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Protein Translocation into Mitochondria
|Protein Translocation into
Mitochondria from different sources such as rat
liver, rabbit brain, or yeast can be isolated as intact
organelles. Isolated mitochondria are able to respire,
maintain a membrane potential across their inner
membrane, possess an active ATP synthase, and
shuttle nucleotides across their membranes. In addition,
even a process as complicated as import of mitochondrial
precursor proteins can be studied outside
the living cell. For this purpose, radiolabeled precursor
proteins, synthesized in an in vitro
translation system, are mixed with isolated mitochondria
(Glick, 1991; Melton et al.
, 1984). In the presence
of ATP, precursor proteins will cross the mitochondrial
membranes, become processed to their mature
form, and fold to their native state. Building on this
basic "import assay," sophisticated experiments have
been developed and the results of these experiments
provide most of what we know about mitochondrial
import today (Neupert, 1997; Pfanner and Geissler,
This article describes a standard protocol for the in
vitro synthesis of a radiolabeled precursor protein and
the subsequent import of this precursor into isolated
yeast mitochondria. As an example, we have selected
the precursor protein yeast malate dehydrogenase
(Dubaqui6 et al.
, 1998). The N-terminal presequence of
the yeast malate dehydrogenase precursor, as is of
most mitochondrial precursor proteins, is removed by
a protease localized in the mitochondrial matrix
(Jensen and Yaffe, 1988). The mRNA of the precursor
protein is transcribed with SP6 RNA polymerase
(Melton et al.
II. MATERIALS AND
SP6 RNA polymerase (Cat. No. 810 274); RNase
inhibitor from human placenta (Cat. No. 799 017); set
of ATP, CTP, GTP, UTP, lithium salts, 100 mM
(Cat. No. 1 277 057); creatine kinase from rabbit muscle
(Cat. No. 127 566); creatine phosphate, disodium salt
(Cat. No. 127 574); and proteinase K (Cat. No. 1092766)
are from Roche. Tris (Cat. No. 108382); KCl (Cat. No.
104936); KOH (Cat. No. 105021); MgCl2
(Cat. No. 822335); 25% NH3
(Cat. No. 105432); ethanol (Cat. No. 100983); and
sodium salicylate (Cat. No. 106602) are from Merck.
Spermidine (Cat. No. S 0266); bovine serum albumin,
essentially fatty acid free (BSA) (Cat. No. A-7511);
dithiothreitol (DTT) (Cat. No. D 5545); HEPES (Cat.
No. H 7523); trypsin (Cat. No. T 1426); trypsin
inhibitor, from soybean (Cat. No. T 9003); α-nicotinamide
adenine dinucleotide disodium salt,
reduced form (NADH) (Cat. No. N 6879); EDTA (Cat.
No. E 9884); (NH4
(Cat. No. A 2939); CaCl2
(Cat. No. C 5080); magnesium acetate tetrahydrate
(Cat. No. M 2545); valinomycin (Cat. No. V 0627);
ATP, disodium salt (Cat. No. A 7699); glycerol (Cat. No.
G 6279); potassium acetate (Cat. No. P 5708); KH2
(Cat. No. P 5379); L
-methionine (Cat. No. M 9625);
urea (Cat. No. U 5128); phenylmethylsulfonyl fluoride
(PMSF) (Cat. No. P 7626); tRNA, from bovine liver
(Cat. No. R 4752); and Triton X-100 (Cat. No. T 9284)
are from Sigma. Rabbit reticulocyte lysate (Cat. No. L
4960) and amino acid mixture, minus methionine (Cat.
No. L 4960), are from Promega. NaCl (Cat. No. 9265.1)
and trichloroacetic acid (TCA) (Cat. No. 8789.1) are from Roth. Sorbitol (Cat. No. 2039) is from Baker. L
S]methionine, >1000Ci/mmol (Cat. No. SJ 235),
mWG(5Ç)ppp(5(Ç)G (G-cap) (Cat. No. 27 4635 02), and
Kodak X-OMAT X-ray film (Cat. No. V1651496) are
from Amersham Biosciences. Sorvall centrifuge RC
M120 GX, Kendro. Sorvall Rotor S100 AT3-204,
Kendro. Eppendorf centrifuge 5417 R, "microfuge"
Eppendorf. Greiner PP-tubes 15ml (Cat. No. 188261),
Greiner. X-ray cassettes (Cat. No Rö 13), GLW. Plasmid
is pSP65mdh1 (Dubaquié et al.
