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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 43, pp.

29126 29134, October 24, 2008


2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

The Redox Environment in the Mitochondrial Intermembrane


Space Is Maintained Separately from the Cytosol and Matrix*
Received for publication, April 21, 2008, and in revised form, July 11, 2008 Published, JBC Papers in Press, August 15, 2008, DOI 10.1074/jbc.M803028200
Jingjing Hu, Lixue Dong, and Caryn E. Outten1
From the Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208

Redox control in the mitochondrion is essential for the proper teinylglycine) and the small protein thioredoxin can serve as
functioning of this organelle. Disruption of mitochondrial reductants themselves or as cofactors for anti-oxidant enzymes
redox processes contributes to a host of human disorders, (3). Glutathione is considered the primary determinant of the
including cancer, neurodegenerative diseases, and aging. To cellular redox environment, because it has a relatively low
better characterize redox control pathways in this organelle, we redox potential (240 mV at pH 7.0) and a high intracellular
have targeted a green fluorescent protein-based redox sensor to abundance (113 mM) (4).
the intermembrane space (IMS) and matrix of yeast mitochon- Measurements of GSH:GSSG levels in subcellular compart-
dria. This approach allows us to separately monitor the redox ments demonstrate that individual organelles have different
state of the matrix and the IMS, providing a more detailed pic- redox requirements. The endoplasmic reticulum maintains a
ture of redox processes in these two compartments. To verify relatively oxidizing environment (170 to 185 mV at pH 7.0,
that the sensors respond to localized glutathione (GSH) redox or a GSH:GSSG ratio of 1:1 to 3:1) (5), whereas the cytosol is
changes, we have genetically manipulated the subcellular redox quite reducing in comparison (290 mV at pH 7.0, or a GSH:
state using oxidized GSH (GSSG) reductase localization GSSG ratio of 3300:1) (6). GSH:GSSG measurements in iso-
mutants. These studies indicate that redox control in the cytosol lated mitochondria indicate a redox potential of 250 mV to
and matrix are maintained separately by cytosolic and mito- 280 mV at pH 7.8 or GSH:GSSG ratios of 20:1 to 40:1 (710).
chondrial isoforms of GSSG reductase. Our studies also demon- However, measuring the GSH:GSSG redox state in isolated
strate that the mitochondrial IMS is considerably more oxidiz- mitochondria has several drawbacks. First, GSH:GSSG levels in
ing than the cytosol and mitochondrial matrix and is not directly the matrix and the intermembrane space (IMS) cannot be
influenced by endogenous GSSG reductase activity. These redox measured separately, because the IMS is quite small (5% of
measurements are used to predict the oxidation state of thiol- the total mitochondria volume), making it difficult to effectively
containing proteins that are imported into the IMS. isolate IMS GSH:GSSG from matrix pools. Second, GSH may
be oxidized during cell lysis and fractionation steps creating an
artificially low GSH:GSSG ratio. Finally, metabolites may be
Maintenance of the thiol-disulfide balance in cells is critical lost or exchanged during the mitochondrial isolation procedure
for the proper functioning of numerous enzymes and proteins thereby altering the physiology and redox state of the organelle.
with functionally important cysteine residues. The cellular Nevertheless, defining redox control in the IMS is critical
redox balance can be disrupted by unregulated production of given the various redox-dependent pathways in this compart-
reactive oxygen species (ROS)2 that interfere in redox signaling ment, including apoptotic signaling (11, 12), assembly of respi-
pathways and oxidatively damage DNA, proteins, and lipids (1). ratory chain components (13), anti-oxidant activation (14), and
To control the cellular redox environment, cells contain two protein import (15). It is not known if the redox state of this
primary redox regulatory systems that utilize thiol-disulfide compartment is relatively oxidizing or reducing in comparison
redox chemistry: the glutathione (GSH)/glutathione disulfide to the mitochondrial matrix and cytosol. On the one hand, this
(GSSG) redox couple and the reduced/oxidized thioredoxin compartment is phylogenetically linked to the oxidizing
redox couple (1, 2). The tripeptide glutathione (-glutamylcys- periplasm of bacteria (16). Furthermore, a substantial number
of IMS proteins have functionally essential disulfide bonds (17,
18). On the other hand, porin channels in the mitochondrial
* This work was supported, in whole or in part, by National Institutes of Health
Grant ES 13780 (to C. E. O.). This work was also supported by the University outer membrane presumably allow free exchange of GSH and
of South Carolina Center for Colon Cancer Research. The costs of publica- GSSG between the IMS and cytosol (15, 19), suggesting that the
tion of this article were defrayed in part by the payment of page charges. GSH:GSSG redox state in the IMS is similar to the reducing
This article must therefore be hereby marked advertisement in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
cytosol.
1
To whom correspondence should be addressed: Dept. of Chemistry and An in vivo method for measuring the subcellular redox state
Biochemistry, 631 Sumter St., University of South Carolina, Columbia, SC of GSH:GSSG is an effective approach to address redox control
29205. Tel.: 803-777-8783; Fax: 803-777-9521; E-mail: caryn.outten@
chem.sc.edu.
in individual compartments. stergaard and coworkers (6)
2
The abbreviations used are: ROS, reactive oxygen species; GSH, reduced have developed a genetically encoded, cytosolic redox sensor
glutathione; GSSG, oxidized glutathione; IMS, intermembrane space; GFP, based on the yellow variant of green fluorescent protein (GFP)
green fluorescent protein; rxYFP, redox-sensitive yellow fluorescent pro- called redox-sensitive YFP (rxYFP). GFP and its derivatives
tein; GRX, glutaredoxin; PMS, post-mitochondrial supernatant; DAPI, 4,6-
diamidino-2-phenylindole; 4-DPS, 4,4-dithiodipyridine; DTT, dithiothrei- provide ideal scaffolds for creating in vivo sensors due to their
tol; Cyb2, cytochrome b2; WT, wild type. protease resistance and high stability in a broad range of pH and

