natants of reticulocyte lysates
(0.5
ml) were applied
to the columns. The samples contained
0.3
M
KC1
and when indicated l'z Triton. The flow rate was
18
ml/h with Sephadex (3-100, and
6
ml/h with Ultro-
gel AcA 34. Fractions of
1.5
ml were collected. These
were analysed by the same procedures as described
for the sucrose density gradient fractions.
Inzmuntjprecll?itation
For immunoprecipitation of ADP/ATP carrier
from mitochondria, mitochondrial pellets were lysed
in
1
ml
1
'%,
Triton,
0.3
M
KCI,
1
mM p-chloro-
mercuribenzoate,
10
mM Tris-HC1
pH
7.5, and freed
from insoluble material by centrifugation for 15 min
at 20000
xg.
100
pl
of a solution containing
2
mg
R.
Zimmermann
and
W.
Neupert
21
9
immunoglobulin fraction, 0.3 M
KCI,
10
mM Tris-
HC1 pH 7.5 was added to the supernatant per 0.25 mg
mitochondrial protein and the mixture was kept at
4
C
for 4 11. The precipitate was collected by centrif-
ugation, and washed twice with
0.3
M KCl,
10
mM
Tris-HC1,
1
%,
Triton pH 7.5 and three times with
10
mM
Tris-HC1 pH 7.5.
Immunoprecipitation of ADP/ATP carrier from
reticulocyte lysates and from fractions of sucrose
density gradients or gel filtration
(1
-
1.4 ml) was per-
formed after addition of KC1 to a final concentra-
tion of 0.3
M
and Triton to final concentration of
1
(if not already present). First, 10 pl of the immuno-
globulin solution (see above) and after 5 min at
4
C
Sepharose-bound protein A (10 mg dry weight, pre-
swollen in
100
pl0.3
M KCl,
10
mM Tris-HC1 pH 7.5,
Pharmacia, Uppsala, Sweden) were added. The mix-
ture was shaken for
5
min and then Sepharose beads
were collected by centrifugation. They were washed
as
described above for direct inmunoprecipitation.
Iminunoprecipitates obtained by direct precipita-
tion or with Sepharose-bound protein A were disso-
ciated by heating for 2 min at 95
'C
in
50
pl
2'%;
sodium dodecylsulfate,
10
mM Tris-HC1 pH 7.5,
2.5
o/,
(v/v) 2-mercaptoethanol.
L,imited Proteolysis
of
Immunoprecipitates
Mitochondria
(2
mg protein) were isolated from
cells labelled by growth on [35S]sulfate. After lysis
with Triton, ADPIATP carrier was immunoprecipi-
tated. The washed immunoprecipitate was dissolved
in 120
~12%
sodium dodecylsulfate, 10 mM Tris-HCI
pH
7.5, and 360 p1 of 10 mM Tris-HC1 pH 7.5 werc
added and the sample was kept at 95 "C for 2 min.
Mitochondria were isolated from unlabelled cells,
immunoprecipitation
was
carried out and the precipi-
tate dissolved in 60 pl
2%,
dodecylsulfate,
10
mM
Tris-HCI pH 7.5. An immunoprecipitate was also
obtained from the postribosomal supernatant
of
a
reticulocyte lysate
(1
ml) which was previously incu-
bated with [35S]methionine and
Neurospora
poly(A)-
containing RNA for 60 min. This immunoprecipitate
was dissolved in
60
pl
2% sodium dodecylsulfate,
10
mM
Tris-HC1 pH 7.5, and mixed with the dissoci-
ated precipitate from the unlabelled mitochondria.
Then
360
pl
10
mM
Tris-HC1 pH 7.5 were added and
the mixture kept at 95
'
C for
2
min.
The two samples were each divided into two equal
portions. To one of each portion were added 30 pl,
to the other two 60
p1
of a solution containing
1
mg/ml
trypsin and
10
mM Tris-HC1 pH 7.5. All four samples
were kept at 23
'C
for 15 min. Proteolysis was stopped
by first adding 30 pl of 2-mercaptoethanol and
30
pl
20% sodium dodecylsulfate in
10
mM
Tris-HCI pH 7.5,
and then boiling for
2
min.
