THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 43, pp.
29126 29134, October 24, 2008
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
functioning of this organelle. Disruption of mitochondrial
redox processes contributes to a host of human disorders,
including cancer, neurodegenerative diseases, and aging. To
better characterize redox control pathways in this organelle, we
have targeted a green fluorescent protein-based redox sensor to
the intermembrane space (IMS) and matrix of yeast mitochon-
dria. This approach allows us to separately monitor the redox
state of the matrix and the IMS, providing a more detailed pic-
ture of redox processes in these two compartments. To verify
that the sensors respond to localized glutathione (GSH) redox
changes, we have genetically manipulated the subcellular redox
state using oxidized GSH (GSSG) reductase localization
mutants. These studies indicate that redox control in the cytosol
and matrix are maintained separately by cytosolic and mito-
chondrial isoforms of GSSG reductase. Our studies also demon-
strate that the mitochondrial IMS is considerably more oxidiz-
ing than the cytosol and mitochondrial matrix and is not directly
influenced by endogenous GSSG reductase activity. These redox
measurements are used to predict the oxidation state of thiol-
containing proteins that are imported into the IMS.
Maintenance of the thiol-disulfide balance in cells is critical
for the proper functioning of numerous enzymes and proteins
with functionally important cysteine residues. The cellular
redox balance can be disrupted by unregulated production of
reactive oxygen species (ROS)2 that interfere in redox signaling
pathways and oxidatively damage DNA, proteins, and lipids (1).
To control the cellular redox environment, cells contain two
primary redox regulatory systems that utilize thiol-disulfide
redox chemistry: the glutathione (GSH)/glutathione disulfide
(GSSG) redox couple and the reduced/oxidized thioredoxin
redox couple (1, 2). The tripeptide glutathione (-glutamylcys-
* 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
of South Carolina Center for Colon Cancer Research. The costs of publica-
tion of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, 631 Sumter St., University of South Carolina, Columbia, SC
29205. Tel.: 803-777-8783; Fax: 803-777-9521; E-mail: caryn.outten@
chem.sc.edu.
2
The abbreviations used are: ROS, reactive oxygen species; GSH, reduced
glutathione; GSSG, oxidized glutathione; IMS, intermembrane space; GFP,
green fluorescent protein; rxYFP, redox-sensitive yellow fluorescent pro-
tein; GRX, glutaredoxin; PMS, post-mitochondrial supernatant; DAPI, 4,6-
diamidino-2-phenylindole; 4-DPS, 4,4-dithiodipyridine; DTT, dithiothrei-
tol; Cyb2, cytochrome b2; WT, wild type.
29126 JOURNAL OF BIOLOGICAL CHEMISTRY
2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
teinylglycine) and the small protein thioredoxin can serve as
reductants themselves or as cofactors for anti-oxidant enzymes
(3). Glutathione is considered the primary determinant of the
cellular redox environment, because it has a relatively low
redox potential (240 mV at pH 7.0) and a high intracellular
abundance (113 mM) (4).
Measurements of GSH:GSSG levels in subcellular compart-
ments demonstrate that individual organelles have different
redox requirements. The endoplasmic reticulum maintains a
relatively oxidizing environment (170 to 185 mV at pH 7.0,
or a GSH:GSSG ratio of 1:1 to 3:1) (5), whereas the cytosol is
quite reducing in comparison (290 mV at pH 7.0, or a GSH:
GSSG ratio of 3300:1) (6). GSH:GSSG measurements in iso-
lated mitochondria indicate a redox potential of 250 mV to
280 mV at pH 7.8 or GSH:GSSG ratios of 20:1 to 40:1 (710).
However, measuring the GSH:GSSG redox state in isolated
mitochondria has several drawbacks. First, GSH:GSSG levels in
the matrix and the intermembrane space (IMS) cannot be
measured separately, because the IMS is quite small (5% of
the total mitochondria volume), making it difficult to effectively
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
lost or exchanged during the mitochondrial isolation procedure
thereby altering the physiology and redox state of the organelle.
Nevertheless, defining redox control in the IMS is critical
given the various redox-dependent pathways in this compart-
ment, including apoptotic signaling (11, 12), assembly of respi-
ratory chain components (13), anti-oxidant activation (14), and
protein import (15). It is not known if the redox state of this
compartment is relatively oxidizing or reducing in comparison
to the mitochondrial matrix and cytosol. On the one hand, this
compartment is phylogenetically linked to the oxidizing
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
outer membrane presumably allow free exchange of GSH and
GSSG between the IMS and cytosol (15, 19), suggesting that the
GSH:GSSG redox state in the IMS is similar to the reducing
cytosol.