, 1998). Highly purified
mitochondria (25 mg/ml) are prepared after Glick and
Solutions used directly as obtained from the supplier
are only listed in Section II. Protocols for the
preparation of solutions used throughout the procedure
are only given once.
A. Transcription Using SP6 Polymerase
- 1M Tris-HCl stock solution, pH 7.5: Dissolve 12.1 g
Tris in 80ml H2O and adjust pH to 7.5 with 5M HCl. Add H2O to 100ml. Autoclave and store at
- 1M HEPES-KOH, pH 7.4: Dissolve 23.8 g HEPES in
80ml H2O and adjust pH to 7.4 using 4M KOH.
Add H2O to 100ml. Autoclave and store at room
- 1M spermidine: Dissolve 145 mg spermidine in 1 ml
H2O. Store at -20°C.
- 100 mg/ml BSA: Dissolve 500 mg BSA in 5 ml H2O.
Store at -20°C.
- 2.5M MgCl2: Dissolve 50.8 g MgCl2 in 100ml H2O.
Autoclave and store at 4°C.
- 2.5 M KCl: Dissolve 18.6 g KCl in 100 ml H2O. Autoclave
and store at 4°C.
- 100 mM DTT: Dissolve 15.4 mg DTT in 1 ml H2O.
Store at -20°C. Make a fresh solution about every
- 5× SP6 reaction buffer: 200mM Tris-HCl, pH 7.5,
30mM MgCl2, 10mM spermidine, and 0.5mg/ml
BSA. To obtain 10ml of a 5× reaction buffer, mix
2 ml 1M Tris-HCl, pH 7.5, 120 µl 2.5M MgCl2,
100µl 1M spermidine and 50µl 100mg/ml BSA. If
necessary, readjust the pH to 7.5. Store in 1-ml
aliquots at -20°C.
- G-cap (m7G(5')ppp(5')G): Dissolve 25 A250 units in
242 µl H2O. Freeze 10-µl aliquots in liquid nitrogen.
Store at -70°C.
- 5 mM NTP-GTP: To make a 500µl stock, add 25 µl
100 mM ATP, 25 µl 100 mM UTP, and 25 µl 100 mM CTP to 425 µl 20mM HEPES-KOH, pH 7.4. Store
in 100-µl aliquots at -70°C.
- 5mM GTP: Mix 475µl 20mM HEPES-KOH, pH
7.4, with 25 µl 100 mM GTP solution.
- RNase inhibitor buffer: 20 mM HEPES-KOH, pH 7.4,
50mM KCl, 10mM DTT, and 50% glycerol. Make
10ml of the buffer by mixing 200µl 1M HEPES-KOH, pH 7.4, 200µl 2.5M KCl, 1 ml
100mM DTT, and 5 ml glycerol. Add H2O to 10ml
and store at -20°C.
- 4 units/µl RNase inhibitor: Add 500µl RNase
inhibitor buffer to 2000 units of RNase inhibitor.
Store at -20°C for up to 6 months.
- 1µg/µl linearized plasmid DNA: Prepare the linearized
plasmid (pSP65mdh1) according to standard
molecular biology procedures.
B. Translation Using Reticulocyte Lysate
- Mix the following solutions carefully, avoiding
the formation of air bubbles. Follow the indicated
order of addition because the DNA might precipitate
in 5x SP6 buffer. Precipitation of DNA can also occur
if the mixture is placed on ice. Incubate the mixture at
40°C for 15 min.
|4 units/µl RNase inhibitor
|1µg/µl linear plasmid (pSP65mdh1)
|5 mM rNTPs minus GTP
|5 mM G-cap
|100 mM DTT
|5× SP6 buffer
- Start transcription by adding 5 µl 5 mM GTP solution
and incubate for 90 min at 40°C.
- Extract the mRNA with phenol/chloroform and
then with chloroform/isoamylalcohol, precipitate with
100% ethanol, and wash with 70% ethanol. Resuspend
the dried pellet in 125 µl H2O.
- The mRNA obtained by this procedure is used
directly in the translation protocol, mRNA can be
stored in 10-µl aliquots at -70°C. If frozen mRNA is
used for translation, thaw rapidly and keep at room
temperature before adding the mRNA to the translation
- 1M DTT: Dissolve 154mg DTT in 1 ml H2O. Store at
-20°C. Make a fresh solution about every 4 weeks.