29126 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 NUMBER 43 OCTOBER 24, 2008
Redox Potential Measurements in Yeast Mitochondria
buffer conditions (20). The rxYFP protein in particular can be C terminus, respectively. The PCR fragment was inserted in-
used to measure the redox potential in live cells via formation of frame at the N terminus of rxYFP at the SpeI and NdeI sites of
an engineered disulfide bond that perturbs the local chro- pHOJ150, forming pJH200.
mophore environment without significantly altering the overall The matrix-rxYFP plasmid pLD207 was constructed by
-can fold (21). The relative ratio of oxidized to reduced rxYFP amplifying the promoter region and mitochondrial targeting
can also be assessed via non-reducing SDS-PAGE in which the signal (codons for amino acids 125) of cytochrome oxidase
two forms have different electrophoretic mobilities. stergaard subunit IV (COX4) with primers that introduced NotI site and
and coworkers (6) have shown both in vivo and in vitro that the NdeI sites at the 5 and 3 ends, respectively. The PCR fragment
cysteines in rxYFP specifically equilibrate with GSH and GSSG was inserted in-frame at the N terminus of rxYFP at the NotI
via rapid disulfide exchange reactions with the cytosolic glu- and NdeI sites of pJH300. The COX4 promoter was later
taredoxins (GRXs). In contrast, the two cytosolic thioredoxins replaced with the manganese superoxide dismutase (SOD2)
are unable to undergo thiol-disulfide exchange with this sensor. promoter by creating a SpeI site just upstream of the Cox4
The ratio of oxidized to reduced rxYFP measured in the cell can mitochondrial targeting signal. The COX4 promoter was
be used to generate an in vivo readout of the GSH:GSSG redox removed by digestion with NotI and SpeI and a PCR fragment
state. Their measurements indicated that the ratio of GSH: containing SOD2 promoter was inserted in its place. This plas-
GSSG in the eukaryotic cytosol was 3300:1, which corresponds mid was then digested with NotI and SacII, and the fragment
to a redox potential of 289 mV (6). This value is considerably containing the SOD2 promoter, the coding sequence of COX4-
more reducing than whole cell redox measurements (221 to rxYFP, and the TDH3 terminator was inserted into the URA3-
236 mV) (5), highlighting the importance of examining sub- CEN vector pRS316, yielding pLD207.
cellular compartments separately. The GLR1-expressing plasmids pJH201 (WT Glr1), pJH202
In this study we have modified rxYFP for expression in the (M17L Glr1), and pJH203 (M1L Glr1) were created by inserting
mitochondrial IMS and matrix of the yeast Saccharomyces cer- the GLR1 gene sequence from the plasmids pCO113 (WT),
evisiae to compare redox differences between these compart- pCO114 (M17L), or pCO116 (M1L) (9) into the SacI and XhoI
ments and the cytosol. We have demonstrated exclusive target- sites of the HIS3-CEN vector pRS413 (23). The sequence integ-
ing of IMS- and matrix-rxYFP to these compartments and have rity of all plasmids was verified by double-stranded DNA
confirmed their dynamic response to an exogenous oxidant and sequencing (USC Environmental Genomics Core Facility).
reductant. We have measured the IMS redox potential, demon- Subcellular FractionationYeast cells were grown aerobi-
strating that this compartment is considerably more oxidizing cally to mid-log phase in selecting SC medium with 2% galac-
than the cytosol or matrix. Furthermore, we have manipulated tose. Mitochondrial and post-mitochondrial supernatant
the subcellular redox potential using GSSG reductase mutants (PMS) fractions were obtained as previously described by con-
to verify that the sensors respond to specific intracellular redox verting cells to spheroplasts followed by gentle lysis by Dounce
changes that are localized to subcellular compartments. Over- homogenization and differential centrifugation (24). Mito-
all, our data suggest that redox control is independently regu- chondrial intermembrane and mitoplast fractions were pre-
lated within these individual compartments in the cell. pared by osmotic shock as previously described (25). Mitoplasts
and IMS fractions were subsequently treated with 50 g/ml
EXPERIMENTAL PROCEDURES proteinase K in the absence or presence of 0.2% Triton X-100.
Yeast Strains, Media, and Growth ConditionsS. cerevisiae Immunoblotting TechniquesYeast extracts were subjected
strains used in this study were BY4741 (MATa his31 leu20 to electrophoresis on Tris-glycine gels (Invitrogen) and ana-
met150 ura30) and BY4741 glr1::kanMX4 obtained from lyzed by Western blotting using an anti-rxYFP antibody (kind
Research Genetics. Yeast transformations were performed by gift of J. Winther) or an anti-GFP antibody (Invitrogen) and a
the lithium acetate procedure (22). Strains were maintained at secondary anti-rabbit IgG (IRDye, LI-COR Lincoln, NE). PMS
30 C on either enriched yeast extract-peptone-based medium fractions were monitored by anti-3-phosphoglycerate kinase
supplemented with 2% glucose (YPD) or synthetic complete (PGK1) antibodies (Invitrogen). Mitochondrial fractions were
medium (SC) supplemented with 2% glucose or 2% galactose monitored by using antibodies directed against Cyb2 in the
and the appropriate amino acids. IMS, mitochondrial processing protease Mas2 in the matrix
PlasmidsThe plasmid pJH208 expressing cytosol-rxYFP (kind gifts of R. Jensen), or mitochondrial NADH kinase
was constructed by digesting the rxYFP yeast expression plas- Pos5 in the matrix (26). Proteins were analyzed by Western
mid pHOJ150 (6) with NotI and SacII. The NotI-SacII-digested blot using an Odyssey Infrared Imaging System (LI-COR).
fragment containing the PGK1 promoter, the coding sequence Protein concentrations were determined using the Bradford
of rxYFP, and the TDH3 terminator was then inserted into the method (Bio-Rad) with bovine serum albumin as the calibra-
URA3-CEN vector pRS316 (23). tion standard.
The IMS-rxYFP plasmid pJH200 was constructed as follows. Fluorescence MicroscopyThe BY4741 parental strain trans-
An NdeI site downstream of the rxYFP coding sequence in formed with pJH208 (cytosol-rxYFP), pJH200 (IMS-rxYFP), or
pHOJ150 was removed by site-directed mutagenesis forming pLD207 (matrix-rxYFP) was grown aerobically to mid-log
pJH300, which leaves one NdeI site at the start codon for rxYFP. phase in selecting SC medium containing 2% galactose. Cells
The mitochondrial targeting sequence of S. cerevisiae cyto- were incubated with 2.5 g/ml 4,6-diamidino-2-phenylindole
chrome b2 (Cyb2) (codons for amino acids 1 88) was amplified (DAPI) (Invitrogen) for 30 min to stain mitochondrial DNA.
by PCR introducing an NheI site and an NdeI site at the N and Live cells were examined with a Zeiss LSM 510 META Confocal