An in vivo method for measuring the subcellular redox state
of GSH:GSSG is an effective approach to address redox control
in individual compartments. stergaard and coworkers (6)
have developed a genetically encoded, cytosolic redox sensor
based on the yellow variant of green fluorescent protein (GFP)
called redox-sensitive YFP (rxYFP). GFP and its derivatives
provide ideal scaffolds for creating in vivo sensors due to their
protease resistance and high stability in a broad range of pH and
VOLUME 283 NUMBER 43 OCTOBER 24, 2008

buffer conditions (20). The rxYFP protein in particular can be
used to measure the redox potential in live cells via formation of
an engineered disulfide bond that perturbs the local chro-
mophore environment without significantly altering the overall
-can fold (21). The relative ratio of oxidized to reduced rxYFP
can also be assessed via non-reducing SDS-PAGE in which the
two forms have different electrophoretic mobilities. stergaard
and coworkers (6) have shown both in vivo and in vitro that the
cysteines in rxYFP specifically equilibrate with GSH and GSSG
via rapid disulfide exchange reactions with the cytosolic glu-
taredoxins (GRXs). In contrast, the two cytosolic thioredoxins
are unable to undergo thiol-disulfide exchange with this sensor.
The ratio of oxidized to reduced rxYFP measured in the cell can
be used to generate an in vivo readout of the GSH:GSSG redox
state. Their measurements indicated that the ratio of GSH:
GSSG in the eukaryotic cytosol was 3300:1, which corresponds
to a redox potential of 289 mV (6). This value is considerably
more reducing than whole cell redox measurements (221 to
236 mV) (5), highlighting the importance of examining sub-
cellular compartments separately.
In this study we have modified rxYFP for expression in the
mitochondrial IMS and matrix of the yeast Saccharomyces cer-
evisiae to compare redox differences between these compart-
ments and the cytosol. We have demonstrated exclusive target-
ing of IMS- and matrix-rxYFP to these compartments and have
confirmed their dynamic response to an exogenous oxidant and
reductant. We have measured the IMS redox potential, demon-
strating that this compartment is considerably more oxidizing
than the cytosol or matrix. Furthermore, we have manipulated
the subcellular redox potential using GSSG reductase mutants
to verify that the sensors respond to specific intracellular redox
changes that are localized to subcellular compartments. Over-
all, our data suggest that redox control is independently regu-
lated within these individual compartments in the cell.
EXPERIMENTAL PROCEDURES
Yeast Strains, Media, and Growth ConditionsS. cerevisiae
strains used in this study were BY4741 (MATa his31 leu20
met150 ura30) and BY4741 glr1::kanMX4 obtained from
Research Genetics. Yeast transformations were performed by
the lithium acetate procedure (22). Strains were maintained at
30 C on either enriched yeast extract-peptone-based medium
supplemented with 2% glucose (YPD) or synthetic complete
medium (SC) supplemented with 2% glucose or 2% galactose
and the appropriate amino acids.
PlasmidsThe plasmid pJH208 expressing cytosol-rxYFP
was constructed by digesting the rxYFP yeast expression plas-
mid pHOJ150 (6) with NotI and SacII. The NotI-SacII-digested
fragment containing the PGK1 promoter, the coding sequence
of rxYFP, and the TDH3 terminator was then inserted into the
URA3-CEN vector pRS316 (23).
The IMS-rxYFP plasmid pJH200 was constructed as follows.
An NdeI site downstream of the rxYFP coding sequence in
pHOJ150 was removed by site-directed mutagenesis forming
pJH300, which leaves one NdeI site at the start codon for rxYFP.
The mitochondrial targeting sequence of S. cerevisiae cyto-
chrome b2 (Cyb2) (codons for amino acids 1 88) was amplified
by PCR introducing an NheI site and an NdeI site at the N and
OCTOBER 24, 2008 VOLUME 283 NUMBER 43
Redox Potential Measurements in Yeast Mitochondria
C terminus, respectively. The PCR fragment was inserted in-
frame at the N terminus of rxYFP at the SpeI and NdeI sites of
pHOJ150, forming pJH200.
