From the
Mitochondrial GSH derives from a mitochondrial transport system
(RmGshT), which translocates cytosol GSH into the mitochondrial matrix.
Mitochondria of oocytes, isolated 3-4 days after microinjection
of total liver mRNA, expressed a RmGshT compared with water-injected
oocytes. The expressed RmGshT exhibited similar functional features as
reported in isolated mitochondria of rat liver such as ATP stimulation,
inhibition by glutamate, and insensitivity to inhibition by
sulfobromophthalein-glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed
RmGshT is localized in the inner mitochondrial membrane since
expression is still observed in mitoplasts prepared from total liver
mRNA-injected oocytes. Fractionation of poly(A)
GSH participates in multiple cell functions, and therefore
intracellular reduced GSH is of major importance in protecting cells
against damage by toxic xenobiotics, reactive oxygen compounds, and
radiation(1, 2, 3) . There is increasing
evidence that GSH within mitochondria is essential to guarantee a
normal functioning organelle that supplies the energy needed to
maintain normal cell functions (4-8). Furthermore, it has been
shown in several cell types and organs that depletion of mitochondrial
GSH results in cell injury and death, which is prevented by the
selective increase of the mitochondrial pool of GSH using GSH
derivatives such as GSH ethyl
ester(4, 5, 6, 7) . Mitochondria do not
synthesize GSH de novo; thus the mitochondrial pool of GSH is
derived from the activity of a mitochondrial transporter
(RmGshT)
Despite
playing a critical role in cell function, very little is known about
the molecular properties of this critical transporter since there have
been no reports, to our knowledge, to purify or express this
transporter to initiate the identification of its molecular properties.
Furthermore, functional characterization of this transporter,
especially in relationship to the functional features of other plasma
membrane GSH carriers, has not been
examined(10, 12, 13, 14) . Therefore,
the purpose of the present study was first to determine the feasibility
to functionally express the hepatic mitochondrial GSH transporter as
the initial stage toward the molecular cloning of this mitochondrial
membrane carrier using Xenopus laevis oocytes and second to
functionally characterize the expressed GSH mitochondrial transporter
in relation to known features of the sinusoidal (RsGshT) and
canalicular (RcGshT) GSH plasma membrane carriers that have been
recently cloned(13, 14) .
Mitoplasts were prepared by fractionation of whole mitochondria with
digitonin (0.15 mg/mg of mitochondrial protein) as described previously
(20, 21).
To examine transport characteristics of
RmGshT, we next studied its properties in isolated mitochondria from
oocytes expressing rat liver poly(A)
Oocytes were
fractionated into cytosol and mitochondria 3 days after microinjection
of water (33 nl) or total rat liver mRNA (33 ng) followed by a second
injection for GSH loading (66 nl of a stock solution of 50 mM GSH) or water (66 nl) 2 h before isolation of mitochondria.
Oocytes were washed three times with Barth's medium after GSH
loading to remove excess GSH. Results are the mean ± S.D. of n = 3 oocyte preparations.
We thank Dr. J. A. Bomb for assistance with the
electron microscopy studies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RNA
identified a single mRNA species of
3-3.5 kilobases encoding
for the RmGshT, which was stimulated by ATP and inhibited by glutamate
but not by BSP-GSH or DNP-GSH. Microinjection of this fraction did not
lead to expression of plasma membrane GSH transport in intact oocytes,
and conversely, oocytes microinjected with cRNA for rat liver
sinusoidal GSH transporter (RsGshT) or rat liver canalicular GSH
transporter (RcGshT) did not express mitochondrial GSH transport
activity. Thus, our results show the successful expression of the rat
hepatic mitochondrial GSH carrier, which is different from RsGshT and
RcGshT, and provide the strategic basis for the cloning of this
important carrier.
(
)that translocates GSH from cytosol
into the mitochondrial
matrix(4, 9, 10, 11) . The exact
mechanism of this transport system is presently unknown and remains
controversial, since recent studies have suggested diffusion of GSH
into mitochondria through a channel (11) and transport via a
high affinity system stimulated by ATP (10) or subject to a
rapid exchange between cytosol and mitochondria(4) .