OCTOBER 24, 2008 VOLUME 283 NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29127
Redox Potential Measurements in Yeast Mitochondria
Scanning Laser Microscope (Instrumentation Resource Facility
at the University of South Carolina School of Medicine).
Redox WesternRedox Western blot analysis of rxYFP was
adapted from previous methods (6). Briefly, cells were grown in
selecting SC medium to mid-log phase. In some experiments,
cells were treated with 180 M 4,4-dithiodipyridine (4-DPS)
(Sigma) or 50 mM dithiothreitol (DTT) (Sigma) and incubated
at 30 C for an additional 20 min. 4-DPS and DTT are both
membrane-permeable. Cell cultures were acid-quenched with
trichloroacetic acid (Sigma) (15% (w/v) final concentration) at
4 C for 20 min. Five A600 units of cells were harvested by cen-
trifugation and resuspended in 1 ml of 10% trichloroacetic acid.
Following glass bead lysis, the lysed cells were transferred to a
new tube and pelleted by centrifugation. The pellet was resus-
pended in 500 l of 1X non-reducing SDS sample buffer con-
taining 40 mM N-ethylmaleimide (Sigma). Following a 10-min
incubation at room temperature, the proteins were separated
on a 16% Tris-glycine gel (Invitrogen). Reduced and oxidized
forms of rxYFP were analyzed by quantitative immunoblot
using an Odyssey Infrared Imaging System (LI-COR).
Redox Potential CalculationsThe rxYFP sensor equili-
brates with GSH:GSSG pools according to the following reac-
tion (Scheme 1) (6).
SH
rxYFP SH GSSG N rxYFPSS] 2GSH
SCHEME 1

The ratio of oxidized to reduced rxYFP and the standard reduc-


tion potentials of rxYFP and GSH were inserted into the Nernst
equation (Equation 1) to estimate the oxidation state of GSH:
GSSG.
RT
E E lnQ (Eq. 1) FIGURE 1. Mitochondria-targeted constructs of rxYFP. A, the gray box rep-
nF resents the rxYFP protein, the black coil represents the amphipathic helix
required for matrix targeting, and the black box represents the hydrophobic
In this equation, R is the gas constant (8.314 J K1 mol1), T is sorting domain required for IMS targeting. The N termini of native mitochon-
drial proteins (Cox4 and Cyb2) were fused in-frame to rxYFP. B and C, sche-
the temperature in K, n is the number of electrons, and F is matics depicting import into the mitochondrial matrix (B) and IMS (C) via
Faradays constant (96,485 C mol1). At 30 C, for the reaction N-terminal targeting sequences. The targeting signals are cleaved during
shown in Scheme 1, Equation 1 can be rewritten as Equation 2, import by matrix and/or inner membrane (IM) proteases and the protein folds
into its native conformation.
60.1 mV GSH2
E GSH E log
GSH 2 GSSG GSH/GSSG AssaysTotal glutathione (GSH GSSG) and
oxidized glutathione (GSSG) in PMS and mitochondrial
60.1 mV rxYFPSH
SH
extracts were measured by the 5,5-dithiobis(2-nitrobenzoic
E log ErxYFP (Eq. 2)
rxYFP 2 rxYFPSS] acid)-GSSG reductase cycling assay as previously described (9).
According to Equation 2, ErxYFP is dependent on the ratio of RESULTS
reduced:oxidized rxYFP but not the absolute concentration, Targeting rxYFP to the Mitochondrial Matrix and Intermem-
[rxYFP]. However, estimation of the GSH:GSSG ratio from brane SpaceMitochondria-targeted versions of rxYFP were
ErxYFP requires the absolute concentrations [GSH] or [GSSG]. created by fusing the mitochondrial targeting signals from

At pH 7.0, the standard reduction potential of rxYFP (ErxYFP ) is native mitochondrial proteins to the N terminus of rxYFP (Fig.
265 mV (6) and the standard reduction potential of GSH 1A). The mitochondrial targeting signal of Cox4 was used to

(EGSH ) is 240 mV (4). The reduction potentials of rxYFP and generate matrix-rxYFP, whereas the mitochondrial targeting
GSH at different pH values were calculated using the expres- signal of Cyb2 was used to make IMS-rxYFP. Both sequences
sion shown in Equation 3 (4), have been successfully employed in the past for targeting non-
E pH E pH 7.0 60.1 mV (Eq. 3) native proteins to the matrix or intermembrane space (2729).
The matrix targeting sequence encodes an amphipathic helix
In this expression, E is the standard reduction potential of that is recognized by the mitochondrial import machinery and
GSH or rxYFP at pH 7.0. subsequently removed by matrix proteases (Fig. 1B) (30). The