The matrix-rxYFP plasmid pLD207 was constructed by
amplifying the promoter region and mitochondrial targeting
signal (codons for amino acids 125) of cytochrome oxidase
subunit IV (COX4) with primers that introduced NotI site and
NdeI sites at the 5 and 3 ends, respectively. The PCR fragment
was inserted in-frame at the N terminus of rxYFP at the NotI
and NdeI sites of pJH300. The COX4 promoter was later
replaced with the manganese superoxide dismutase (SOD2)
promoter by creating a SpeI site just upstream of the Cox4
mitochondrial targeting signal. The COX4 promoter was
removed by digestion with NotI and SpeI and a PCR fragment
containing SOD2 promoter was inserted in its place. This plas-
mid was then digested with NotI and SacII, and the fragment
containing the SOD2 promoter, the coding sequence of COX4-
rxYFP, and the TDH3 terminator was inserted into the URA3-
CEN vector pRS316, yielding pLD207.
The GLR1-expressing plasmids pJH201 (WT Glr1), pJH202
(M17L Glr1), and pJH203 (M1L Glr1) were created by inserting
the GLR1 gene sequence from the plasmids pCO113 (WT),
pCO114 (M17L), or pCO116 (M1L) (9) into the SacI and XhoI
sites of the HIS3-CEN vector pRS413 (23). The sequence integ-
rity of all plasmids was verified by double-stranded DNA
sequencing (USC Environmental Genomics Core Facility).
Subcellular FractionationYeast cells were grown aerobi-
cally to mid-log phase in selecting SC medium with 2% galac-
tose. Mitochondrial and post-mitochondrial supernatant
(PMS) fractions were obtained as previously described by con-
verting cells to spheroplasts followed by gentle lysis by Dounce
homogenization and differential centrifugation (24). Mito-
chondrial intermembrane and mitoplast fractions were pre-
pared by osmotic shock as previously described (25). Mitoplasts
and IMS fractions were subsequently treated with 50 g/ml
proteinase K in the absence or presence of 0.2% Triton X-100.
Immunoblotting TechniquesYeast extracts were subjected
to electrophoresis on Tris-glycine gels (Invitrogen) and ana-
lyzed by Western blotting using an anti-rxYFP antibody (kind
gift of J. Winther) or an anti-GFP antibody (Invitrogen) and a
secondary anti-rabbit IgG (IRDye, LI-COR Lincoln, NE). PMS
fractions were monitored by anti-3-phosphoglycerate kinase
(PGK1) antibodies (Invitrogen). Mitochondrial fractions were
monitored by using antibodies directed against Cyb2 in the
IMS, mitochondrial processing protease Mas2 in the matrix
(kind gifts of R. Jensen), or mitochondrial NADH kinase
Pos5 in the matrix (26). Proteins were analyzed by Western
blot using an Odyssey Infrared Imaging System (LI-COR).
Protein concentrations were determined using the Bradford
method (Bio-Rad) with bovine serum albumin as the calibra-
tion standard.
Fluorescence MicroscopyThe BY4741 parental strain trans-
formed with pJH208 (cytosol-rxYFP), pJH200 (IMS-rxYFP), or
pLD207 (matrix-rxYFP) was grown aerobically to mid-log
phase in selecting SC medium containing 2% galactose. Cells
were incubated with 2.5 g/ml 4,6-diamidino-2-phenylindole
(DAPI) (Invitrogen) for 30 min to stain mitochondrial DNA.
Live cells were examined with a Zeiss LSM 510 META Confocal
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 2. Subcellular localization of cytosol- (A), matrix- (B), and IMS-
rxYFP (C). WT yeast cells (BY4741) expressing cytosol-, matrix-, or IMS-rxYFP
were grown to mid-log phase in SC galactose media. Cells were lysed and
fractionated, and fractions were analyzed by SDS-PAGE and immunoblotting
using antibodies directed against rxYFP, PGK1 (cytosol marker), Pos5 or Mas2
(mitochondrial matrix markers), or Cyb2 (mitochondrial IMS marker). In the
left panel of AC, 75 g of total cell protein (Total) was fractionated into post-
mitochondrial supernatant (PMS) and mitochondria (Mito), and the entire
amount of each fraction was analyzed. In the right panel of B, mitochondria
(15 g of protein) from cells expressing matrix-rxYFP were further fraction-
ated into IMS and mitoplast components, and the entire amount of each
fraction was analyzed. In the right panel of C, mitochondria (15 g of protein)
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-
cated. rxYFP-expressing plasmids utilized include: pJH208 (cytosol), pLD207
(matrix), and pJH200 (IMS).
IMS targeting sequence includes an additional hydrophobic
sorting domain, which is cleaved by an inner membrane prote-
ase (Fig. 1C) (31).
To confirm the correct localization of the rxYFP constructs,
we conducted a Western blot analysis of crude mitochondria
and largely cytosolic (PMS) fractions from WT strains trans-
formed with cytosol-, matrix-, or IMS-rxYFP. As shown in Fig.
