Biochem. J. (2008) 411, 115–122 (Printed in Great Britain)
115
doi:10.1042/BJ20071476
Oxidative folding competes with mitochondrial import of the small
Tim proteins
Bruce MORGAN and Hui LU1
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, U.K.
All small Tim proteins of the mitochondrial intermembrane space
contain two conserved CX3 C motifs, which form two intramolecular disulfide bonds essential for function, but only the
cysteine-reduced, but not oxidized, proteins can be imported
into mitochondria. We have shown that Tim10 can be oxidized
by glutathione under cytosolic concentrations. However, it was
unknown whether oxidative folding of other small Tims can
occur under similar conditions and whether oxidative folding
competes kinetically with mitochondrial import. In the present
study, the effect of glutathione on the cysteine-redox state of
Tim9 was investigated, and the standard redox potential of Tim9
was determined to be approx. − 0.31 V at pH 7.4 and 25 ◦C with
both the wild-type and Tim9F43W mutant proteins, using reversephase HPLC and fluorescence approaches. The results show that
reduced Tim9 can be oxidized by glutathione under cytosolic
concentrations. Next, we studied the rate of mitochondrial import
INTRODUCTION
Understanding the molecular mechanism of mitochondrial protein
import is a fundamentally important issue in biochemistry and
cell biology, because the mitochondrion is an essential organelle
of the cell. Approx. 99 % of the total mitochondrial proteins
are synthesized in the cytosol and have to be imported into
mitochondria for biogenesis of this organelle [1–3]. The small
Tim proteins of the mitochondrial IMS (intermembrane space)
play an essential role during the import of the mitochondrial innerand outer-membrane proteins [4,5]. There are five homologous
small Tim proteins in yeast, and the proteins are evolutionarily
conserved throughout the eukaryotic kingdom. All members of
the small Tim family have a molecular mass of approx. 10 kDa
and contain a strictly conserved twin CX3 C zinc-finger motif [6].
Tim9 and Tim10 are the two most important members of the
small Tim family in yeast, which form a hexameric complex
possessing a chaperone-like activity probably to prevent aggregation of the hydrophobic membrane proteins in the aqueous IMS.
We have previously shown that both Tim9 and Tim10 are imported
individually from the cytosol in a cysteine-reduced form, and the
oxidized (disulfide-bonded) proteins are import-incompetent [7].
While keeping the protein in a reduced form is essential for its
mitochondrial import, only the oxidized proteins can form the
Tim9–Tim10 complex in the mitochondrial IMS [7]. The complex
consists of three molecules of Tim9 and Tim10, with each subunit
having two intramolecular disulfide bonds [7–9]. Interestingly,
it has been shown that zinc can bind to the reduced but not to
the oxidized protein, at a molar ratio of 1:1 in vitro, with a sub-
and oxidative folding of Tim9 under identical conditions. The
rate of import was approx. 3-fold slower than that of oxidative
folding of Tim9, resulting in approx. 20 % of the precursor protein
being imported into an excess amount of mitochondria. A similar
correlation between import and oxidative folding was obtained
for Tim10. Therefore we conclude that oxidative folding and
mitochondrial import are kinetically competitive processes. The
efficiency of mitochondrial import of the small Tim proteins is
controlled, at least partially in vitro, by the rate of oxidative
folding, suggesting that a cofactor is required to stabilize the
cysteine residues of the precursors from oxidation in vivo.
Key words: cysteine redox potential, disulfide bond formation,
glutathione, mitochondrial import, protein folding, Tim
protein.
nanomolar dissociation constant, suggesting a potential role for
zinc in protecting the reduced protein from oxidation [10,11].
Detailed mitochondrial import studies have shown that import
of the small Tim proteins does not require the TOM (translocase
of the outer membrane) receptors of the outer membrane and
it is ATP-independent [12]. It has been suggested that import
may be a passive translocation process with the reduced proteins
traversing the protein-conducting channel of the TOM complex
freely [12,13]. On the other hand, recent studies have shown that
import of the small Tim proteins and many other IMS proteins
that could potentially form disulfide bonds is mediated by a newly
identified Mia40 import machinery in a redox-sensitive manner
[14–17] (Mia40 is an essential protein of the mitochondrial
IMS; synonyms YKL195W, FMP15 and Tim40). Mia40-substrate
import intermediates linked by an intermolecular disulfide bond
are observed during import of the substrate proteins. Import
studies with cysteine mutants of Tim9 and Tim10, mutated
systematically one by one, demonstrate that Mia40 specifically
recognizes the first cysteine of the proteins during their import
and thus acts as an import receptor in the mitochondrial IMS
[18,19]. Taken together, all these results are consistent with only
the reduced small Tim proteins being import-competent.