29128 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 NUMBER 43 OCTOBER 24, 2008
Redox Potential Measurements in Yeast Mitochondria

FIGURE 3. Differential interference contrast (DIC) and fluorescence


FIGURE 2. Subcellular localization of cytosol- (A), matrix- (B), and IMS- microscopy of WT yeast cells (BY4741) expressing cytosol-, matrix, or
rxYFP (C). WT yeast cells (BY4741) expressing cytosol-, matrix-, or IMS-rxYFP IMS-rxYFP. Cells were grown to mid-log phase in SC galactose media and
were grown to mid-log phase in SC galactose media. Cells were lysed and incubated with DAPI for 30 min to stain DNA. Live cells were examined with
fractionated, and fractions were analyzed by SDS-PAGE and immunoblotting Zeiss LSM 510 META confocal scanning laser microscope at a magnification of
using antibodies directed against rxYFP, PGK1 (cytosol marker), Pos5 or Mas2 605. Merge merged images of DAPI and YFP fluorescence with white areas
(mitochondrial matrix markers), or Cyb2 (mitochondrial IMS marker). In the indicating overlap. Plasmids and strains are the same as in Fig. 2.
left panel of AC, 75 g of total cell protein (Total) was fractionated into post-
mitochondrial supernatant (PMS) and mitochondria (Mito), and the entire it is located on the outside of the mitochondrial inner mem-
amount of each fraction was analyzed. In the right panel of B, mitochondria brane in the IMS compartment. We note that, unlike the Cyb2
(15 g of protein) from cells expressing matrix-rxYFP were further fraction-
ated into IMS and mitoplast components, and the entire amount of each control, rxYFP in the soluble IMS fraction is only partially
fraction was analyzed. In the right panel of C, mitochondria (15 g of protein) digested (Fig. 2C, lane 6), even though all proteins in this frac-
from cells expressing IMS-rxYFP were fractionated as in B. Mitoplast and IMS
fractions were further treated with proteinase K and/or Triton X-100 as indi-
tion are soluble and therefore accessible to proteinase K. This
cated. rxYFP-expressing plasmids utilized include: pJH208 (cytosol), pLD207 observation may be explained by the fact that GFP-based pro-
(matrix), and pJH200 (IMS). teins have an unusually stable core structure that is resistant to
complete digestion by proteases (20).
IMS targeting sequence includes an additional hydrophobic Localization and fluorescence signal of the rxYFP sensors
sorting domain, which is cleaved by an inner membrane prote- were further verified by fluorescence microscopy. Live yeast
ase (Fig. 1C) (31). cells expressing cytosol-, matrix-, or IMS-rxYFP were stained
To confirm the correct localization of the rxYFP constructs, with DAPI for co-localization experiments. DAPI staining for
we conducted a Western blot analysis of crude mitochondria DNA highlights both mitochondria (string-like structures) and
and largely cytosolic (PMS) fractions from WT strains trans- the nucleus (large, rounded structure) as shown in Fig. 3.
formed with cytosol-, matrix-, or IMS-rxYFP. As shown in Fig. Matrix- and IMS-rxYFP specifically co-localized with DAPI
2A, cytosol-rxYFP is localized to the cytosol as previously deter- staining of the mitochondrial, but not nuclear DNA, indicating
mined (6). In contrast, matrix-rxYFP and IMS-rxYFP are local- that these sensors are exclusively localized to the mitochondria
ized to mitochondria (Fig. 2, B and C, left panels). The molecu- and are correctly folded with the expected fluorescence
lar weights of the rxYFP bands in Fig. 2 (B and C) are consistent properties.
with the mature, processed forms of both sensors. Mitochon- Matrix- and IMS-rxYFP Register Dynamic Subcellular Redox
dria were further fractionated into intermembrane space and ChangesTo test the reactivity and response of the targeted
mitoplasts, revealing that matrix-rxYFP is correctly localized to rxYFP sensors, a strong oxidant or reductant was added to cells
the matrix (Fig. 2B, right panel), whereas IMS-rxYFP is located expressing cytosol-, matrix-, or IMS-rxYFP. The redox state of
in the soluble IMS fraction (Fig. 2C, right panel, lane 5). How- rxYFP can be monitored by redox Western blot or fluorescence
ever, a portion of IMS-rxYFP is also found associated with spectroscopy as described for cytosol-rxYFP (6). Both of these
mitoplasts (Fig. 2C, lane 2). To determine whether IMS-rxYFP techniques avoid the artificial oxidation problems and lengthy
in this fraction is located on the outside (IMS side) or inside subcellular fractionation steps encountered by conventional
(matrix side) of the mitoplast membrane, proteinase K was methods for measuring subcellular redox potential.
added to intact mitoplasts to digest exposed (IMS side) pro- The redox Western blots shown in Fig. 4 indicate that the
teins. IMS-rxYFP was largely degraded by this protease (Fig. 2C, mitochondrial rxYFP constructs are responsive to redox
lane 3) in a similar manner to soluble IMS-rxYFP (Fig. 2C, lane changes generated by exogenous reagents, as previously dem-
6), whereas the control matrix protein Pos5 (26) was unaffected. onstrated for cytosol-rxYFP (6). Addition of the thiol-oxidant
The Pos5 matrix control is only digested (indicated by a change 4-DPS or the disulfide reductant DTT shifts all three sensors
in molecular weight) upon addition of Triton X-100, which dis- toward the oxidized state (Fig. 4A, lane 2) or reduced state (Fig.
rupts the mitoplast membrane allowing access to matrix pro- 4A, lane 3), respectively. Fluorescence measurements were also
teins (Fig. 2C, lane 4). In contrast, proteolysis of mitoplast- performed using live cells expressing cytosol-, matrix-, and
associated IMS-rxYFP is similar with and without the addition IMS-rxYFP. The fluorescence response of cells expressing
of Triton X-100 (compare Fig. 2C, lanes 3 and 4), indicating that cytosol-rxYFP upon addition of DTT or 4-DPS was similar to

OCTOBER 24, 2008 VOLUME 283 NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29129
Redox Potential Measurements in Yeast Mitochondria

FIGURE 5. rxYFP redox response in WT and glr1 cells. WT (BY4741) and


glr1 (BY4741 glr1) yeast cells expressing cytosol-, matrix-, or IMS-rxYFP
were grown to mid-log phase in SC glucose media. Redox Western blot was
performed as described in Fig. 4.