Materials
GSH, GSSG, acivicin, L-methionine, L-cystathionine, actinomycin D,
collagenase type IA, emetine, chloramphenicol, and sucrose were
obtained from Sigma. ATP and dithiothreitol were from Boehringer
Mannheim. 145 Ci/mmol [S]GSH was purchased from
DuPont NEN. S-(2,4-dinitrophenyl)glutathione (DNP-GSH) and
sulfobromophthalein-glutathione (BSP-GSH) were enzymatically
synthesized and purified as described previously(15) . The
conjugates were collected, lyophilized, and stored at -20 °C
until use.
Animals
Male Sprague-Dawley rats weighing
180-220 g were obtained from PanLab (Barcelona, Spain) and fed adlibitum until used. Mature X. laevis females were purchased from CRBM (CNRS, Montpelier, France) and
kept under standard conditions(12, 16) .
Isolation of Rat Liver Poly(A)
mRNA isolation and
fractionation on sucrose density gradient (30-10%) were performed
as described previously(17) . Stage 5 and 6 defolliculated
oocytes were selected and maintained in modified Barth's solution
at room temperature with daily changes of
medium(12, 13, 16) . Mature stage 6 oocytes were
injected (33-66 nl/oocyte) with mRNA (0.1-3 ng/nl) using a
computerized pressure-controlled microinjector (Atto Instruments,
Potomac, MD).
RNA,
Oocyte, and Microinjection of mRNA
Isolation of Mitochondria and Mitoplast from
Oocytes
Water or total mRNA-microinjected oocytes were
fractionated to isolate the mitochondrial fraction(18) . Batches
of 150-200 microinjected oocytes were washed 3 times with
Barth's medium and transferred to 190 mM mannitol, 1
mM CaCl, 0.5 mM EDTA, and 10 mM TES, pH 7.4, and homogenized in a loose-fitting glass homogenizer
with 10 strokes at 4 °C. The crude mitochondrial pellet was
separated from the pigment and yolk on a sucrose density gradient as
described(19) . The mitochondrial fraction was collected and
washed in 195 mM mannitol, 1 mM EDTA, 5 mM TES, pH 7.4, and kept on ice until use for GSH transport.
cRNA Synthesis from RsGshT and RcGshT Clones
cDNA
clones from RsGshT and RcGshT were described
previously(13, 14) . cDNA were used for in vitro transcription with T7 RNA polymerase in the presence of
mG(5`)ppp(5`)G (Pharmacia Biotech, Barcelona, Spain), using
a protocol supplied with the Riboprobe transcription system (Promega
Biotec, Madison, WI). Complementary RNA (cRNA) from each clone was
injected into oocytes and kept at room temperature for 3 days before
transport experiments. Plasma membrane transport of
[
S]GSH was performed as described
previously(12, 13, 14) .
Mitochondrial GSH Transport
To check the
expression of mitochondrial GSH transport, initial rates of GSH uptake
into mitochondria isolated from total or size-fractionated
mRNA-injected oocytes was determined in uptake buffer containing 5
mM Hepes, pH 7.2, 220 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 0.1% bovine serum albumin (fatty
acid-free), 5 mM succinate, 1 mM potassium phosphate,
and 1 µCi of [
S]GSH (0.025-50
mM) in a final volume of 250 µl. Uptake was initiated by
the addition of mitochondrial suspension (0.5-0.7 mg/ml, final
concentration) and after 15 s terminated by addition of 1.0 ml of
ice-cold uptake buffer followed by vacuum filtering (Millipore, 0.45
µm) to separate medium from mitochondria. Retained mitochondria on
filter were washed twice with 2.5 ml of ice-cold buffer(15) . In
some cases, to check for the molecular form of GSH associated with the
mitochondria, filters with retained mitochondria were placed in
borosilicate glass tubes with 1 ml of distilled water to release
intramitochondrial contents retained on filters. Samples were treated
with 10% trichloroacetic acid and spun 13,000
g for 2
min, and the supernatants were analyzed for HPLC as
described(22) . To study the ATP-dependent uptake of
mitochondrial GSH, ATP (2 mM) was added to the uptake medium
and transport experiments for 15 s and was terminated as described
above. For a longer period of transport, ATP and an ATP-regenerating
system (10 mM creatine phosphate and 100 µg/ml creatine
kinase) were included in the incubation.