Glutathione, present in its reduced (GSH) and oxidized (GSSG)
forms, is considered to be the major thiol–disulfide redox buffer of
the cell [20]. Most of the glutathione in cells is usually found in the
cytosol, which is the principal site of GSH biosynthesis, with
the glutathione concentration in cells typically between 1 and
13 mM [20–22]. The ratio of GSH to GSSG has been reported
to range between 30:1 to 3000:1 in the cytosol. Our recent redox
Abbreviations used: AMS, 4-acetamido-4 -maleimidylstilbene-2,2 -disulfonic acid; CBB, Coomassie Brilliant Blue; DTT, dithiothreitol; ER, endoplasmic
reticulum; GST, glutathione transferase; IMS, intermembrane space; Mia40, an essential protein of the mitochondrial IMS (synonyms YKL195W, FMP15
and Tim40); OxDTT, oxidized form of DTT; PDI, protein disulfide-isomerase; RP-HPLC, reverse-phase HPLC; TCEP, tris-(2-carboxyethyl)phosphine; TFA,
trifluoroacetic acid; TOM, translocase of the outer membrane.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
116
B. Morgan and H. Lu
stability studies on Tim10 have shown that thermodynamically
Tim10 can be oxidized by glutathione under cytosolic concentrations, and the presence of zinc ions can inhibit the oxidative
folding of Tim10 kinetically [23]. Thus zinc binding is probably
required to stabilize the precursor proteins in a reduced and
import-competent form in the cytosol. However, oxidative folding
studies with purified apoTim10 showed that the rate of the
oxidation, under the cytosolic glutathione conditions, seems
much slower than that of the protein import. Thus it is unclear
whether import of the small Tim proteins can actually be inhibited
by oxidative folding. So far, no study on the correlation between
oxidative folding and mitochondrial import of the small Tim
proteins has been reported; therefore, this is one of the main aims
of the present study. In addition, all the small-Tim-protein-import
studies so far have demonstrated that import of these proteins was
typically approx. 10–20 % of the total material or less [7,8,12,15],
which is much less efficient than that of other mitochondrial
subcompartment proteins, such as AAC (ADP–ATP carrier) and
many matrix proteins [24–26]. Why import of the IMS small Tim
proteins is so inefficient is an important issue that needs to be
addressed.
In the present study, we investigated the effect of glutathione on
the redox states of recombinant yeast Saccharomyces cerevisiae
Tim9 and the redox stability of the protein, using a thiol
modification assay. The standard redox potential of Tim9 was
determined to be − 0.31 V at pH 7.4, showing that oxidized Tim9
is the thermodynamically stable form under cytosolic glutathione
conditions. To determine whether oxidative folding will compete
directly with mitochondrial import of the small Tim proteins
kinetically, time courses of import and oxidative folding of Tim9
in the absence of Zn2+ were studied at identical conditions. The
data analyses showed that the rate of oxidation was approx. 3-fold
faster than that of mitochondrial import. Import was inhibited
once the reduced Tim9 was oxidized, and only approx. 20 %
of the total precursor protein was imported into mitochondria
added in excess. The same results were obtained with different
preparations and with Nycodenz-gradient-purified mitochondria,
showing that the fast oxidative folding was not due to the
effect of a contamination in mitochondria. A similar correlation
between import and oxidative folding for Tim10 was obtained.
We conclude that oxidative folding is a strong competitor and
can inhibit mitochondrial import of apoTim9 and apoTim10
kinetically. The efficiency of mitochondrial import of the small
Tim proteins is controlled, at least partially, by the rate of oxidative
folding.
and lysed by sonication at 4 ◦C in buffer A (50 mM Tris/HCl,
pH 7.4, 150 mM NaCl and 1 mM EDTA) plus 10 mM DTT
(dithiothreitol). Most of the GST–Tim9 was isolated in inclusion
bodies (the pellet), which was solubilized in 10 ml of buffer B
(buffer A containing 8 M urea and 10 mM DTT) for 1 h at room
temperature, and renatured for 2 h at 4 ◦C by 10-fold dilution with
buffer A. The solubilized Tim9 was incubated overnight at 4 ◦C
with approx. 2 ml of glutathione–Sepharose 4B beads equilibrated with buffer A. The beads were washed and incubated
with buffer A containing 5 units/ml thrombin at 4 ◦C to release
Tim9. Tim9 was further purified using an FPLC gel filtration
column (Superdex 75; Amersham Biosciences) with buffer A at
0.5 ml/min.