- 8mg/ml creatine kinase: Dissolve 8mg creatine
kinase in 475 µl H2O. Add 20 µl 1M HEPES-KOH,
pH 7.4, 5µl 1M DTT, and 500µl glycerol. Freeze
in 10-µl aliquots in liquid nitrogen and store at
- 5mg/ml tRNA: Dissolve 10mg tRNA from bovine
liver in 2 ml H2O. Store in 100-µl aliquots at -20°C.
- 400mM HEPES-KOH, pH 7.4: Mix 6ml H2O with
4 ml 1M HEPES-KOH, pH 7.4. If necessary, readjust
- 10 mM GTP: Mix 450 µl 20 mM HEPES-KOH, pH
7.4, with 50µl 100mM GTP. Store at -20°C.
- 100mM ATP: Dissolve 55.1mg ATP in 900µl
H2O. Adjust to pH -7 using 4M NaOH and pH indicator
paper. Adjust volume to 1 ml and store at
- 600 mM creatine phosphate: Dissolve 153.06 mg creatine
phosphate in 1 ml H2O. Store at -20°C.
- 4M potassium acetate: Dissolve 3.92g potassium
acetate in 10 ml H2O. Do not adjust the pH. Store at
- 50 mM magnesium acetate: Dissolve 10.7 mg magnesium
acetate tetrahydrate in 1 ml H2O. Store at
C. Denaturation of Radiolabeled Precursor
- Prepare the reticulocyte lysate mix and the tRNA
mix fresh. To obtain a 100-µl translation reaction, mix
the following solutions.
- Use the mRNA obtained in Section III,A. It is
possible to use mRNA produced in a different transcription
system, e.g., with T7 RNA polymerase. Mix
60 µl reticulocyte lysate mix, 10 µl tRNA mix, 2 µl
50 mM magnesium acetate, 18 µl mRNA, 10 µl
[35S]methionine, and 2µl 1M DTT.
- Incubate this mixture for 60min at 30°C. Shield
it from light to prevent heme-induced photooxidation
of the precursor proteins. Remove ribosomes after the
translation reaction by centrifugation for 15min at
150,000g (65,000rpm with S100 AT3-204 rotor in
Sorvall centrifuge). Remove the supernatants, being
careful not to disturb the ribosomal pellet.
- Saturated (NH4)2SO4 solution: Weigh 100 g (NH4)2SO4 and add H2O to a final volume of 100ml. Stir for 30
min at room temperature. The (NH4)2SO4 will not
dissolve entirely. Remove the supernatant and keep
at room temperature.
- 8M urea: Dissolve 4.85g urea in a final volume of
10ml 25 mM Tris-HCl, pH 7.5, containing 25 mM DTT.
D. Import of Denatured Radiolabeled
- Proteins synthesized in reticulocyte lysate are
either folded or bound to chaperone proteins present
in the lysate (Wachter et al., 1994). In order to unfold
the protein prior to import, it can be precipitated by
high concentrations of ammonium sulfate and subsequently
denatured in 8M urea.
- Add 200µl of the (NH4)2SO4 solution to the
100-µl translation reaction. Mix well and allow precipitation
of the protein for 30 min on ice. Collect precipitate
by centrifugation in an Eppendorf centrifuge at 20,000 g
for 10 min.
- Discard the supernatant and dissolve the pellet
in 100 µl of 8 M urea solution. Keep the denatured precursor
at room temperature for 10-30 min. This precursor
solution is used for the import reaction (Section
III,D) and preparation of the precursor standard
For additional solutions required, see Sections III,A
- 2.4M sorbitol: Dissolve 43.7 g of sorbitol in a final
volume of 100ml H2O. Autoclave and store at 4°C.
- 1M KH2PO4: Dissolve 1.36 g KH2PO4 in 10 ml
H2O. Filter sterilize and keep at room temperature.
- 1M HEPES-KOH, pH 7.0: Dissolve 23.8 g HEPES
in 80ml H2O and adjust pH to 7.0 with 4M KOH. Add
H2O to a final volume of 100ml. Filter sterilize and
store at room temperature.
- 250 mM EDTA, pH 7.0: Resuspend 7.3 g of EDTA
in 70ml H2O. Adjust pH to 7.0 using 5 M NaOH. Add
H2O to a final volume of 100ml. Filter sterilize and
keep at room temperature.