Expression of GSH Transport in Mitochondria of Oocytes
Injected with Total mRNA from Rat Liver
Xenopus oocytes
contain about 10 times as many mitochondria as a typical
somatic cell(19) . The final mitochondrial band was enriched in
succinic dehydrogenase activity (4-5-fold) relative to
homogenate. Electron microscopy examination revealed sealed
mitochondria uncontaminated with other intracellular structures. For
purposes of comparison of the basal status of mitochondrial GSH with
that of hepatocytes, we expressed our results as per 8 oocytes, since
their volume is equivalent to that of 10
hepatocytes(12) . Most of the GSH content, which was
comprised of >95% reduced GSH, was found in cytosol (11-13
nmol/8 oocytes), which is 3-4 times lower relative to hepatocytes
(30-40 nmol/10
cells). Mitochondria of basal oocytes
were found to have also a lower GSH content (<1.0 nmol/8 oocytes)
than hepatic mitochondria (5-7 nmol/10
cells). These
values of basal GSH content in total mRNA-injected oocytes did not
differ from water-injected oocytes. Therefore, to discern if injection
of total rat liver-mRNA leads to expression of GSH transport in
mitochondria, we loaded cytosol of oocytes with GSH to provide
substrate for the operation of the mitochondrial transporter. As shown
in , mitochondria isolated from oocytes 3 days after mRNA
injection and preloaded with GSH 2 h before mitochondria isolation
showed a much greater level of GSH (2.0-2.5 nmol/8 oocytes
(3-4-fold increase)) compared with mRNA-injected oocytes not
loaded with GSH or water-injected GSH-loaded oocytes. These results
strongly suggest that injection of total rat liver mRNA leads to
expression of the mitochondrial transport system leading to an
increased steady-state level of mitochondrial GSH when cytosol GSH was
available as a substrate.
RNA. Three days
after microinjection of total mRNA, isolated mitochondria exhibited a
time-dependent uptake of [
S]GSH, with an initial
linear rate up to 1 min, compared with mitochondria from water-injected
control oocytes (not shown). The molecular form of
[
S]GSH associated with retained mitochondria on
filters was checked by HPLC and confirmed as >90% reduced GSH.
However, when the uptake of labeled GSH was examined in the presence of
added ATP, the initial rate of transport increased significantly
compared with mitochondria from mRNA-injected oocytes without addition
of ATP (not shown). This ATP stimulation of GSH uptake into
mitochondria from mRNA-injected oocytes was a unique feature of
mitochondrial GSH transport since neither RsGshT nor RcGshT displays
ATP stimulation(15) . The small endogenous uptake of GSH in
control mitochondria from water-injected oocytes was not stimulated by
ATP (not shown). Mitoplasts from mRNA-injected oocytes exhibited the
same magnitude of transport compared with whole mitochondria,
suggesting that the expressed mitochondrial GSH carrier is located in
the inner mitochondrial membrane (Fig. 1). In addition, our
studies indicated that for the stimulation of transport observed by
ATP, this molecule has to be hydrolyzed since the presence of ADP or
non-hydrolyzable ATP analogues did not result in stimulation of GSH
uptake (not shown). Further studies revealed that the expression of GSH
transport in mitochondria from oocytes following injection of total
mRNA from rat liver was dependent on time of culture of oocytes
injected with total rat liver mRNA, taking at least 3 days to process
the delivered mRNA into a functional protein inserted in mitochondria,
without much further increase after 5 days of culture of microinjected
oocytes (not shown). In addition, increasing the amount of total mRNA
(10-100 µg) from liver injected into the oocyte led to a
proportional increase in the expression of activity of transport in
mitochondria compared with control water-injected oocytes (not shown).
Expression of the mitochondrial GSH carrier in oocytes microinjected
with total rat liver mRNA was unaffected by pretreatment of oocytes
with actinomycin D and was abrogated by treatment with emetine, a
specific inhibitor of the cytoplasmic translation process, indicating
that the expression of the mitochondrial GSH transporter was not the
result of activation of an endogenous Xenopus gene but rather
the consequence of processing and translation of the rat liver mRNA
(not shown).