Preparation of reduced Tim9
Oxidized Tim9 was typically incubated with 2 mM TCEP for
approx. 1 h at 25 ◦C and pH 7.4, followed by gel filtration
(Superdex 75) to remove TCEP. The reduced protein was always
prepared freshly just before use.
AMS alkylation assay
The purified oxidized or reduced Tim9 (typically approx. 10 µM)
in buffer A was incubated with TCEP, GSSG and GSH under
various conditions according to the individual experiment,
followed by addition of 5–20 mM thiol-specific reagent AMS
and non-reducing SDS/PAGE sample buffer at 25 ◦C for approx.
15 min in the dark. Then samples were analysed by Tricine/
SDS/16 %-(w/v)-PAGE followed by CBB (Coomassie Brilliant
Blue) staining. In this assay, the alkylation agent AMS, a 500
Da molecule, reacts specifically and efficiently with free thiols
of the reduced protein, but not disulfide bonds of the oxidized
protein. AMS modification increases the mass of the protein and
thus decreases its mobility on SDS/PAGE. To avoid any potential
oxidation of GSH, samples were prepared in freshly deoxygenated
buffer A.
Fluorescence measurements
Fluorescence spectra were acquired using a Cary Eclipse fluorescence spectrophotometer. A fluorescence cell of 10 mm × 2 mm
was used. Fluorescence spectra at λexcitation = 280 nm and
λemission = 290–500 nm were recorded. A slit width of 5 nm was
used for both excitation and emission.
EXPERIMENTAL
Materials
TCEP [tris-(2-carboxyethyl)phosphine] and AMS (4-acetamido4 -maleimidylstilbene-2,2 -disulfonic acid) were obtained from
Invitrogen Molecular Probes. EDTA was from BDH, and all other
chemicals were obtained from Sigma at the highest grade.
Protein purification
Wild-type Tim9 and Tim9F43W mutant were purified from
Escherichia coli using the same protocol for Tim10 as described
previously [23,27]. Briefly, BL21-Codon plus cells (Stratagene)
containing a plasmid expressing GST (glutathione transferase)–
Tim9 were grown in LB (Luria–Bertani) medium containing
ampicillin (0.1 mg/ml) at 30 ◦C until the D600 (attenuance at
600 nm) was approx. 0.4 and then the cells were induced for 3 h by
IPTG (isopropyl β-D-thiogalactoside). The cells were harvested
c The Authors Journal compilation c 2008 Biochemical Society
RP-HPLC (reverse-phase HPLC)
RP-HPLC (the Pharmacia Biotech SMART system) was used to
separate the reduced and oxidized forms of Tim9. All samples
were acid-quenched with 0.1 % TFA (trifluoroacetic acid), both
to prepare the samples for RP-HPLC and to prevent any further
disulfide exchange, prior to loading on to an Ace 5 C4-300 reversephase column. Buffer 1 was 0.1 % TFA in water, and buffer 2 was
0.1 % TFA in acetonitrile. The column was equilibrated with 10 %
buffer 2 prior to sample injection. Separation was performed by
running a linear gradient from 10–60 % buffer 2, with a flow rate
of 200 µl/min. The injection volume was 100 µl.
Determination of redox potential
Oxidized Tim9 in buffer A plus 1 mM EDTA was incubated
with DTT and OxDTT (oxidized form of DTT) at various ratios:
Oxidative folding and mitochondrial import of Tim9
117
0.5 mM OxDTT plus 0.5 µM–20 mM DTT, at 25 ◦C for 2 h to
overnight in the cases where reduction was slow (low DTT concentration). For the wild-type Tim9, reactions were acidified
by the addition of 0.1 % TFA and subjected to RP-HPLC.
The percentage of Tim9 in the oxidized form was calculated
as area under the curve of absorbance at 215 nm. For the
Tim9F43W, fluorescence spectra before and after incubation with
DTT/OxDTT were measured. Fluorescence intensity changes at
350 +
− 5 nm were used for further data analyses. The equilibrium
constant K eq for redox reaction 1:
Tim9Ox + 2DTT ↔ Tim9Red + 2OxDTT
(Reaction 1)
was calculated according to eqn (1) :
K eq = [Tim9Red ] × [OxDTT]2 /([Tim9Ox ] × [DTT]2 )
Y = YO + (YR − YO ) × [K eq × ([DTT]/[OxDTT])2 ]/
{K eq × ([DTT]/[OxDTT])2 + 1}
(1)
Y is the measured fluorescence intensity of mutant Tim9 at various
ratios of DTT/OxDTT; Y R and Y O are the calculated fluorescence
intensity of reduced (R) and oxidized (O) states respectively. In
the case of wild-type proteins, Y is the fractions of the oxidized
protein at various DTT/OxDTT concentrations, and Y O and Y R
are set at 100 % and 0 respectively.