(3336), so 7.0 was used for our calculations. The redox poten-

tial of rxYFP at pH 7.0 (ErxYFP 265 mV) (6) was inserted into
equation 2 along with the ratio of reduced-to-oxidized sensor
to calculate the cytosolic redox potential. Similarly, the redox
potential of rxYFP at pH 7.4 (E7.4 ErxYFP

24 mV according
to Equation 3) was used to calculate the matrix redox potential,
FIGURE 4. rxYFP redox response to an exogenous oxidant or reductant. because this matrix pH value was reported for yeast cells grown
WT yeast cells expressing cytosol-, matrix-, or IMS-rxYFP were grown to mid- in glucose (36). Using these values, the cytosolic redox potential
log phase in SC glucose media. Cells were treated with 4-DPS or DTT as corresponds to 286 mV, whereas the matrix redox potential is
described under Experimental Procedures. A, redox Western blot of the
samples separated by non-reducing SDS-PAGE and immunoblotted with 296 mV (Table 1). Studies in mammalian cells suggest that
anti-GFP antibodies. B, reduced (red) and oxidized (ox) forms of rxYFP were the IMS pH is typically 0 0.7 pH units lower than cytosolic pH
quantified using an Odyssey Infrared Imaging System. The reported values
are the mean of three to four independent experiments. Error bars are the (37, 38). By using pH 7.0 as a conservative estimate, the IMS
means S.D. Plasmids and strains are the same as in Fig. 2. redox potential calculated with IMS-rxYFP is 255 mV (Table
1). If the IMS pH is lower than 7.0, the calculated E value will
published reports (6) (data not shown). However, fluorometer increase by 6.0 mV for every 0.1 unit of decrease in pH (see
measurements of whole cells expressing matrix- or IMS-rxYFP Equation 3). Therefore it is possible that the IMS redox state is
were more difficult to obtain over the background autofluores- even more oxidizing than our estimate at pH 7.0. In any case,
cence. This difficulty may be explained by the relatively small these values are considerably more oxidizing than both the
mitochondrial volume of these cells, which constitutes only cytosol and mitochondrial matrix.
3% of the total cell volume under these growth conditions Deletion of GLR1 Alters Redox Status in the Cytosol and
(32). Matrix but Not the IMSTo examine how the GSH:GSSG ratio
The IMS Redox State Is More Oxidizing Than the Matrix or influences redox status within submitochondrial compart-
CytosolUsing the differently targeted versions of rxYFP, we ments, we tested the sensors in glr1 mutant cells that lack the
compared the redox state in the cytosol, mitochondrial matrix, GSSG reductase gene (GLR1). Because this enzyme is respon-
and mitochondrial IMS. As shown in Fig. 5 and Table 1, cytosol- sible for regenerating GSH from GSSG, deletion of this gene
rxYFP is 16% oxidized under steady-state conditions, which is creates a higher than normal intracellular GSSG:GSH ratio (6,
similar to published results (6), whereas matrix-rxYFP is 38% 9, 39). As shown in Fig. 5 and Table 1, cytosol-rxYFP undergoes
oxidized. Inserting these values into Equation 2 and assuming dramatic redox changes (74% oxidized) in glr1 mutant cells
an intracellular [GSH] of 13 mM (6), we estimate that the GSH: compared with WT cells (16% oxidized), as previously dem-
GSSG ratio in the cytosol and matrix are 3000:1 and 900:1, onstrated (6). Likewise, the matrix-rxYFP sensor shifts from
respectively (Table 1). Interestingly, IMS-rxYFP is significantly 38% oxidized to 84% oxidized in WT versus glr1 strains.
more oxidized (68%) under steady-state conditions com- Glr1 is localized to the cytosol and mitochondrial matrix (9),
pared with both cytosol- and matrix-rxYFP. Assuming that the therefore deletion of this gene should have direct consequences
GSH concentration in this compartment is similar to the for both of these compartments. In contrast, there is very little
cytosol (i.e. 13 mM) (6), this value corresponds to a GSH:GSSG change in the percent oxidized IMS-rxYFP upon deletion of the
ratio of 250:1 (see Discussion). GLR1 gene (Fig. 5 and Table 1). We have previously shown that
The in vivo redox state of rxYFP was also used to generate Glr1 is not localized to the IMS (9), and these results support
redox potentials for each subcellular compartment using pub- that conclusion. Taken together, these experiments suggest
lished cytosol and matrix pH values (Table 1). Yeast cytosolic that redox control in the IMS is maintained separately from the
pH measurements for glucose-grown cells vary from 6.7 to 7.3 cytosol and mitochondrial matrix.

29130 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 NUMBER 43 OCTOBER 24, 2008
Redox Potential Measurements in Yeast Mitochondria
TABLE 1
GSH:GSSG ratios and redox potential measurements in the cytosol, mitochondrial matrix, and mitochondrial IMS of yeast cells
Cytosol Matrix IMS
Strains
Oxidized rxYFPa GSH:GSSGb Ec Oxidized rxYFP GSH:GSSG Ed Oxidized rxYFP GSH:GSSG Ec
% mV % mV % mV
WT 16 5 3000:1 286 5 38 9 900:1 296 5 68 4 250:1 255 3
glr1 74 2 200:1 252 2 84 3 100:1 267 3 62 10 300:1 259 6
a
The reported values of percent oxidized rxYFP are the mean S.D. for 3 8 independent experiments.
b
GSH:GSSG ratios were calculated using intracellular GSH 13 mM (6).
c
Cytosolic and IMS redox potentials were calculated from Equation 2 using ErxYFP 265 mV at pH 7.0 (6).
d
Matrix redox potentials were calculated from Equation 2 using ErxYFP 289 mV at pH 7.4.