- 2× import buffer: 1.2M sorbitol, 100mM HEPES-KOH, pH 7.0, 100 mM KCl, 20 mM MgCl2, 5 mM EDTA, pH 7.0, 4 mM KH2PO4, 2 mg/ml BSA, and
1.5mg/ml methionine. To make 100ml of 2× import
buffer, mix 50ml 2.4M sorbitol, 400µl 1M KH2PO4 solution, 4ml 2.5M KCl, 10ml 1M HEPES-KOH, pH
7.0, 0.8ml 2.5M MgCl2, 2ml 250mM EDTA, pH 7.0,
150 mg methionine, and 200 mg BSA. Adjust pH to 7.0
and add H2O to 100ml. Store at -20°C.
- 1× import buffer minus BSA: Prepare 2× import
buffer, but without BSA. To obtain 1× import buffer
minus BSA, mix 2ml 2× import buffer with 2ml
- 500mM NADH: Dissolve 35.5 mg of NADH in a
final volume of 100µl 20mM HEPES-KOH, pH 7.0.
Store at -20°C.
- 1 mg/ml valinomycin: Dissolve 2mg valinomycin
in 2ml ethanol. Store at -20°C.
- Purified yeast mitochondria: 25mg mitochondrial
protein/ml. Store at -70°C in 0.6M sorbitol, 20mM HEPES-KOH, pH 7.4, and 10mg/ml BSA. Thaw
rapidly at 25°C immediately before the experiment. Do
not refreeze. A detailed protocol of the purification
procedure is given in Glick and Pon, (1995).
- Import into the matrix of mitochondria requires
a membrane potential across the inner mitochondrial
membrane. Therefore, the most thorough control for
the specificity of an import reaction is to determine its
dependence on a membrane potential. Adding ATP
and the respiratory substrate NADH generates this
potential. (Note that mammalian mitochondria cannot
oxidize added NADH.)
- Perform two import reactions, one in the absence
and one in the presence of valinomycin. Preincubate
the import reaction in a 15-ml Greiner tube at 25°C for
- Add 50µl of the denatured precursor protein
solution (see Section III,C) containing denatured
malate dehydrogenase to each reaction (reactions 1
and 2). Intact mitochondria should be handled gently.
However, it is essential to mix the denatured precursor
protein into the import reaction rapidly. Agitate
the import reaction gently on a vortex mixer while
adding the denatured precursor mixture dropwise. If
mixing is performed only after addition, the precursor protein tends to aggregate and becomes import
- Incubate at 25°C for 10 min. Agitate gently every
other minute to facilitate gas exchange. Stop the
import reaction by transferring the tubes onto ice. Add
1 µl of 1 mg/ml valinomycin to reaction 1.
- Remove 200µl each from reactions 1 and 2 and
put the samples on ice. Spin down mitochondria in an
Eppendorf centrifuge and remove the supernatant
(be careful, the pellet will be very small). Resuspend
the mitochondrial pellets of reactions 1 and 2 in each
200 µl of 1× import buffer. These samples represent the
total of the two import reactions (Fig. 1, lanes 2 and 5).
Add 22µl 50% TCA to each. Keep on ice and process
further after all samples have been acid denatured (for
the method of TCA precipitation, see Section III, G).
|FIGURE 1 Import of radiolabeled yeast malate dehydrogenase
into isolated yeast mitochondria. Lane 1, 10% of the material added
to each import reaction; lanes 2-4, import in the presence of ATP and
a membrane potential across the inner mitochondrial membrane;
and lanes 5-7, import in the absence of ATP and a membrane potential
across the inner mitochondrial membrane. Lanes 2 and 5: total;
material isolated together with the mitochondrial pellet. Lanes 3 and
6: import; material protease protected in intact mitochondria. Lanes
4 and 7: folded; material protease resistant even after solubilization
of the mitochondria with Triton X-100. pMdh1, precursor form of
Mdh1; mMdh1, mature form of Mdh1. For experimental details, see
E. Protease Treatment of Intact Mitochondria
- 10mg/ml trypsin: Dissolve 3 mg of trypsin in 300µl
H2O. Make fresh.
- 20mg/ml trypsin inhibitor: Dissolve 6 mg of trypsin
inhibitor in 300 µl H2O. Make fresh.
Perform the following steps in parallel with both
F. Inherent Protease Resistance of
the Imported Protein
- To digest precursor proteins that stick to the surface
of the mitochondria, add 8µl 10mg/ml trypsin
(final concentration 100µg/ml). Incubate for 30min
- Add 8µl 20mg/ml trypsin inhibitor (final concentration
200µg/ml) and incubate on ice for 5min.
- Transfer the sample into a new Eppendorf tube.