Then, the standard redox potential was calculated according to
the Nernst equation (eqn 2) at 25 ◦C with the number of electrons
n = 4 (eqn 2):
E 0 ,Tim9 = E 0 DTT/OxDTT + (59.1/4) log(K eq ) mV
(2)
The standard redox potential of DTT (E 0 ,DTT/OxDTT ), − 330 mV, at
25 ◦C and pH 7.4 was used, based on the equilibrium constant for
the reaction GSSG + DTT=2GSH + OxDTT, which is approx.
200 M [28,29].
Mitochondria isolation
The wild-type yeast strain D273-10B (MATα) was grown at 30 ◦C,
and the T9ts strain was grown at 24 ◦C for 30 h, followed by a
further 8 h growth at 37 ◦C. Both strains were grown in media
containing 2.2 % (v/v) lactic acid and 0.3 % (w/v) yeast extract,
with the pH adjusted to 5.5, until the D600 was ∼ 1.5–2.0. Then,
mitochondria were isolated as previously described [30].
Mitochondria import
35
S-labelled proteins were synthesized using the TNT SP6
coupled transcription/translation kit (Promega). Typically, 5 µl
of lysate was added to 150 µl of import reaction mixture
containing mitochondria at 0.5 mg/ml in sorbitol-based import
buffer, consisting of 0.6 M sorbitol, 50 mM KCl, 0.75 mg/ml Lmethionine, 1 mg/ml fatty acid-free BSA, 50 mM Hepes/KOH
(pH 7.4) and 2 mM EGTA. Import was performed at 25 ◦C for the
times indicated and stopped by decanting an aliquot into a test
tube containing 5 vol. of ice-cold 0.6 M sorbitol plus 50 µg/ml
trypsin to stop import and to digest any surface-bound unimported materials for 10 min. The mitochondria were re-isolated
followed by resuspension in gel sample buffer for Tris/Tricine
SDS/PAGE analysis and visualized by autoradiography. For
analysis of the redox state of un-imported proteins, aliquots
were removed from the import reaction and mitochondria
were removed by centrifugation. The supernatant was added to
sample buffer containing 10 mM AMS for thiol modification.
Then, samples were subjected to non-reducing Tris/Tricine
SDS/PAGE separation and visualized by autoradiography. The
Figure 1
assays
Effect of glutathione on the redox state of Tim9 measured by AMS
CBB-stained non-reducing Tricine SDS/PAGE of (a) untreated oxidized Tim9 (lane 1) and
AMS-treated Tim9 (lanes 2–7). The protein was pre-incubated with 1 mM TCEP (lane 2) or
0–10 mM GSH (lanes 3–7) respectively at 25 ◦C for 2 h prior to AMS treatment. The reduced
(R) and oxidized (O) states are indicated. (b) Reduced Tim9 was pre-incubated with 5 mM GSH
plus 2–1000 µM GSSG (lanes 2–10), followed by the AMS assay. Lane 1 is the AMS-treated
starting material of reduced Tim9. The reduced (R) and oxidized (O) states are indicated. 2.5 k,
2500.
result was quantified by two-dimensional densitometry using
Aida Image Analyser V.4.00, and the data were further
analysed using a single or a double exponential function:
y = y0 + A1 × exp(− k1 × x) + A2 × exp(− k2 × x).
RESULTS AND DISCUSSION
Effects of glutathione on the cysteine-redox state of Tim9
Glutathione (GSH/GSSG) is considered the major thiol-disulfide
redox buffer of the cytosol and has a standard redox potential
of approx. − 0.26 V at pH 7.4 [20]. Therefore it is important to
know the relative redox stability of a protein to glutathione, in
order to ascertain whether disulfide bonds could form within the
cytosol. To address this issue, the effects of GSH and GSSG on
the redox state of purified Tim9 were investigated. First, oxidized
Tim9, containing two native intramolecular disulfide bonds, was
incubated with GSH at various concentrations for 2 h. Then,
excess amount of thiol-specific reagent AMS was added to react
covalently with reduced cysteine thiol groups of the protein. Under
these conditions, reduced but not oxidized Tim9 was modified,
with an increased molecular mass of approx. 0.5 kDa per thiol.