Localized rxYFP Sensors Are Responsive to Redox Changes in


Distinct Subcellular CompartmentsWe next determined
whether the targeted rxYFP sensors are responsive to Glr1-
induced redox changes that specifically alter either cytosolic or
mitochondrial matrix redox status. The GLR1 gene encodes
both a cytosolic and mitochondrial form of Glr1 via two differ-
ent start codons in the mRNA sequence. Translation from the
first start codon (encoding M1) generates the long mitochon-
drial form with a mitochondrial targeting signal, while transla-
tion from the second start codon (encoding M17) generates a
shorter cytosolic form that lacks this signal peptide (9). Muta-
tion of the first Met to a Leu (M1L) results in exclusive expres-
sion of cytosolic Glr1, while mutation of Met-17 to a Leu
(M17L) primarily generates the mitochondrial form. However,
mitochondrial Glr1 is somewhat overexpressed in the M17L
mutant compared with WT, resulting in incomplete mitochon-
drial import. Consequently, some residual Glr1 is present in the
cytosol in M17L Glr1 strains (9).
To test whether rxYFP is responsive to redox changes within
distinct subcellular compartments, matrix-rxYFP, IMS-rxYFP,
or cytosol-rxYFP was expressed in glr1 yeast strains harboring
plasmid-encoded WT Glr1, no Glr1 (vector), cytosolic Glr1
(M1L), or mitochondrial Glr1 (M17L). When Glr1 is exclu-
sively localized to the cytosol (M1L), the oxidation state of the
cytosolic redox sensor is similar to cells expressing WT Glr1
(Fig. 6A, compare lanes 1 and 3), whereas the matrix sensor
measurements are similar to glr1 strains (compare lanes 2 and
3). This result confirms that the matrix and cytosolic sensors
are registering redox changes in distinct compartments.
Accordingly, expression of mitochondrial Glr1 (M17L) shifts
the matrix sensor equilibrium back toward the reduced form
(lane 4), which is similar to cells expressing WT Glr1 (lane 1).
However, the cytosolic sensor is also more reduced in this strain FIGURE 6. Cytosolic and mitochondrial GSH:GSSG pools are maintained
(lane 4) compared with glr1 with the vector control (lane 2). separately. The glr1 strain was doubly transformed with an rxYFP expres-
sion plasmid (pJH208 (cytosol-rxYFP), pLD207 (matrix-rxYFP), or pJH200 (IMS-
This observation can be explained by the fact that the M17L rxYFP)) and a Glr1 expression plasmid (pJH201 (WT Glr1), pRS413 (vector con-
Glr1 mutant exhibits some cytosolic localization due to ineffi- trol), pJH203 (M1L Glr1), or pJH202 (M17L Glr1)). Cells were grown to mid-log
cient mitochondrial import (9). This small cytosolic amount phase in selecting SC glucose media. A, redox Western blot and quantification
(B) were performed as described in Fig. 4. C, the percent of oxidized glutathi-
may be sufficient to maintain cytosolic GSH:GSSG in a more one (percent GSS/(GSH GSS)) was calculated from total GSH and GSSG
reduced form compared with the strain with no Glr1 expressed. levels in each extract. GSS 2XGSSG. For B and C, the reported values are the
mean of three independent experiments. Error bars are the means S.D.
To confirm the rxYFP sensor responses, we also measured
GSH and GSSG levels in cytosolic and whole mitochondrial
extracts using conventional subfractionation methods. As These extracts were assayed for GSH and GSSG by enzyme
pointed out in the introduction, this technique has several cycling and spectrophotometric analysis (9). We cannot assess
drawbacks, but it provides a rough estimation for comparison matrix and IMS GSH:GSSG separately with this method, so the
to the sensor measurements. The same yeast strains shown in data shown represent whole mitochondria. However, because
Fig. 6 (A and B) were subjected to lysis and subcellular fraction- the matrix volume is much larger than the IMS, these values are
ation to generate cytosolic and whole mitochondrial extracts. primarily a reflection of the matrix redox state. As shown in