- Spin for 3 min in an Eppendorf microfuge at 10,000 g.
Remove the supernatant carefully by aspiration.
- Carefully resuspend the mitochondrial pellet in
800µl 1× import buffer minus BSA. As it is
extremely important to resuspend the pellet completely,
it should be done as follows. First add 100
µl of l x import buffer minus BSA and resuspend
mitochondria by pipetting up and down. Then add
another 700µl of 1× import buffer minus BSA to
yield 800 µl final volume.
- Remove 200µl of each sample and add 22µl 50%
TCA. Keep on ice. These samples represent the
material that has crossed the outer membrane completely
(import, Fig. 1, lanes 3 and 6).
- 1M Tris-HCl stock solution, pH 8.0: Dissolve 12.1 g
Tris in 80ml H2O and adjust pH to 8.0 using 5M HCl. Add H2O to a final volume of 100ml. Autoclave
and store at room temperature.
- 2 M CaCl2: Dissolve 14.7 g CaCl2 in 50 ml H2O. Autoclave
and store at room temperature.
- 10% Triton X-100 (w/v): Dissolve 10g of Triton X-100
in a final volume of 100ml H2O. Store at room temperature
in the dark.
- 10% NaN3: Dissolve 1 g NaN3 in a final volume of
10ml H2O. Store at room temperature.
- Proteinase K buffer: 50mM Tris-HCl, pH 8.0, 1 mM CaCl2, and 0.02 % NaN3. To make 10ml of proteinase
K buffer, mix 500µl 1M Tris-HCl, pH 8.0,
5µl 2M CaCl2, and 20µl 10% NaN3. Add H2O to a
final volume of 10ml. Store at room temperature.
- 10mg/ml proteinase K stock: Dissolve 5mg of proteinase
K in 500 µl proteinase K buffer. Store at 4°C for up to 1 week without loss of activity.
- 200µg/ml proteinase K solution: Mix 10µl of 10mg/ml
proteinase K stock with 390 µl H2O. Add 100 µl 10%
Triton X-100. Make fresh.
- 200mM PMSF: Make a fresh solution of PMSF by
dissolving 34.85 mg of PMSF in 1 ml of ethanol.
G. Final Processing of Samples and
Preparation of a Precursor Standard Steps
- Transfer 200 µl from the remainder of the import
reaction into a fresh Eppendorf tube. Add 200 µl of the
200-µg/ml proteinase K solution and mix rapidly.
Leave the tube on ice for 15 min.
- Add 2µl 200mM PMSF while agitating on a
vortex mixer. Keep on ice for 5min. Add 44µl 50%
TCA. Add 300µl of acetone to dissolve the Triton X-
100 that precipitates in the presence of TCA. These
samples measure the fraction of the precursor protein
that has completely crossed the outer membrane
and has reached the folded state (folded, Fig. 1, lanes
4 and 7).
H. SDS-Gel Electrophoresis and Processing
of the Gel
- To inactivate proteases, incubate the TCAprecipitated
samples (total, import, folded) at 65°C for 5 min. Place on ice for 5 min and subsequently
collect the TCA precipitate by spinning for 10min
- Remove supernatant by aspiration and dissolve the
pellets in 30 µl 1× sample buffer. If the sample buffer
turns yellow, overlay the sample with NH3 gas
taken from above a 25% NH3 solution. Agitate to
mix the gaseous NH3 gas into the sample buffer
until the color turns blue again.
- Incubate the samples for 5 min at 95°C.
- To estimate the efficiency of the import reaction, the
amount of precursor protein added to the import
reaction has to be determined. The efficiency of
import for most precursor proteins is between 5 and
30%. Here we use a 10% standard (Fig. 1, lane 1).
- To obtain a 10% standard, mix 4µl of purified yeast
mitochondria (see Section III,D) with 30µl 1× sample buffer. Incubate at 95°C for 3 min.
- Add 1 µl of the precursor protein solution (Section
III,C) and incubate for 5 min at 95°C.
- 5% TCA: To make 5 liter, add 250g of TCA to 5 liter
- 1M Tris base: Dissolve 121 g of Tris in 1 liter H2O.
- 1M sodium salicylate: Dissolve 160 g of sodium salicylate
in 1 liter H2O.
- Run samples on a 10% Tris-tricine gel (Schägger and
von Jagow, 1987) stabilized by the addition of 0.26%
linear polyacrylamide prior to polymerization.
- To reduce radioactive background, boil 5% TCA in
a beaker under the hood. Add the gel to the boiling
TCA and incubate for 5 min.