Figure 1:
Expression of transport of GSH in
mitoplast from oocytes microinjected with total mRNA. Mitochondria were
prepared and fractionated with digitonin to obtain a matrix plus inner
membrane structure (as described under ``Experimental
Procedures'') checking monoamine oxidase activity to monitor
disappearance of outer membrane. Mitochondria or mitoplasts were
incubated with [S]GSH (5 mM) to
determine expression of GSH transport activity. When present, ATP was
added at 2.5 mM in the medium.
Inhibitor Specificity and Identification of the mRNA Size
Fraction Encoding the Mitochondrial GSH Transporter
We further
characterized the functional features of the expressed mitochondrial
GSH transport activity by examining cis-competition with known
inhibitors of the RsGshT, RcGshT, and preferred substrates of the
ATP-driven multispecific organic anion
transporter(15, 23) . We examined the cis-effects on 5
mM [S]GSH uptake of BSP-GSH, DNP-GSH,
BSP, methionine, and cystathionine, previously shown to interact with
and inhibit plasma membrane GSH carriers to determine substrate
specificity of the expressed mitochondrial GSH
carrier(12, 13, 14, 15, 24, 25) .
As seen in Fig. 2, GSH thioether adducts, BSP-GSH and DNP-GSH,
did not lead to inhibition of mitochondrial GSH transport in total
mRNA-injected oocytes with or without ATP. The lack of effect by
DNP-GSH contradicts the possibility that the expressed GSH transporter
in mitochondria from oocytes is due to expression and mitochondrial
insertion of RcGshT or multispecific organic anion carrier. BSP and
GSSG, similar to DNP-GSH, did not inhibit mitochondrial GSH transport
(not shown). On the other hand, results with BSP-GSH, a specific
inhibitor of RsGshT and the high affinity canalicular GSH
carrier(24) , indicated that the GSH transport into mitochondria
is not mediated by expression of these carriers. In addition, other
specific inhibitors of RsGshT, such as methionine and cystathionine,
which do not interact with the high affinity canalicular GSH
carrier(26) , did not inhibit the transport of GSH into
mitochondria (not shown). However, as shown in Fig. 2, glutamate
inhibited the activity of basal and ATP-stimulated mitochondrial GSH
transport expressed in total mRNA-injected oocytes, confirming its
effects on rat liver mitochondria(10) . Endogenous GSH uptake by
mitochondria from water-injected oocytes was not inhibited by glutamate
(not shown). In contrast, RsGshT and RcGshT are not affected by
glutamate (24, 25) (Fig. 3). Other GSH S-conjugates including S-methylglutathione, S-butylglutathione, and S-octylglutathione (1-5
mM) did not inhibit the basal or the ATP-stimulated transport
in mitochondria from mRNA-injected oocytes (not shown).
Figure 2:
Expression of mitochondrial GSH transport
following injection of size-fractionated rat liver mRNA. Total mRNA
(80-90 µg) was fractionated by continuous sucrose gradient
centrifugation. Each size fraction was injected into oocytes (3-5
ng/oocyte) and incubated in modified Barth's medium at room
temperature. Mitochondria were then isolated and incubated with
[S]GSH (5 mM (A) or 0.1 mM (B)) for 15 s to determine the initial rate of GSH
uptake. For the ATP dependence, ATP was included in the incubation
medium at 2.5 mM. For cis-competition studies, mitochondria
were incubated in the presence of labeled GSH plus BSP-GSH, DNP-GSH (1
mM each), or glutamate (10 mM) with and without ATP.
The inset of B shows the effect of inhibitors on the
expression of GSH transport encoded by fraction 5. The molecular form
taken up into mitochondria was checked by HPLC and was >95% reduced
GSH. Results are mean ± S.D. of n = 3 for A and n = 2 for B.
Figure 3:
GSH uptake by intact oocytes and isolated
mitochondria. Oocytes were injected with full-length cRNA prepared from
cloned RsGshT or RcGshT (0.5 ng/oocyte) or 5 ng of mRNA size fraction 5
that expressed mitochondrial GSH transport activity as determined in
Fig. 2. 3 days later, cell membrane transport of
[S]GSH (10 mM) was determined (A) in intact oocytes (cell uptake) in the presence of BSP-GSH
(10 mM), ATP (2.5 mM), or glutamate (10 mM).