OCTOBER 24, 2008 VOLUME 283 NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29131
Redox Potential Measurements in Yeast Mitochondria
Fig. 6C, direct measurement of GSH:GSSG showed a close cor- stergaard and coworkers have demonstrated that rxYFP
relation with the cytosol and matrix sensor readouts. The per- largely fulfills these requirements. It specifically registers the
cent oxidized GSSG was high in both the cytosol and mitochon- intracellular GSH:GSSG redox state via disulfide exchange
dria in glr1 strains with no Glr1 expressed, whereas reactions with cytosolic GRXs. This reaction has a similar equi-
expression of cytosolic Glr1 exclusively lowered cytosolic librium constant between pH 6.7 and 7.9 and occurs rapidly in
GSSG, but not mitochondrial matrix GSSG. Expression of pri- the cytosol (within 20 min) (6). However, pulse-chase analysis
marily mitochondrial matrix Glr1 (which also has some cytoso- revealed that steady-state rxYFP redox measurements slightly
lic Glr1) reduced mitochondrial matrix GSSG as well as cyto- underestimated the redox potential (by 6 10 mV) in rapidly
solic GSSG to some extent. However, the percent oxidized growing cells, because rxYFP is synthesized in the reduced
GSSG in the cytosol was not quite at WT levels in this M17L form. If the oxidation rate is comparable to the protein synthe-
strain. Likewise, our cytosolic sensor was slightly more oxidized sis rate, steady-state pools may be slightly skewed toward
in the M17L strain (Fig. 6B), indicating that it is sensitive to this reduced rxYFP (6).
small cytosolic redox potential difference between WT and Similarly, in the mitochondria, rxYFP is imported in the
M17L Glr1 strains. Overall, these results confirm that cytosol- reduced form and must interact with the local redox environ-
and matrix-rxYFP are functional and provide localized read- ment to become oxidized. Are GRXs available in the mitochon-
outs of the subcellular redox potential. drial matrix and IMS to catalyze the rapid equilibration of
The IMS sensor readout in glr1 mutants was quite different rxYFP? Grx1 and Grx2 are primarily localized in the cytosol,
from both the cytosol and matrix. As seen in Figs. 5 and 6, the however, a portion of Grx2 is also located in the mitochondrial
redox state of IMS-rxYFP did not change dramatically in a matrix (43), where it is available to catalyze equilibration of
glr1 strain. Accordingly, expression of WT or cytosolic Glr1 rxYFP with matrix GSH:GSSG pools. In fact, matrix-rxYFP and
(M1L) in a glr1 strain did not significantly alter the redox state GSH:GSSG measurements in Glr1 localization mutants indi-
of IMS-rxYFP. However, we observed a slight decrease in the cate a close correlation between the mitochondrial GSH:GSSG
percent oxidized IMS-rxYFP upon overexpression of matrix redox state and the matrix-rxYFP redox state (see Fig. 6, B and
Glr1 (M17L) compared with cytosolic Glr1. We postulated that C). Grx2 is also localized to the mitochondrial outer membrane,
overexpression of matrix Glr1 could cause some Glr1 to mislo- however the active site of this form is proposed to face the
calize to the IMS. However, using subcellular fractionation and cytosolic side (43). The presence of yeast Grx1 in the IMS has
Western blotting techniques we determined that M17L Glr1 not been addressed, but a recent publication suggests that a
was not found in IMS extracts (data not shown). Overall these fraction of human Grx1 is found in the IMS (44). Therefore, it is
results indicate that endogenous Glr1 expression does not possible that yeast Grx1 is available in the IMS to catalyze equil-
strongly influence the redox state of the mitochondrial IMS. ibration of IMS-rxYFP with the local GSH:GSSG pool. The fact
Additionally, our data suggest that the IMS redox state is largely that IMS-rxYFP is predominantly in the oxidized form argues
insulated from redox shifts that occur in both the matrix and that the equilibration rate is on the same order as the rate of
cytosol. protein synthesis and import. Future studies will focus on the
role of Grx1 and Grx2 in IMS redox control and the kinetics of
DISCUSSION IMS-rxYFP oxidation.
Developing Sensors for Mitochondria Redox Measurements Comparing the Redox State of the Cytosol and MatrixIn
Numerous studies have established that the mitochondrial WT cells, cytosol- and matrix-rxYFP were 16 and 38% oxidized,
redox state and the GSH:GSSG pool in particular play impor- respectively, reflecting substantial differences in the thiol-di-
tant roles in the cell (1, 40, 41). To measure GSH:GSSG in the sulfide equilibrium in these individual compartments. In the
matrix versus the IMS, we created GFP-based redox sensors mitochondrial matrix, the balance is shifted more toward disul-
that are separately targeted to these compartments. We tested fide formation than in the cytosol. This shift may reflect higher
the redox functionality of matrix- and IMS-rxYFP by verifying levels of ROS in the matrix versus the cytosol, which can oxidize
their in vivo response to addition of an oxidant (4-DPS) or free thiols to disulfides. Using published estimates of pH in the
reductant (DTT). Furthermore, we manipulated the subcellular yeast cytosol (7.0) (3336) and matrix (7.4) (36), the cyto-
GSH:GSSG ratio genetically via mutations in Glr1 that favor solic and matrix reduction potentials measured with rxYFP are
cytosolic versus mitochondrial expression of this reductase. 286 mV and 296 mV. Because reduction potential is
These results demonstrate that the mitochondrial and cytosolic strongly dependent on pH (4), the matrix value is actually more
rxYFP sensors are selectively registering redox variations reducing that the cytosol despite the higher percentage of oxi-
within subcellular compartments. dized sensor in this compartment. A similar trend was observed
Equilibration of IMS- and Matrix-rxYFP with Mitochondrial with in vivo redox measurements taken in mammalian cells.
GSH:GSSG PoolsTo obtain an accurate in vivo redox meas- The redox sensor roGFP1 was 16% oxidized in the cytosol (45)
urement, a targeted redox sensor must meet several require- and 33% oxidized in the matrix (46), yet the calculated matrix
ments: 1) the redox couple with which the sensor equilibrates redox potential (360 mV at pH 7.9) was more reducing than
must be clearly identified, 2) the sensor must rapidly reach the cytosol (315 mV at pH 7.0) due to the large pH difference
equilibrium with this couple, 3) the reduced and oxidized forms between these compartments.
of the sensor must have equivalent stabilities at different pH The IMS Redox State and Implications for IMS Thiol-disul-
values, and 4) the sensors redox potential must be tuned to fide EquilibriumThe critical role that thiol-disulfide balance
accommodate the subcellular redox environment (6, 42). plays in the IMS has been highlighted in some recent work on

29132 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 NUMBER 43 OCTOBER 24, 2008
Redox Potential Measurements in Yeast Mitochondria
TABLE 2
Predicted oxidation states of cysteine pairs in IMS-imported proteins in the cytosol versus the IMS
Protein Published E Ref. E at pH 7.0a Oxidation in cytosolb Oxidation in IMSb
mV mV %
Erv1 (C30-C33) 320 at pH 7.0 (57) 320 93 99
Tim10 (twin CX3C) 320 at pH 7.4 (58) 296 68 96
Tim9 (twin CX3C) 310 at pH 7.4 (52) 286 49 91
Cox17 (twin CX9C) 340 at pH 7.6 (54) 304 79 98
a
E values at pH 7.0 were calculated using Equation 3.
b
The percent oxidized protein in the cytosol and IMS was calculated from Equation 2 using Ecyto 286 mV and EIMS 255 mV from Table 1.