50
pl
of each sample was
then subjected to gel electrophoresis. Vertical gel
electrophoresis was performed according to Laemmli
[29]. The concentrations of
acrylamide/bisacrylamide
were 17.5/0.12% (w/v).
Transfer
in vitro
of
ADPIATP Currier
Protein synthesis
in
vitro
was performed in a re-
ticulocyte lysate in the presence of [35S]methionine
and
Neurospora
poly(A)-containing
RNA
for
1
h.
Samples were cooled to
0°C
and centrifuged for
1
h
at 166 000
x
g.
Methionine and sucrose were added to
the supernatant to final concentrations of 50
pM
and
0.3
M, respectively. Mitochondria isolated from
spheroplasts were gently resuspended in this mixture
(0.5
-
1 mg protein/ml). The suspension was incubated
for the times indicated in the individual experiments at
25°C.
Reaction was terminated by cooling to O"C,
then mitochondria and supernatant were separated
again by
12
min centrifugation at 17300
xg.
The
mitochondrial pellet was washed once with
a
solution
containing 0.44
M
sucrose, 2 mM EDTA,
100
mM
Tris-HC1 pH 7.8 (sucrose buffer). Then proteinase
treatment was performed when indicated. For this
purpose the mitochondrial pellets were resuspended
in sucrose buffer and proteinase
K
(Boehringer, Mann-
heiin) (100 pg/ml) was added and samples were incu-
bated at 4 'C for 60 min. Proteolysis was stopped by
the addition of
0.1
pmol/ml phenylmethylsulfonyl
fluoride. Then to
1
ml
of the mitochondrial suspen-
sions
100
pl
3
M
KCI,
100
mM Tris-HC1 pH 7.5,
100
pl
of
10 mM
p-chloromercuribenzoate,
and
60
p1
of 20% Triton
X-100
were added. After a clarifying
spin, direct immunoprecipitation was performed.
RESULTS
Synthesis ofADP/ATP Carrier
in
Reticulocyte
Lysates
and
Release
into
the Postrihosomal Supernatant
Protein synthesis was carried out in rabbit re-
ticulocyte lysates supplemented with
Netirospnrn
mes-
senger RNA in the presence of [35S]methionine. ADP/
ATP carrier was immunoprecipitated from the post-
ribosomal supernatant. A single radioactive band is
secn after gel electrophoresis in the presence
of
do-
decylsulfate (Fig.
1,
lane
1).
It is absent when mes-
senger RNA was omitted, both in lysates pretreated
with niicrococcal nuclease and not pretreated (lanes
2
and 3). The apparent molecular weight is the same as
that of the authentic protein (Fig.
1,
lane
9)
[17,20].
To make sure that the product
in
vitro
actually re-
presents
ADP/ATP carrier, immunoprecipitates from
reticulocyte supernatants and from Triton lysates of
mitochondria from 35S-labelled cells were subjected
to limited proteolysis with trypsin. The patterns of
labelled fragments from product
in
vitrn
and authentic
protein are identical (Fig.
1,
lanes
5-
8).
Essentially,
two major bands are observed. This agrees with the
220
94
-
67
-
Biogenesih of Mitochondria1 ADP ATP Carrier
-
123456789
Origin
L5
-
43
-
c
B
30-
20
-
14.4
-
Fig.
1.
Synf/wsis
of'ADP,ATP
carrier
in
(1
rrtic,ulucyte
ceN-/rci~
system.
A rabbit reticulocyte lysate was treated with micrococcal nuclease
[28]
and then incubatcd together with
Nrurospora
poly(A)-containing RNA and [35S]inethionine in the prcscncc
of
an energy regenerating
system
for
60 min. The postribosomal supernatant was prepared
and
immunoprecipitation with anti-(ADP/ATP carrier) immunoglobulin
was performed. Iminunoprecipitates were analysed by gel electrophoresis in the presence of dodecylsulfate and autoradiographs of the dried
gels were obtained.