Mitochondria were isolated from other oocytes from the same
preparations of injected oocytes, and the initial rate of transport of
[
S]GSH was determined (B) in the
absence or presence of ATP (2.5 mM) or glutamate (10
mM). Results are mean ± S.D. of n = 3
oocyte preparations.
In order to
enrich the mRNA encoding selectively for the mitochondrial GSH
transport system total mRNA was size-fractionated in a continuous
sucrose density gradient. Of all fractions tested, only one (fraction
5) resulted in transport of GSH in mitochondria, which was 8-10
times over that shown by oocytes injected with water using low (0.1
mM) and high (5 mM) GSH (Fig. 2). These two
concentrations were employed to ensure not missing high and low
affinity components since both have been reported(10) . The
presence of ATP in the medium resulted in a further increase of
transport of GSH in mitochondria from oocytes expressing fraction 5,
which was greater at lower GSH concentrations. Similar to the transport
activity expressed in oocytes after injection of total mRNA, fraction 5
mediated expression of mitochondrial GSH transport, which was
insensitive to the presence of BSP-GSH, DNP-GSH, and other GSH S-conjugates (1-5 mMS-methylglutathione, S-butylglutathione, and S-octylglutathione) and thioether amino acids (methionine and
cystathionine). However, the enriched activity expressed by fraction 5
was inhibited by glutamate (5 mM). Despite the difference of
amount of mRNA injected (33 ng for total mRNA versus 3-5
ng of fraction 5) there was a 2-3-fold increase in the activity
of transport of GSH in mitochondria expressing fraction 5, indicating
an overall enrichment of 20-30-fold compared with total mRNA.
Based on agarose gel electrophoresis (data not shown) fraction 5
corresponded to a mRNA size class between 3 and 3.5 kb; this differs
from RcGshT (4 kb) and RsGshT (2.7 kb). The expressed GSH transport
system in mitochondria was unaffected by the presence of aspartate (10
mM, not shown), suggesting that GSH transport expressed in
mitochondria is not mediated via the glutamate/aspartate antiporter.
Hepatic Mitochondrial GSH Transporter Is Distinct from
RsGshT and RcGshT
To determine directly the relationship between
plasma membrane and mitochondrial GSH transporters, we examined
expression of both activities (cell uptake and isolated mitochondrial
uptake) in oocytes injected with either fraction 5 mRNA or cRNA
prepared from the two recently cloned plasma membrane transporters,
RcGshT and RsGshT (13, 14). As shown on Fig. 3, injection of
RsGshT and RcGshT resulted in expression of GSH uptake in intact
oocytes that was unaffected by ATP and glutamate. However, the presence
of BSP-GSH in the medium resulted in inhibition of GSH uptake only when
the sinusoidal clone was injected into oocytes. In addition, methionine
and cystathionine also inhibited transport by RsGshT (not shown).
Oocytes injected with RcGshT displayed sensitivity to inhibition of
transport by DNP-GSH (Fig. 3), BSP, and di-BSP (not shown).
However, injection of mRNA fraction 5 did not lead to expression of GSH
transport in intact oocytes. Moreover, injection of the cRNA from
RsGshT and RcGshT did not result in expression of GSH transport in
isolated mitochondria (Fig. 3B), whereas the injection
of the mRNA size fraction 5 resulted in expression of GSH transport in
mitochondria sensitive to inhibition by glutamate and stimulated by
ATP. Furthermore, since previous studies have identified only one
transcript when rat liver RNA is hybridized with full-length cDNA
probes for RsGshT (2.7 kb) and RcGshT (4.0 kb), it is unlikely that
alternate processing of a transcript of a gene common to one of these
transporters could account for RmGshT. Thus, on the basis of ATP
stimulation, inhibitor specificity, and mRNA size and expression in
oocytes, it is likely that the mitochondrial GSH transporter is encoded
by a distinct gene. In addition, the studies reported here provide the
basis for a cloning strategy for this transporter.
Table: Expression of mitochondrial GSH transport in
oocytes microinjected with mRNA from rat liver
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.