The redox state was examined using non-reducing SDS/PAGE
(Figure 1a). A clear difference in mobility of approx. 2 kDa
between the oxidized (unmodified) and the TCEP-reduced Tim9
(Figure 1, lanes 1 and 2) was shown, which is consistent with
our previous observation of reductive unfolding in the presence
of DTT. More importantly, the result showed that oxidized Tim9
was stable against glutathione reduction; no reduced Tim9 was
observed even in the presence of 10 mM GSH. Next, reduced Tim9
was incubated with GSH/GSSG at various conditions, followed by
the AMS assay as described above (Figure 1b). The results showed
that reduced Tim9 can be oxidized by GSSG under physiological
glutathione redox conditions, even in the presence of as little as
2 µM GSSG and 2500-fold of GSH (Figure 1). Taken together,
these results show that oxidized Tim9 is the thermodynamically
stable form under the cytosolic glutathione conditions. The newly
c The Authors Journal compilation c 2008 Biochemical Society
118
Figure 2
B. Morgan and H. Lu
Determination of the standard redox potential of Tim9
(a) RP-HPLC profiles of the wild-type Tim9 after incubation with 0.5 mM OxDTT and various DTT concentrations, from 0.5 µM to 20 mM, for 16 h at 25 ◦C (see the Experimental section). (b) The
fractions of the oxidized Tim9 were calculated based on the areas of the oxidized and reduced proteins shown in (a) and plotted against the ratio of DTT/OxDTT. The data were analysed as described in
the Experimental section. The equilibrium constant (K eq ) of Tim9 reduction and the standard redox potential of Tim9, at 25 ◦C and pH 7.4, were determined to be 90 +
− 30 and − 0.3 V respectively. (c)
The differential fluorescence spectra of Tim9F43W, after and before the protein was incubated with the various DTT/OxDTT buffers as described above. (d) Fluorescence intensity change at 350 nm
was plotted against the ratio DTT/OxDTT, and the data were analysed as described in the Experimental section. The equilibrium constant and the standard redox potential for Tim9F43W, at 25 ◦C and
pH 7.4, were determined to be 33 +
− 6 and − 0.31 V respectively.
synthesized reduced protein can be oxidized in the cytosol in the
absence of a cofactor to stabilize it.
Determination of the standard redox potential of Tim9
The above studies show that Tim9 is a stronger reductant than
glutathione and probably has a standard redox potential below
− 0.3 V. Next, in the present study, two alternative methods were
developed to determine the standard redox potential of Tim9.
We chose a stronger disulfide-bond reducing agent, DTT, and its
oxidized form (OxDTT) (E0 ,DTT/OxDTT = − 0.33 V) [23,28] as the
redox buffer, coupled with the use of RP-HPLC or fluorescence
measurements in determining the redox potential of Tim9.
First, the oxidized wild-type Tim9 was incubated with various
ratios of DTT/OxDTT in nitrogen-saturated buffer A (50 mM Tris,
150 mM NaCl and 1 mM EDTA, pH 7.4). Then, the mixtures of
the oxidized and reduced proteins were acidified and separated by
RP-HPLC (Figure 2a and the Experimental section). The relative
amounts of the oxidized and reduced proteins were calculated
based on the area of the corresponding peaks at approx. 25.8 ml
for the oxidized form (O) and at 30.5 ml for the reduced form
(R) respectively. There were also a few very small peaks that
were eluted between the two major ones, possibly due to impurity
and/or presence of single-disulfide-bond species, which were not
included in our data analysis. The fractions of oxidized Tim9 were
plotted against ratios of DTT/OxDTT as shown in Figure 2(b).
The redox equilibrium constant (K eq ) for Reaction 1 (see above)
was determined based on eqn (1) (see the Experimental section) to
be 90 +
− 30. Then, the standard redox potential of Tim9 (E0 ,Tim9 )
was calculated according to the Nernst equation (eqn 2 in the
Experimental section) to be approx. − 0.3 V. A potential problem
c The Authors Journal compilation c 2008 Biochemical Society
for the E0 ,Tim9 determination is that the acid and organic solvents
used for the protein separation by RP-HPLC may cause loss of
some reduced and oxidized Tim9 to a different extent. Thus an
alternative approach was required to confirm the redox potential
of Tim9.
Protein fluorescence is a sensitive, convenient and well-defined
technique and can be used to study protein folding at native
conditions; however, there is no tryptophan residue in Tim9, and
fluorescence of the single tyrosine of Tim9 is negligible. We
have shown previously that cleavage of Tim9 disulfide bonds
is accompanied by protein unfolding. Therefore, in order to
measure the standard redox potential and study the oxidative
folding using the fluorescence technique, we made a tryptophan
mutant by mutating the Phe43 at the loop between the two CX3 C
motifs. As expected, the Tim9F43W mutant showed the same
characteristic as the wild-type protein in terms of far-UV CD
spectra, complex formation with Tim10 and effects of glutathione
on the redox state as studied by the AMS assay (results not
shown). Importantly, Tim9F43W showed a significant difference
in fluorescence intensities between the oxidized and reduced
forms, with the intensity of the oxidized form being approximately
three times higher than the reduced form (Figure 2c). Next,
fluorescence spectra of the mutant before and after incubation with
various DTT/OxDTT redox buffers for 16 h were measured, and
the standard redox potential of Tim9F43W was calculated based
on the tryptophan fluorescence intensity change at 350 +
− 5 nm
(Figures 2c and 2d). The data were analysed using eqn (1) (see
the Experimental section for details) and the K eq was calculated to
be 33 +
− 6. The standard redox potential obtained for Tim9F43W
was − 0.31 V, which is highly similar to that for wild-type Tim9.