2A, cytosol-rxYFP is localized to the cytosol as previously deter-
mined (6). In contrast, matrix-rxYFP and IMS-rxYFP are local-
ized to mitochondria (Fig. 2, B and C, left panels). The molecu-
lar weights of the rxYFP bands in Fig. 2 (B and C) are consistent
with the mature, processed forms of both sensors. Mitochon-
dria were further fractionated into intermembrane space and
mitoplasts, revealing that matrix-rxYFP is correctly localized to
the matrix (Fig. 2B, right panel), whereas IMS-rxYFP is located
in the soluble IMS fraction (Fig. 2C, right panel, lane 5). How-
ever, a portion of IMS-rxYFP is also found associated with
mitoplasts (Fig. 2C, lane 2). To determine whether IMS-rxYFP
in this fraction is located on the outside (IMS side) or inside
(matrix side) of the mitoplast membrane, proteinase K was
added to intact mitoplasts to digest exposed (IMS side) pro-
teins. IMS-rxYFP was largely degraded by this protease (Fig. 2C,
lane 3) in a similar manner to soluble IMS-rxYFP (Fig. 2C, lane
6), whereas the control matrix protein Pos5 (26) was unaffected.
The Pos5 matrix control is only digested (indicated by a change
in molecular weight) upon addition of Triton X-100, which dis-
rupts the mitoplast membrane allowing access to matrix pro-
teins (Fig. 2C, lane 4). In contrast, proteolysis of mitoplast-
associated IMS-rxYFP is similar with and without the addition
of Triton X-100 (compare Fig. 2C, lanes 3 and 4), indicating that
OCTOBER 24, 2008 VOLUME 283 NUMBER 43
FIGURE 3. Differential interference contrast (DIC) and fluorescence
microscopy of WT yeast cells (BY4741) expressing cytosol-, matrix, or
IMS-rxYFP. Cells were grown to mid-log phase in SC galactose media and
incubated with DAPI for 30 min to stain DNA. Live cells were examined with
Zeiss LSM 510 META confocal scanning laser microscope at a magnification of
605. Merge merged images of DAPI and YFP fluorescence with white areas
indicating overlap. Plasmids and strains are the same as in Fig. 2.
it is located on the outside of the mitochondrial inner mem-
brane in the IMS compartment. We note that, unlike the Cyb2
control, rxYFP in the soluble IMS fraction is only partially
digested (Fig. 2C, lane 6), even though all proteins in this frac-
tion are soluble and therefore accessible to proteinase K. This
observation may be explained by the fact that GFP-based pro-
teins have an unusually stable core structure that is resistant to
complete digestion by proteases (20).
Localization and fluorescence signal of the rxYFP sensors
were further verified by fluorescence microscopy. Live yeast
cells expressing cytosol-, matrix-, or IMS-rxYFP were stained
with DAPI for co-localization experiments. DAPI staining for
DNA highlights both mitochondria (string-like structures) and
the nucleus (large, rounded structure) as shown in Fig. 3.
Matrix- and IMS-rxYFP specifically co-localized with DAPI
staining of the mitochondrial, but not nuclear DNA, indicating
that these sensors are exclusively localized to the mitochondria
and are correctly folded with the expected fluorescence
properties.
Matrix- and IMS-rxYFP Register Dynamic Subcellular Redox
ChangesTo test the reactivity and response of the targeted
rxYFP sensors, a strong oxidant or reductant was added to cells
expressing cytosol-, matrix-, or IMS-rxYFP. The redox state of
rxYFP can be monitored by redox Western blot or fluorescence
spectroscopy as described for cytosol-rxYFP (6). Both of these
techniques avoid the artificial oxidation problems and lengthy
subcellular fractionation steps encountered by conventional
methods for measuring subcellular redox potential.
The redox Western blots shown in Fig. 4 indicate that the
mitochondrial rxYFP constructs are responsive to redox
changes generated by exogenous reagents, as previously dem-
onstrated for cytosol-rxYFP (6). Addition of the thiol-oxidant
4-DPS or the disulfide reductant DTT shifts all three sensors
toward the oxidized state (Fig. 4A, lane 2) or reduced state (Fig.
4A, lane 3), respectively. Fluorescence measurements were also
performed using live cells expressing cytosol-, matrix-, and
IMS-rxYFP. The fluorescence response of cells expressing
cytosol-rxYFP upon addition of DTT or 4-DPS was similar to
JOURNAL OF BIOLOGICAL CHEMISTRY 29129
Redox Potential Measurements in Yeast Mitochondria
FIGURE 4. rxYFP redox response to an exogenous oxidant or reductant.