(1,4)
Incubation in the presence of poly(A)-containing RNA; (2) incubation in the absence
of
poly(A)-containing
RNA;
(3)
as in 2, but without nuclease treatment;
(5,6)
ADPiATP carricr immunoprecipitated from a reticulocytc lysate incubated as in
4:
the immunoprecipitates were treated with
0.1
mg/ml and 0.2 mg/rnl trypsin, respectively;
(7,8)
ADPiATP carrier immunoprecipitated froin
mitochondria as in
9;
the immunoprecipitates were treated with 0.2
ing/ml
and
0.1
nig/ml trypsin. respectively;
(9)
ADP
ATP
carrier
immunoprecipitated from mitochondria, which were isolated
rrom
cells grown in the presence
of
[35S]sulfate. Arrow indicates position
of
ADPiATP carrier stained with Coomassie blue
earlier observation that the cleavage products have
a
very high tendency to aggregate even in the presence
of dodecylsulfate
(M.
Klingenberg, personal com-
munication) [20,30].
Properti1.s
OJ'
ADPIA
TP
Carrier
in
tiit.
Postrihosomul
Supernatant
In order to find out in which form the newly syn-
thesized carrier released from the ribosomes is present
in the postribosomal fraction, the following experi-
ments were carried
out.
From the postribosomal supernatant of a reticulo-
cyte lysate the carrier was immunoprecipitated in the
absence and presence of Triton
X-100
(Fig.
2,
lanes
4
and
5).
Without addition of Triton there is unspecific
coprecipitation of inany other proteins. This un-
specific reaction is also seen with non-immune serum
in the absence of Triton but not
in
its presence (Fig.
2,
lanes
6
and
7).
Despite of this coprecipitation
it
is
obvious that antibodies against the ADP/ATP carrier
precipitate the same amount of carrier protein in the
presence and absence of Triton (lanes
4
and
5).
Furthermore,
if
the supernatant is treated with trypsin,
the carrier is digested in the absence as well as in the
presence of Triton
X-I
00, so that immunoprecipitation
of
intact protein or even fragments is not any longer
observed (Fig.2, lanes
1-3).
The same experiments
were performed with the supernatant of a homologous
cell-free system
[5,20]
and the results were the same
(not shown).
It
is concluded from these observations that the
newly synthesized protein
is
accessible
to
immuno-
globulins as well as
to
proteinase. Therefore
it
cannot
be enclosed in vesicles or protected in a lipid phase, as
is the functional protein (see below, Fig.
6).
The reticulocyte supernatant was subjected to
sucrose density gradient centrifugation and ADP:
ATP carrier was immunoprecipitated from gradient
fractions. In a first experiment centrifugation was
carried out for short time
(15
h). Fig.3A shows total
acetone-precipitable proteins in the gradient fractions
analysed
by
gel electrophoresis and autoradiography.
Hemoglobin, which is present in the reticulocyte
supernatant in very high amounts, forms
a
band
in
the middle
of
the gradient. The immunoprecipitable
ADP/ATP carrier is found as
a
single band in the
gradient and sediments slightly faster than hemo-
globin (Fig. 3
B).
When centrifugation
was
performed
for
60
h,
ADP/ATP carrier was found in the two
lowest fractions of the gradient (Fig. 3
C). Apparently,
it has a density higher than
1.21
g/cm3. The gradients
R.
Zimtnerrnann and W. Neupert
221
-0
W
>
E
8
c
0
ul
0
L
._
Fig. 2.
/icccw.Sihi!il,i~
io
mti/m/y
andprotcinusc
of
ADfl
A
TP
uirriw
nlhr.sizrc/
in vitro
in
reticulocytr
Iy.sciles.
Protein synthesis was car-
ried
out
in a rabbit reticulocyte lysate in the presence
ol
["SI-
methioninc and
Nrurospnra
poly(A)-containing RNA for
60
min.
Then the postribosomal supernatant was prepared. Samples of the
supernatant were treated as follows and then subjected
to
immuno-
precipitation with immunoglobulins specific for ADP/ATP carrier
or non-immune immunoglobulins and then with Sepharose-bound
protein
A
(as indicated below). The iminunoprecipitates were
analyscd by electrophoresis and autoradiography.