IMS protein import (for recent reviews see Refs. 15, 17, and 47). the IMS. By using IMS-rxYFP as a localized gauge of GSH:
Several IMS proteins with highly conserved cysteines are GSSG, as well as other markers of redox status, these factors will
imported through the outer membrane in a vectorial fashion via be identified in future studies.
the Erv1-Mia40 disulfide relay system (18, 48 50). This system The question still remains: what is oxidizing rxYFP and pre-
catalyzes disulfide bond formation in the imported proteins, sumably GSH:GSSG in the IMS? One potential clue may be the
which facilitates their folding and retention in the IMS. How lack of GSSG reductase (Glr1) in this compartment (9). With-
does the IMS redox state influence this process? Because porin out this antioxidant factor, the IMS may be prone to ROS-in-
channels in the outer mitochondrial membrane are thought to duced oxidation of reactive thiols. As demonstrated by redox
allow free exchange of small molecules like GSH and GSSG proteomic studies in yeast (55), molecular oxygen is the pri-
between the cytosol and IMS (51), a logical assumption is that mary source of oxidizing equivalents for cellular thiols, includ-
the redox state of these two compartments is similar. There- ing GSH. In the cytosol and matrix, Glr1 counteracts the oxi-
fore, it has been proposed that, once oxidized, these disulfide- dizing effects of aerobic metabolism by continuously catalyzing
containing IMS proteins must be resistant to reduction by GSH reduction of GSH to maintain a high GSH:GSSG ratio. Without
(52). The data presented herein provide an alternate explana- Glr1 in the IMS, GSSG produced via ROS oxidation may accu-
tion. Our redox measurements suggest that the IMS redox state mulate, leading to a lower GSH:GSSG ratio, which in turn influ-
in WT cells is more oxidizing (255 mV) compared with both ences the rxYFP redox state. In support of this argument, it is
the cytosol (286 mV) and matrix (296 mV). This redox envi- interesting to note that, in a glr1 mutant, the redox states of
ronment, in turn, may support the oxidative folding of IMS the cytosol and matrix are very similar to the IMS (see Table 1).
imported proteins, rather than opposing it. The redox poten- Future studies will determine whether factors that influence
tials of structural disulfides in several proteins imported via the
ROS production, such as oxygen tension (anaerobic versus aer-
Erv1-Mia40 system have been determined, thus allowing us to
obic growth), carbon source (i.e. fermentative versus non-fer-
test this argument by predicting the redox state of these cys-
mentative), and growth phase, alter the subcellular redox state.
teine pairs in the cytosol versus the IMS, assuming that they are
Independent Redox Control in Subcellular Compartments
in equilibrium with local GSH:GSSG pools. These predictions,
On a broader scale, our redox measurements provide some
shown in Table 2, indicate that the structural disulfide of Erv1-
interesting insights into redox control in subcellular compart-
Mia40 substrates are all 90% oxidized in the IMS, but their
ments. Our redox measurements in Glr1 localization mutants
putative oxidation state in the cytosol ranges from 46 to 92%
(Fig. 6) suggest that redox homeostasis is primarily maintained
oxidized. Whereas several structural studies on disulfide-con-
taining IMS proteins have proposed this type of redox differen- independently in the cytosol, matrix, and IMS, because the
tial between the cytosol and IMS (53, 54), we provide here direct thiol-disulfide balance in each of these compartments is quite
evidence for a more oxidizing IMS environment. different. Furthermore, alteration of the matrix redox state by
According to Equation 2, the IMS redox measurements abrogating Glr1 mitochondrial import has little influence on
reflect the [GSH]2/[GSSG] ratio, but do not provide the abso- the cytosolic and IMS redox state. We were unable to test the
lute concentration of GSH or the GSH:GSSG ratio. Therefore reciprocal experiment (selective expression of matrix Glr1)
the relatively oxidizing IMS environment may be due to a lower because our matrix Glr1 construct (M17L) exhibits some cyto-
GSH:GSSG ratio or lower overall GSH levels in this compart- solic localization due to inefficient mitochondrial import (see
ment compared with the cytosol. The latter possibility implies Ref. 9). However, we did observe that matrix Glr1 completely
that the IMS has a lower redox buffering capacity than the restores GSH:GSSG balance in the matrix but not the cytosol.
cytosol. Our measurements also assume that the IMS-rxYFP Interestingly, we also noted a subtle effect of matrix Glr1 over-
redox state is primarily influenced by GSH:GSSG pools. Alter- expression on the IMS redox state, because our IMS-rxYFP was
natively, rxYFP may interact with another redox pathway, such slightly less oxidized in these strains compared with cytosol
as the Mia40-Erv1 disulfide relay system, which may play a Glr1 expression. This results suggests that the matrix GSH:
more dominant role in this compartment than GSH:GSSG. GSSG pool has more influence over the IMS redox state than
However, previous studies have shown that this relay system cytosol GSH:GSSG pools. In fact, exchange of GSH between the
selectively interacts with substrates that possess a twin CX9C or matrix and IMS has been suggested to occur in purified rat liver
CX3C motif (18), which is absent in rxYFP. In any case, the mitochondria (56). The specific role that matrix versus cytoso-
factors that influence the IMS redox state are apparently more lic GSH:GSSG pools play in influencing the IMS redox state will
complex that simple diffusion of GSH:GSSG from the cytosol to be addressed in future studies.

OCTOBER 24, 2008 VOLUME 283 NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29133
Redox Potential Measurements in Yeast Mitochondria
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