- Recover the gel and place it into a tray. Wash briefly
with water. Neutralize by incubation in 1M Trisbase
for 5 min on a shaker.
- Wash briefly with water. Add 1M sodium salicylate
and incubate for 20min on a shaker.
- Dry gel on a Whatman filter paper and expose to a
Kodak X-OMAT X-ray film for the desired time.
Exposure time for the experiment shown in Fig. 1
was 12 h.
The method describes a standard experiment to test
a precursor protein that has not been used in mitochondrial
import before. Most importantly, as demonstrated
here for malate dehydrogenase, the protocol
will reveal if import is dependent on a membrane
potential (compare Fig. 1 lanes 3 and 6). This is essential,
as sometimes protease-resistant precursor proteins
tend to stick to the outside of mitochondria, thereby
The efficiency of import can be deduced by a comparison
of the amount of imported material with a
precursor standard (compare Fig. 1 lanes 1 and 3). In
addition, the experiment reveals if a precursor protein
folds to a protease-resistant conformation after its
import into the mitochondrial matrix. Under the
conditions chosen here, complete protease resistance
was obtained for malate dehydrogenase (Fig. 1, lanes
3 and 4).
The quality of the DNA used for transcription is
essential for efficiency. Use a clean, RNA- and RNasefree
plasmid preparation (e.g., purified with a Qiagen
plasmid kit, Qiagen). Linearize plasmid by cutting
with a restriction enzyme behind the coding region of
the gene of interest. Extract with phenol/chloroform
and then with chloroform/isoamylalcohol, precipitate
with 100% ethanol, and wash with 70% ethanol.
Resuspend the dried pellet in H2
O at a concentration
of 1 µg/µl and store at 4°C. Never freeze DNA templates
used for transcription.
To avoid RNase contamination, solutions used for
transcription and translation have to be prepared with
special caution. Always wear gloves even when
loading pipette tips into boxes. If initiation at downstream
AUG codons is a problem, try diluting the reticulocyte
lysate up to fourfold.
It is important to establish that import is linear with
time. To establish those conditions it is necessary to
perform time course experiments of the import reaction
and to try import at different temperatures.
Methods for determining the intramitochondrial
localization of an imported precursor protein (Glick,
1991), investigating the energy requirements of mitochondrial
import (Glick, 1995), and detecting interaction
between imported precursor proteins and matrix
chaperones (Rospert and Hallberg, 1995) have been
Dubaquié, Y., Looser, R., Fünfschilling, U., Jenö, E, and Rospert, S.
(1998). Identification of in vivo
substrates of the yeast mieochondrial
chaperonins reveals overlapping but non-identical requirement
for hsp60 and hspl0. EMBO J
Glick, B. S. (1991). Protein import into isolated yeast mitochondria. Methods Cell Biol
Glick, B. S. (1995). Pathways and energetics of mitochondrial protein
import in Saccharomyces cerevisiae. Methods Enzymol
Glick, B. S., and Pon, L. A. (1995). Isolation of highly purified mitochondria
from Saccharomyces cerevisiae. Methods Enzymol
Jensen, R. E., and Yaffe, M. P. (1988). Import of proteins into yeast
mitochondria: The nuclear MAS2 gene encodes a component of
the processing protease that is homologous to the MASl-encoded
subunit. EMBO J
Melton, D. A., Krieg, E A., Rebagliati, M. R., Maniatis, T., Zinn, K.,
and Green, M. R. (1984). Efficient in vitro
synthesis of biologically
active RNA and RNA hybridization probes from plasmids containing
a bacteriophage SP6 promoter. Nucleic Acids Res
Neupert, W. (1997). Protein import into mitochondria. Annu. Rev.
Pfanner, N., and Geissler, A. (2001). Versatility of the mitochondrial
protein import machinery. Nature Rev. Mol. Cell. Biol
Rospert, S., and Hallberg, R. (1995). Interaction of HSP 60 with proteins
imported into the mitochondrial matrix. Methods Enzymol
Sch/igger, H., and von Jagow, G. (1987). Tricine-sodium dodecyl
sulfate-polyacrylamide gel elektrophoresis for the separation of
proteins in the range from 1 to 100kDa. Anal. Biochemi
Wachter, C., Schatz, G., and Glick, B. S. (1994). Protein import into
mitochondria: The requirement for external ATP is precursorspecific
whereas intramitochondrial ATP is universally needed
for translocation into the matrix. Mol. Biol. Cell