Thus the results for both the wild-type and the mutant showed
Oxidative folding and mitochondrial import of Tim9
119
that Tim9 is a much stronger reductant than glutathione. Based
on the K eq of 33 and the standard redox potential for GSH/GSSG
and DTT/OxDTT of − 0.26 and − 0.33 V respectively at 25 ◦C and
pH 7.4, the equilibrium constant for the reaction:
Tim9Red + 2GSSG ↔ Tim9Ox + 4GSH
was calculated to be 1.7 × 103 M2 . This confirms that oxidized
Tim9 is the thermodynamically stable and dominant form under
cytosolic glutathione conditions. The standard redox potential
of Tim9 is similar to that of Tim10 (− 0.32 V) as we reported
previously [23], suggesting that the small Tim proteins have very
similar redox stability and may share the same oxidative folding
mechanism. The fact that the standard redox potentials of these
small Tim proteins are lower than that of PDI (protein disulfideisomerase) (− 0.18 V) [31] is thus consistent with the fact that
PDI can catalyse the oxidative folding of these proteins in vitro
[23]. Similarly, other thiol oxidoreductases, such as thioredoxin
(− 0.27 V) [32] and glutaredoxin (− 0.2 to − 0.23 V) [33], may
be able to catalyse the oxidative folding of the small Tim proteins
as well.
Oxidative folding competes with mitochondria import of Tim9
and Tim10
The above study shows that newly synthesized reduced Tim9
can be oxidized under physiological glutathione concentrations.
Therefore we tested whether both oxidized and reduced Tim9
can be imported into mitochondria isolated from a temperaturesensitive yeast strain, tim9ts, in which both Tim9 and Tim10 are
not detectable [34]. After incubation of the oxidized or reduced
Tim9 with the mitochondria for 30 min, import was stopped
and the mitochondria were treated with proteinase to remove
any surface-bound Tim9. Then, imported Tim9 was analysed
by SDS/PAGE followed by Western blotting. As shown in
Figure 3(a), the reduced Tim9 can be imported into mitochondria,
but the oxidized protein cannot. The result was the same as that
demonstrated previously for Tim10 [7].
Next, we asked whether the folding and import processes are
kinetically controlled. In other words, if oxidative folding is
a kinetically unfavoured slow process compared with the rate
of protein import, it will have little effect on the mitochondrial
import. On the other hand, protein import will be inhibited by fast
folding.
To understand the correlation between oxidative folding and
mitochondria import in a biologically relevant condition, a cellfree import system was used. It allows us to study the rate
of mitochondrial import and oxidative folding of Tim9 under
identical conditions. Radioactive labelled Tim9 ([35 S]Tim9)
synthesized in the rabbit reticulocyte lysate was incubated with
mitochondria isolated from yeast in a standard import buffer. After
various times of incubation, import was stopped and mitochondria
were separated from the un-imported material. While the
isolated mitochondria were treated with trypsin to remove any
surface-bound materials, the un-imported materials were treated
immediately with AMS for the protein redox-state analysis. Then,
both imported (Figure 3b) and un-imported (Figure 3c) Tim9 were
analysed using SDS/PAGE and autoradiography. As expected,
the level of import increased with time and reached a plateau by
approx. 30 min (Figure 3b). However, only approx. 20 % of the
total material was imported into mitochondria. Nearly all of
the un-imported Tim9 was fully (O) or partially (I) oxidized
by 30 min (Figure 3c), showing that import was accompanied by
protein oxidation. The data were quantified and further analysed
using a single exponential function for the mitochondrial import
(Figure 3d), as well as the reduced and fully oxidized un-imported
Figure 3 Mitochondrial import and oxidative folding of purified (a) and
35
S-radiolabelled (b–e) Tim9
(a) Western blotting of Tim9 imported using reduced or oxidized recombinant protein and mitochondria isolated from tim9ts yeast. (b) Time course of import of [35 S]Tim9 into isolated
mitochondria from yeast, analysed by reducing SDS/PAGE. (c) AMS assay of the un-imported
35
S-Tim9 during the time course experiment shown in (b). The reduced (R), partially
oxidized intermediate (I) and fully oxidized (O) states are indicated. (d) The quantified level
of import as shown in (b) was plotted against time, and the data were analysed with a
−1
single exponential function (continuous line) giving a rate constant of 0.07 +
− 0.02 min .
