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. 2008 Oct 24;283(43):29126-34.
doi: 10.1074/jbc.M803028200. Epub 2008 Aug 15.

The redox environment in the mitochondrial intermembrane space is maintained separately from the cytosol and matrix

Jingjing Hu  1 , Lixue Dong, Caryn E Outten
Affiliations

The redox environment in the mitochondrial intermembrane space is maintained separately from the cytosol and matrix

Jingjing Hu et al. J Biol Chem. .

Abstract

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 mitochondria. This approach allows us to separately monitor the redox state of the matrix and the IMS, providing a more detailed picture 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 mitochondrial isoforms of GSSG reductase. Our studies also demonstrate that the mitochondrial IMS is considerably more oxidizing 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.

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Figures

FIGURE 1.
FIGURE 1.
Mitochondria-targeted constructs of rxYFP. A, the gray box represents the rxYFP protein, the black coil represents the amphipathic helix required for matrix targeting, and the black box represents the hydrophobic sorting domain required for IMS targeting. The N termini of native mitochondrial proteins (Cox4 and Cyb2) were fused in-frame to rxYFP. B and C, schematics depicting import into the mitochondrial matrix (B) and IMS (C) via N-terminal targeting sequences. The targeting signals are cleaved during import by matrix and/or inner membrane (IM) proteases and the protein folds into its native conformation.
FIGURE 2.
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 A–C, 75 μg 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 μg of protein) from cells expressing matrix-rxYFP were further fractionated into IMS and mitoplast components, and the entire amount of each fraction was analyzed. In the right panel of C, mitochondria (15 μg 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 indicated. rxYFP-expressing plasmids utilized include: pJH208 (cytosol), pLD207 (matrix), and pJH200 (IMS).
FIGURE 3.
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.
FIGURE 4.
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.
FIGURE 5.
FIGURE 5.
rxYFP redox response in WT and glr1Δ cells. WT (BY4741) and glr1Δ (BY4741 glr1Δ) 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.
FIGURE 6.
FIGURE 6.
Cytosolic and mitochondrial GSH:GSSG pools are maintained separately. The glr1Δ strain was doubly transformed with an rxYFP expression plasmid (pJH208 (cytosol-rxYFP), pLD207 (matrix-rxYFP), or pJH200 (IMS-rxYFP)) and a Glr1 expression plasmid (pJH201 (WT Glr1), pRS413 (vector control), pJH203 (M1L Glr1), or pJH202 (M17L Glr1)). Cells were grown to mid-log 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 glutathione (percent GSS/(GSH + GSS)) was calculated from total GSH and GSSG 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.
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