WT yeast cells expressing cytosol-, matrix-, or IMS-rxYFP were grown to mid-
log phase in SC glucose media. Cells were treated with 4-DPS or DTT as
described under Experimental Procedures. A, redox Western blot of the
samples separated by non-reducing SDS-PAGE and immunoblotted with
anti-GFP antibodies. B, reduced (red) and oxidized (ox) forms of rxYFP were
quantified using an Odyssey Infrared Imaging System. The reported values
are the mean of three to four independent experiments. Error bars are the
means S.D. Plasmids and strains are the same as in Fig. 2.
published reports (6) (data not shown). However, fluorometer
measurements of whole cells expressing matrix- or IMS-rxYFP
were more difficult to obtain over the background autofluores-
cence. This difficulty may be explained by the relatively small
mitochondrial volume of these cells, which constitutes only
3% of the total cell volume under these growth conditions
(32).
The IMS Redox State Is More Oxidizing Than the Matrix or
CytosolUsing the differently targeted versions of rxYFP, we
compared the redox state in the cytosol, mitochondrial matrix,
and mitochondrial IMS. As shown in Fig. 5 and Table 1, cytosol-
rxYFP is 16% oxidized under steady-state conditions, which is
similar to published results (6), whereas matrix-rxYFP is 38%
oxidized. Inserting these values into Equation 2 and assuming
an intracellular [GSH] of 13 mM (6), we estimate that the GSH:
GSSG ratio in the cytosol and matrix are 3000:1 and 900:1,
respectively (Table 1). Interestingly, IMS-rxYFP is significantly
more oxidized (68%) under steady-state conditions com-
pared with both cytosol- and matrix-rxYFP. Assuming that the
GSH concentration in this compartment is similar to the
cytosol (i.e. 13 mM) (6), this value corresponds to a GSH:GSSG
ratio of 250:1 (see Discussion).
The in vivo redox state of rxYFP was also used to generate
redox potentials for each subcellular compartment using pub-
lished cytosol and matrix pH values (Table 1). Yeast cytosolic
pH measurements for glucose-grown cells vary from 6.7 to 7.3
29130 JOURNAL OF BIOLOGICAL CHEMISTRY
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,
because this matrix pH value was reported for yeast cells grown
in glucose (36). Using these values, the cytosolic redox potential
corresponds to 286 mV, whereas the matrix redox potential is
296 mV (Table 1). Studies in mammalian cells suggest that
the IMS pH is typically 0 0.7 pH units lower than cytosolic pH
(37, 38). By using pH 7.0 as a conservative estimate, the IMS
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
increase by 6.0 mV for every 0.1 unit of decrease in pH (see
Equation 3). Therefore it is possible that the IMS redox state is
even more oxidizing than our estimate at pH 7.0. In any case,
these values are considerably more oxidizing than both the
cytosol and mitochondrial matrix.
Deletion of GLR1 Alters Redox Status in the Cytosol and
Matrix but Not the IMSTo examine how the GSH:GSSG ratio
influences redox status within submitochondrial compart-
ments, we tested the sensors in glr1 mutant cells that lack the
GSSG reductase gene (GLR1). Because this enzyme is respon-
sible for regenerating GSH from GSSG, deletion of this gene
creates a higher than normal intracellular GSSG:GSH ratio (6,
9, 39). As shown in Fig. 5 and Table 1, cytosol-rxYFP undergoes
dramatic redox changes (74% oxidized) in glr1 mutant cells
compared with WT cells (16% oxidized), as previously dem-
onstrated (6). Likewise, the matrix-rxYFP sensor shifts from
38% oxidized to 84% oxidized in WT versus glr1 strains.
Glr1 is localized to the cytosol and mitochondrial matrix (9),
therefore deletion of this gene should have direct consequences
for both of these compartments. In contrast, there is very little
change in the percent oxidized IMS-rxYFP upon deletion of the
GLR1 gene (Fig. 5 and Table 1). We have previously shown that
Glr1 is not localized to the IMS (9), and these results support
that conclusion. Taken together, these experiments suggest
that redox control in the IMS is maintained separately from the
cytosol and mitochondrial matrix.