(1.5)
Lysis with
Triton: addition of specific immunoglobulins;
(2)
lysis with Triton;
then treatment with trypsin
(1
mgiml)
for
60
min at 23
C
followed
by addition
of
trypsin soybean inhibitor
(1
mglml)
and cooling to
0
'C;
addition of specific immunoglobulins; (3) treatment with
trypsin
(1
inglml)
for
60
min at 23
C,
then addition of trypsin
inhibitor
as
in
2;
the sample was cooled and Triton was added;
then specific immunoglobulins were added;
(4)
addition of specific
immunoglobulins without lysis with detergent;
(6)
lysis with Triton
and addition
of
non-immune immunoglobulins;
(7)
addition of
non-imninne immunoglobulins without lysis with detergent. Sam-
ples
1
-
3
and samples
4-7
were obtained from two difl'erent
ex-
periments. Arrow indicates position
or
ADPiATP carrier
did not contain visible pellets and no immunoprc-
cipitable radioactivity could be washed off from the
bottom of the tubes (not shown).
Sedimentation was then carried out employing a
gradient which contained Triton
X-100 abovc the
critical micellar concentration. The applied reticulo-
cyte supernatant contained Triton. After
60
h
cen-
trifugation the ADP/ATP carrier formed a distinct
band in the upper third of the gradient (Fig.3D).
Apparently, the carrier protein binds detergent, which
leads to
a
reduced specific density. This binding
of
detergent to the carrier synthesized
in
vitro
is a specific
property not shared by other proteins such as hemo-
globin, as seen from its position in the detergent-
containing gradient. It was also found that the sedi-
mentation behaviour
of
Neurosporu
isocitrate lyase,
an enzyme of the glyoxysomal matrix, did not differ
in detergent-free and detergent-containing gradients
(not shown here).
For further analysis of the ADP/ATP carrier syn-
thesized
in
vitro,
the supernatant of reticulocyte lysates
was subjected to gel filtration. When Sephadex
G-100
was
employed, the carrier was eluted with the void
volume (Fig.
4).
No
indication of
a
monomer or of
a dimer was found. In the electrophoretic patterns
of
the total proteins in the column fractions hemoglobin
can be used as an internal marker. In contrast
to
the
ADP/ATP carrier it enters the Sephadex gel.
In order to achieve resolution in the higher molec-
ular weight region, chromatography was performed
on Ultrogel AcA
34
(Fig.5). The column was cali-
brated with marker proteins (Fig.
5C).
The ADP/ATP
carrier is detected in two bands in the elution diagram.
The apparent molecular weights of the two bands
were determined to be about
120000
and 500000 or
higher. The second species was eluted close to the
exclusion limit and therefore a precise value for its
molecular weight cannot be given. In different experi-
ments the distribution
of
ADP/ATP carrier between
these two bands varied in the range
of
2:
1
or
1
:
2.
Detergent binding was also studied by gel filtra-
tion. The postribosomal supernatant of the reticulo-
cyte lysates was made
1
in Triton X-100 and chro-
matographed on Sephadex
G-100
and on Ultrogel
AcA
34, equilibrated with Triton-containing buffer.
From Sephadex
G-100
the carrier was eluted with the
void volume as in the absence of Triton (Fig.4). The
analysis of acetone-precipitated column fractions
shows that thc elution behaviour
of
hemoglobin is
not affected by the detergent. This shows that Triton
does not effect dissociation into monomers. From
Ultrogel the ADP/ATP carrier was eluted as a broad
band corresponding
to
a molecular weight of
200000
-
400000.
Labelled Triton
X-100
was eluted from the
column with an apparent molecular weight of
90000
in agreement with the reported size
of
Triton micelles
[31]. Elution behaviour
of
marker proteins was not
affected by the inclusion of Triton into the elution
buffer. Apparently, the soluble protein complex con-
taining the ADP/ATP carrier forms mixed micelles
with Triton X-100. Gel filtration was
also
performed
on Ultrogel AcA 34 in the presence of
1
'%,
sodium
deoxycholate, 0.3 M
KCI,
10
mM
Tris-HC1 pH
8.0.
As with Triton, the carrier was eluted corresponding
to an apparent molecular weight
of
200000-
400000
(not shown).
Transfer
of
ADPIATP Carrier
fronq
Postribosomal Supernatant
of
Reticulocgte
Lyrates
into Isolated
Mitochondriu
Mitochondria were isolated from
Neurosporu
spheroplasts and were resuspended in a postribosomal