(e) The percentage of un-imported Tim9 as reduced (R), intermediate (I), fully oxidized (O) or
intermediate plus fully oxidized (I + O) was plotted against time and analysed with a single- or
double-exponential function. The calculated rate constant for disappearance of the reduced Tim9
−1
−1
+
was 0.2 +
− 0.06 min , and formation of the fully oxidized Tim9 was 0.025 − 0.005 min . The
error bars shown in (d, e) represent S.E.M. for three sets of repeat experiments.
Tim9 (Figure 3e). The analyses show that the level of reduced
−1
protein decreased with a rate constant of 0.2 +
− 0.06 min and
0.005
min−1 ,
the fully oxidized Tim9 formed at a rate of 0.025 +
−
−1
whereas the rate of import was 0.07 +
− 0.02 min . Thus the import
was approximately three times slower than the first step of the
oxidative folding, and approximately three times faster than
the formation of fully oxidized protein. For the intermediate
(I), the data were not clear enough to give an independent kinetic
c The Authors Journal compilation c 2008 Biochemical Society
120
Figure 4
B. Morgan and H. Lu
Import and AMS assay of the un-imported [35 S]Tim9
Time course of oxidation of [35 S]Tim9 during import into mitochondria isolated (a) without or
(b) with further purification by Nycodenz gradient. (c) Time course of [35 S]Tim9 mitochondrial
import in the absence (control) or presence of 1 mM TCEP (‘+TCEP’), analysed by reducing
SDS/PAGE and autoradiography. (d) AMS assay of the un-imported material from the time
courses shown in (c). An observed degradation band of [35 S]Tim9 seen in the presence of
TCEP is indicated by ‘*’. (e) The quantified level of import for the control (grey) and in the
presence of TCEP (black) in (c) plotted against time. Error bars represent the S.E.M. for three
independent experiments. (f) Time course of [35 S]Tim9 import into mitochondria in the presence
of 10 mM GSH; samples were analysed by SDS/PAGE and autoradiography. (g) AMS assay of
the un-imported materials in (f). The reduced, intermediate and oxidized states in the AMS
assays are indicated by R, I and O respectively.
parameter, but it was well described by the double exponential
function using the two rate constants obtained from the reduced
and fully oxidized proteins (Figure 4e). Thus the fully oxidized
Tim9 was formed after formation of a single-disulfide-bond
intermediate, and its formation was approx. 10-fold slower than
that of the partially oxidized form. The increase in partially and
fully oxidized proteins (I + O), which fitted the same rate constant
as the reduced Tim9, was shown as well (Figure 3e).
Clearly, the import was inhibited as soon as the fully reduced
Tim9 was gone, although the partially oxidized Tim9 was still
populated. Together with the result that only approx. 20 %
of the total protein was imported into the excess amount of
mitochondria used in the experiment, our study suggests that the
c The Authors Journal compilation c 2008 Biochemical Society
partially oxidized Tim9 intermediate(s) may be largely importincompetent as well. If the partially oxidized Tim9 were
import-competent, a higher level of import would be expected
as the fully oxidized Tim9 was formed approx. three times slower
than Tim9 import; statistically, up to 75 % of the total precursor
protein would be expected.
We have shown previously that oxidative folding of the small
Tim proteins can be accelerated by PDI in vitro [23]. There
are many PDI-like oxidoreductases in the ER (endoplasmic
reticulum) responsible for disulfide bond formation within this
organelle [35–38]. Thus one potential reason for the fast oxidative
folding during the mitochondrial import experiments is the
presence of contaminants from the ER and/or other organelles
in the isolated mitochondria, which can accelerate the oxidation
of these proteins. To address this issue, we carried out the
import experiment using different preparations of mitochondria
and mitochondria isolated with or without further purification
by Nycodenz gradient, and very similar results were obtained
(Figure 4). This confirmed that the observed oxidative folding
of the un-imported protein was not due to contamination of
other cellular components. On the other hand, the presence
of the disulfide-reducing agent, TCEP, maintains Tim9 in the
reduced form and enhances the import efficiency, with approx.