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-
relation with the cytosol and matrix sensor readouts. The per-
cent oxidized GSSG was high in both the cytosol and mitochon-
dria in glr1 strains with no Glr1 expressed, whereas
expression of cytosolic Glr1 exclusively lowered cytosolic
GSSG, but not mitochondrial matrix GSSG. Expression of pri-
marily mitochondrial matrix Glr1 (which also has some cytoso-
lic Glr1) reduced mitochondrial matrix GSSG as well as cyto-
solic GSSG to some extent. However, the percent oxidized
GSSG in the cytosol was not quite at WT levels in this M17L
strain. Likewise, our cytosolic sensor was slightly more oxidized
in the M17L strain (Fig. 6B), indicating that it is sensitive to this
small cytosolic redox potential difference between WT and
M17L Glr1 strains. Overall, these results confirm that cytosol-
and matrix-rxYFP are functional and provide localized read-
outs of the subcellular redox potential.
The IMS sensor readout in glr1 mutants was quite different
from both the cytosol and matrix. As seen in Figs. 5 and 6, the
redox state of IMS-rxYFP did not change dramatically in a
glr1 strain. Accordingly, expression of WT or cytosolic Glr1
(M1L) in a glr1 strain did not significantly alter the redox state
of IMS-rxYFP. However, we observed a slight decrease in the
percent oxidized IMS-rxYFP upon overexpression of matrix
Glr1 (M17L) compared with cytosolic Glr1. We postulated that
overexpression of matrix Glr1 could cause some Glr1 to mislo-
calize to the IMS. However, using subcellular fractionation and
Western blotting techniques we determined that M17L Glr1
was not found in IMS extracts (data not shown). Overall these
results indicate that endogenous Glr1 expression does not
strongly influence the redox state of the mitochondrial IMS.
Additionally, our data suggest that the IMS redox state is largely
insulated from redox shifts that occur in both the matrix and
cytosol.
DISCUSSION
Developing Sensors for Mitochondria Redox Measurements
Numerous studies have established that the mitochondrial
redox state and the GSH:GSSG pool in particular play impor-
tant roles in the cell (1, 40, 41). To measure GSH:GSSG in the
matrix versus the IMS, we created GFP-based redox sensors
that are separately targeted to these compartments. We tested
the redox functionality of matrix- and IMS-rxYFP by verifying
their in vivo response to addition of an oxidant (4-DPS) or
reductant (DTT). Furthermore, we manipulated the subcellular
GSH:GSSG ratio genetically via mutations in Glr1 that favor
cytosolic versus mitochondrial expression of this reductase.
These results demonstrate that the mitochondrial and cytosolic
rxYFP sensors are selectively registering redox variations
within subcellular compartments.
Equilibration of IMS- and Matrix-rxYFP with Mitochondrial
GSH:GSSG PoolsTo obtain an accurate in vivo redox meas-
urement, a targeted redox sensor must meet several require-
ments: 1) the redox couple with which the sensor equilibrates
must be clearly identified, 2) the sensor must rapidly reach
equilibrium with this couple, 3) the reduced and oxidized forms
of the sensor must have equivalent stabilities at different pH
values, and 4) the sensors redox potential must be tuned to
accommodate the subcellular redox environment (6, 42).
29132 JOURNAL OF BIOLOGICAL CHEMISTRY
stergaard and coworkers have demonstrated that rxYFP
largely fulfills these requirements. It specifically registers the
intracellular GSH:GSSG redox state via disulfide exchange
reactions with cytosolic GRXs. This reaction has a similar equi-
librium constant between pH 6.7 and 7.9 and occurs rapidly in
the cytosol (within 20 min) (6). However, pulse-chase analysis
revealed that steady-state rxYFP redox measurements slightly
underestimated the redox potential (by 6 10 mV) in rapidly
growing cells, because rxYFP is synthesized in the reduced
form. If the oxidation rate is comparable to the protein synthe-
sis rate, steady-state pools may be slightly skewed toward
reduced rxYFP (6).
Similarly, in the mitochondria, rxYFP is imported in the
reduced form and must interact with the local redox environ-
ment to become oxidized. Are GRXs available in the mitochon-
drial matrix and IMS to catalyze the rapid equilibration of
rxYFP? Grx1 and Grx2 are primarily localized in the cytosol,
however, a portion of Grx2 is also located in the mitochondrial
matrix (43), where it is available to catalyze equilibration of
rxYFP with matrix GSH:GSSG pools. In fact, matrix-rxYFP and
GSH:GSSG measurements in Glr1 localization mutants indi-
cate a close correlation between the mitochondrial GSH:GSSG
redox state and the matrix-rxYFP redox state (see Fig. 6, B and
C). Grx2 is also localized to the mitochondrial outer membrane,
however the active site of this form is proposed to face the
cytosolic side (43). The presence of yeast Grx1 in the IMS has
not been addressed, but a recent publication suggests that a
fraction of human Grx1 is found in the IMS (44). Therefore, it is
possible that yeast Grx1 is available in the IMS to catalyze equil-
ibration of IMS-rxYFP with the local GSH:GSSG pool. The fact
that IMS-rxYFP is predominantly in the oxidized form argues
that the equilibration rate is on the same order as the rate of
protein synthesis and import. Future studies will focus on the
role of Grx1 and Grx2 in IMS redox control and the kinetics of
IMS-rxYFP oxidation.