2-fold increase in import level at 60 min (Figures 4c–4e). After
approx. 30 min, the reduced protein was not stable and degraded
(Figure 4d). Furthermore, the presence of 10 mM GSH does not
have an obvious effect on the rates of both import and oxidation
of Tim9 (Figures 4f and 4g), suggesting that the accelerator
for Tim9 oxidation is independent of GSH. The factor(s) that
is responsible for the fast folding is currently unknown, and it
would be interesting to test, in future, whether the reactive oxygen
species generated by mitochondria are responsible.
Next, to find out whether oxidative folding is a strong
competitor for import of the small Tim proteins in general, we
performed the same experiment with [35 S]Tim10. As shown in
Figure 5, Tim10 was imported into mitochondria with a similar
time course to that of Tim9. Data analysis showed that the rate
constant for Tim10 import was approx. 0.13 min−1 , slightly faster
than that of Tim9, whereas the overall level of import was approx.
11 % and thus less efficient than that of Tim9 (approx. 20 %).
Unfortunately, the oxidative folding of the un-imported Tim10
was poorly resolved, and the reason is unclear. Although we were
not able to get a quantitative measurement of the rate of Tim10
oxidation, the result of the AMS assay did suggest that the unimported Tim10 was rapidly oxidized, with the reduced form
disappearing in less than 15 min, at which point the import was
inhibited. Thus the correlation between import and oxidation of
Tim10 is consistent with that of Tim9 in general, demonstrating
that oxidative folding can compete with and inhibit mitochondrial
import of the small Tim proteins kinetically.
It is noteworthy that import of the small Tim proteins is typically
approx. 10–20 % as shown previously by us and other research
groups, based on the use of radioactive precursor and excess
amount of mitochondria. In the present study, an explanation for
the inefficient import of the small Tim proteins was provided. We
demonstrated that oxidative folding is a significant factor limiting
the mitochondrial import of small Tim proteins. Consistent with
this hypothesis, in the presence of TCEP, the import level of Tim9
was increased approx. 2-fold. Although other factors may also
play a role, the efficiency of the import of the small Tim proteins
is at least partially controlled by the rate of oxidative folding.
This result suggests that a cofactor is present in vivo to stabilize
the cysteine from oxidation. Since zinc binding can stabilize the
reduced Tim10 [23] and reduced Tim9 (B. Morgan and H. Lu,
unpublished work) from oxidation in vitro, zinc ions as well as
Oxidative folding and mitochondrial import of Tim9
121
Research in H. L.’s laboratory is financially supported by The Royal Society and
BBSRC (Biotechnology and Biological Sciences Research Council grants BB/C514323/1
and BBS/S/A/2004/10901). B. M. is sponsored by a BBSRC Committee Ph.D. Studentship
(BBS/S/A/2004/10901).
REFERENCES
Figure 5
Tim10
Mitochondrial import and oxidative folding of
35
S-radiolabelled
(a) Time course of import of [35 S]Tim10 into isolated mitochondria from yeast, analysed by
reducing SDS/PAGE. (b) AMS assay of the un-imported [35 S]Tim10 during the time course
experiment shown in (a). (c) The quantified level of import as shown in (a) was plotted against
time, and the data were analysed with a single exponential function (continuous line) giving a
−1
rate constant of 0.13 +
− 0.02 min . The error bars represent the S.E.M. for three sets of repeat
experiments.
other components may play a role during import of the proteins,
which is under investigation.
In summary, the present study has demonstrated that the newly
synthesized reduced Tim9 in the absence of a cofactor is not
stable and can be oxidized thermodynamically under the cytosolic
glutathione conditions. The standard redox potential of Tim9
was determined, using two alternative methods developed in
the present study, to be approx. − 0.31 V at pH 7.4, and the
equilibrium constant for the reaction between reduced Tim9
and GSSG was determined to be 1.7 × 103 M2 . These results
confirm that Tim9 is a stronger reductant than glutathione.
Most importantly, we showed that for both Tim9 and Tim10,
oxidative folding is a major competitive process that can inhibit
the import of these proteins kinetically. Our results also suggest
that both partially and fully oxidized proteins are incompetent
for mitochondrial import, and cofactors are required to stabilize
the precursors of the small Tim proteins in the reduced form
in vivo.
We are grateful to Professor Neil Bulleid and Dr Martin Pool (both of the Faculty of
Life Sciences, University of Manchester, Manchester, U.K.) for helpful comments on this
paper, to Professor Kostas Tokatlidis (Institute of Molecular Biology and Biotechnology,
Foundation of Research and Technology Hellas, Heraklion, Crete, Greece) for protein
plasmids and to Professor Carla Koehler (Department of Chemistry and Biochemistry,
University of California at Los Angeles, Los Angeles, CA, U.S.A.) for the yeast strain
tim9ts. H. L. is supported by a Royal Society University Research Fellowship (2003).
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