Comparing the Redox State of the Cytosol and MatrixIn
WT cells, cytosol- and matrix-rxYFP were 16 and 38% oxidized,
respectively, reflecting substantial differences in the thiol-di-
sulfide equilibrium in these individual compartments. In the
mitochondrial matrix, the balance is shifted more toward disul-
fide formation than in the cytosol. This shift may reflect higher
levels of ROS in the matrix versus the cytosol, which can oxidize
free thiols to disulfides. Using published estimates of pH in the
yeast cytosol (7.0) (3336) and matrix (7.4) (36), the cyto-
solic and matrix reduction potentials measured with rxYFP are
286 mV and 296 mV. Because reduction potential is
strongly dependent on pH (4), the matrix value is actually more
reducing that the cytosol despite the higher percentage of oxi-
dized sensor in this compartment. A similar trend was observed
with in vivo redox measurements taken in mammalian cells.
The redox sensor roGFP1 was 16% oxidized in the cytosol (45)
and 33% oxidized in the matrix (46), yet the calculated matrix
redox potential (360 mV at pH 7.9) was more reducing than
the cytosol (315 mV at pH 7.0) due to the large pH difference
between these compartments.
The IMS Redox State and Implications for IMS Thiol-disul-
fide EquilibriumThe critical role that thiol-disulfide balance
plays in the IMS has been highlighted in some recent work on
VOLUME 283 NUMBER 43 OCTOBER 24, 2008

TABLE 2
Predicted oxidation states of cysteine pairs in IMS-imported proteins in the cytosol versus the IMS
Protein Published E
mV
Erv1 (C30-C33) 320 at pH 7.0
Tim10 (twin CX3C) 320 at pH 7.4
Tim9 (twin CX3C) 310 at pH 7.4
Cox17 (twin CX9C) 340 at pH 7.6
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).
Several IMS proteins with highly conserved cysteines are
imported through the outer membrane in a vectorial fashion via
the Erv1-Mia40 disulfide relay system (18, 48 50). This system
catalyzes disulfide bond formation in the imported proteins,
which facilitates their folding and retention in the IMS. How
does the IMS redox state influence this process? Because porin
channels in the outer mitochondrial membrane are thought to
allow free exchange of small molecules like GSH and GSSG
between the cytosol and IMS (51), a logical assumption is that
the redox state of these two compartments is similar. There-
fore, it has been proposed that, once oxidized, these disulfide-
containing IMS proteins must be resistant to reduction by GSH
(52). The data presented herein provide an alternate explana-
tion. Our redox measurements suggest that the IMS redox state
in WT cells is more oxidizing (255 mV) compared with both
the cytosol (286 mV) and matrix (296 mV). This redox envi-
ronment, in turn, may support the oxidative folding of IMS
imported proteins, rather than opposing it. The redox poten-
tials of structural disulfides in several proteins imported via the
Erv1-Mia40 system have been determined, thus allowing us to
test this argument by predicting the redox state of these cys-
teine pairs in the cytosol versus the IMS, assuming that they are
in equilibrium with local GSH:GSSG pools. These predictions,
shown in Table 2, indicate that the structural disulfide of Erv1-
Mia40 substrates are all 90% oxidized in the IMS, but their
putative oxidation state in the cytosol ranges from 46 to 92%
oxidized. Whereas several structural studies on disulfide-con-
taining IMS proteins have proposed this type of redox differen-
tial between the cytosol and IMS (53, 54), we provide here direct
evidence for a more oxidizing IMS environment.
According to Equation 2, the IMS redox measurements
reflect the [GSH]2/[GSSG] ratio, but do not provide the abso-
lute concentration of GSH or the GSH:GSSG ratio. Therefore
the relatively oxidizing IMS environment may be due to a lower
GSH:GSSG ratio or lower overall GSH levels in this compart-
ment compared with the cytosol. The latter possibility implies
that the IMS has a lower redox buffering capacity than the
cytosol. Our measurements also assume that the IMS-rxYFP
redox state is primarily influenced by GSH:GSSG pools. Alter-
natively, rxYFP may interact with another redox pathway, such
as the Mia40-Erv1 disulfide relay system, which may play a
more dominant role in this compartment than GSH:GSSG.
However, previous studies have shown that this relay system
selectively interacts with substrates that possess a twin CX9C or
CX3C motif (18), which is absent in rxYFP. In any case, the
factors that influence the IMS redox state are apparently more
complex that simple diffusion of GSH:GSSG from the cytosol to
OCTOBER 24, 2008 VOLUME 283 NUMBER 43
Redox Potential Measurements in Yeast Mitochondria
Ref. E at pH 7.0a Oxidation in cytosolb Oxidation in IMSb
mV %
(57) 320 93 99
(58) 296 68 96
(52) 286 49 91
(54) 304 79 98
the IMS. By using IMS-rxYFP as a localized gauge of GSH:
GSSG, as well as other markers of redox status, these factors will
be identified in future studies.
The question still remains: what is oxidizing rxYFP and pre-
sumably GSH:GSSG in the IMS? One potential clue may be the
lack of GSSG reductase (Glr1) in this compartment (9). With-
out this antioxidant factor, the IMS may be prone to ROS-in-
duced oxidation of reactive thiols. As demonstrated by redox
proteomic studies in yeast (55), molecular oxygen is the pri-
mary source of oxidizing equivalents for cellular thiols, includ-
ing GSH. In the cytosol and matrix, Glr1 counteracts the oxi-
dizing effects of aerobic metabolism by continuously catalyzing
reduction of GSH to maintain a high GSH:GSSG ratio. Without
Glr1 in the IMS, GSSG produced via ROS oxidation may accu-
mulate, leading to a lower GSH:GSSG ratio, which in turn influ-
ences the rxYFP redox state. In support of this argument, it is
interesting to note that, in a glr1 mutant, the redox states of
the cytosol and matrix are very similar to the IMS (see Table 1).
Future studies will determine whether factors that influence
ROS production, such as oxygen tension (anaerobic versus aer-
obic growth), carbon source (i.e. fermentative versus non-fer-
mentative), and growth phase, alter the subcellular redox state.
Independent Redox Control in Subcellular Compartments
On a broader scale, our redox measurements provide some
interesting insights into redox control in subcellular compart-
ments. Our redox measurements in Glr1 localization mutants
(Fig. 6) suggest that redox homeostasis is primarily maintained
independently in the cytosol, matrix, and IMS, because the
thiol-disulfide balance in each of these compartments is quite
different. Furthermore, alteration of the matrix redox state by
abrogating Glr1 mitochondrial import has little influence on
the cytosolic and IMS redox state. We were unable to test the
reciprocal experiment (selective expression of matrix Glr1)
because our matrix Glr1 construct (M17L) exhibits some cyto-
solic localization due to inefficient mitochondrial import (see
Ref. 9). However, we did observe that matrix Glr1 completely
restores GSH:GSSG balance in the matrix but not the cytosol.
Interestingly, we also noted a subtle effect of matrix Glr1 over-
expression on the IMS redox state, because our IMS-rxYFP was
slightly less oxidized in these strains compared with cytosol
Glr1 expression. This results suggests that the matrix GSH:
GSSG pool has more influence over the IMS redox state than
cytosol GSH:GSSG pools. In fact, exchange of GSH between the
matrix and IMS has been suggested to occur in purified rat liver
mitochondria (56). The specific role that matrix versus cytoso-
lic GSH:GSSG pools play in influencing the IMS redox state will
be addressed in future studies.
JOURNAL OF BIOLOGICAL CHEMISTRY 29133
Redox Potential Measurements in Yeast Mitochondria
Overall, the existence of independent GSH:GSSG pools in
subcellular compartments parallels previous studies on the sep-
arate nature of NADPH pools in the cytosol and mitochondrial
matrix (26). Taken together, these experiments suggest that the
redox status of the mitochondrial matrix, IMS, and cytosol are
influenced separately by factors that are specifically targeted to
these compartments.
AcknowledgmentsWe thank Jakob Winther (University of Copen-
hagen) for providing rxYFP antibodies and technical advice, Jakob
Winther and the Carlsberg Laboratory (Copenhagen Valby, Den-
mark) for providing the rxYFP expression vector pHOJ150, and Robert
Jensen (Johns Hopkins University School of Medicine) for providing
Cyb2 and Mas2 antibodies. We also thank F. Wayne Outten for crit-
ical reading of the manuscript.
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