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Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins¹

ALFRED HANSEL, LIOBA KUSCHEL, SOLVEIG HEHL, CORNELIUS LEMKE^*, HANS-JÜRGEN AGRICOLA, TOSHINORI HOSHI and STEFAN H. HEINEMANN²

Molecular and Cellular Biophysics, Medical Faculty of the Friedrich Schiller University Jena, D-07747 Jena, Germany;
^* Institute of Anatomy I, Friedrich Schiller University Jena, D-07743 Jena, Germany;
Institute of General Zoology and Animal Physiology, Friedrich Schiller University Jena, D-07743 Jena, Germany; and
Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

²Correspondence: Molecular and Cellular Biophysics, Medical Faculty of the Friedrich Schiller University Jena, Drackendorfer Str. 1, D-07747 Jena, Germany. E-mail: stefan.h.heinemann{at}uni-jena.de

SPECIFIC AIMS

The enzyme peptide methionine sulfoxide reductase (MSRA) catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine and is a repair mechanism for oxidatively damaged proteins. It may also be involved in regulatory processes. A prerequisite for meeting these specific functions is close physical contact with its various targets. The aim of this study was therefore to elucidate the subcellular localization of this enzyme.

PRINCIPAL FINDINGS

1. EGFP fusions of human MSRA (hMSRA) were transiently transfected into mammalian cells and localization was examined using laser scanning microscopy
Whereas hMSRA fused carboxyl-terminally to EGFP resulted in fluorescence evenly distributed in the transfected cells, hMSRA amino-terminally fused to EGFP showed a distinct localization (Fig. 1 a). Staining with organelle-specific dyes demonstrated that the enzyme is targeted to mitochondria. The localization was not dependent on the cell type. It could be observed in all cell lines used for transfection: human neuroblastoma (SH-SY5Y), lung (A549), embryonic kidney (HEK 293), liver (HEP-G2), melanoma (IGR-1), T cell leukemia (JURKAT), Chinese hamster ovary (CHO-K1), and Swiss mouse embryo (NIH-3T3) cell lines.

View larger version (102K): [in this window] [in a new window]   Figure 1. The amino-terminal 23 amino acids constitute the signal for mitochondrial targeting. The amino-terminal sequences of human (1), bovine (2), mouse (3), rat (4), and Escherichia coli MSRA (5) are aligned and constructs used for the localization experiments are indicated. SH-SY5Y cells were transfected with the following constructs: a) hMSRA-EGFP (image width/height: 52.2 µm); b) hMSRA(24–235)-EGFP (29.2 µm); c) hMSRA(1–22)-EGFP (29.2 µm); d) hMSRA(R6A*R7A)-EGFP (26.6 µm). Mitochondria were stained with MitoTracker Orange. Fusion of the first 23 amino acids of hMSRA to EGFP (b) resulted in the same mitochondrial localization as hMSRA-EGFP (a), whereas deletion of the hMSRA-NH2 terminus prevented targeting (c). Substitution of either Arg residue (indicated by arrows; R22A not shown) abolished mitochondrial localization as well (d).

2. The signal for the mitochondrial targeting resided within the amino-terminal 23 amino acids
Fusion of this amino-terminal sequence to EGFP led to a mitochondria-localized signal in transfected SH-SY5Y cells (Fig. 1 b), whereas removal of this putative signal sequence prevented localization of hMSRA-EGFP (Fig. 1 c).

3. Removal of the signal sequence did not affect the function of hMSRA
Full-length recombinant, His-tagged hMSRA and a version lacking the first 22 amino acids were active in an enzyme assay with a synthetic peptide containing methionine sulfoxide as a substrate. Reduction was monitored by analyzing the mass of the peptide using MALDI-TOF mass spectrometry.

4. Substitution of positively charged amino acids (R6, R7, R22) within the signal sequence with alanine residues led to an impairment of localization (Fig. 1) d

5. Disturbance of a hydrophobic stretch within the signal sequence led to a prevention of localization as well
In analogy to a variant of hMSRA found in the databases, which lacks three of four consecutive Leu residues (L11-L13), a mutant was constructed that displayed localization in only a few of the transfected cells (2 of 30 analyzed CHO-K1 cells).

6. All mammalian msrA genes known to date (human, rat, mouse, bovine) encode proteins with an amino-terminal extension showing high homology to the hMSRA signal peptide (Fig. 1)
Rat MSRA fused amino-terminally to EGFP was also targeted to the mitochondria in transiently transfected CHO-K1 and SH-SY5Y cells.

7. Mitochondrial targeting was verified in native tissue by detecting MSRA in rat and mouse liver slices with an hMSRA-specific antibody in relation to a mitochondria-specific anti-HSP60 antibody and fluorescence microscopy
Immunoperoxidase staining and electron microscopy showed that the enzyme resides in the mitochondria matrix.

CONCLUSIONS AND SIGNIFICANCE

Reactive oxygen species generated during aerobic metabolism have the potential to damage a variety of intracellular targets. The enzyme MSRA, which seems to be expressed in most living organisms, has been postulated to play an important role in the repair of oxidatively damaged proteins, as it can reduce methionine sulfoxide to methionine. Some targets of the enzyme, such as calmodulin or the 1-proteinase inhibitor, lose their function on oxidation of specific methionine residues. MSRA can revert this functional loss. Other enzymes such as glutamine synthetase do not lose their activity as long as only surface-exposed Met residues are oxidized. It was therefore suggested that cyclic oxidation/reduction of these Met residues might constitute a scavenger system for reactive oxygen species, which are produced intracellularly mainly through the respiratory chain. A disturbance of this oxidation/reduction balance may lead to accumulation of oxidized Met, which is observed during aging and under several pathological conditions. In fact, a decrease in MSRA activity is measurable in the brains of Alzheimer’s disease patients and in detoxifying organs of aged rats.

The oxidative state of specific Met residues may modulate the function of certain proteins, as was shown for the Drosophila Shaker C/B potassium channel. As MSRA has to come into close physical contact with its various targets for meeting these diverse functions, we studied the subcellular localization of the enzyme. We addressed this question using EGFP fusions of MSRA and found that the enzyme is not cytosolically localized, as previously postulated, but instead targeted to mitochondria. The signal for this targeting was located in the amino-terminal part of the protein, as the first 23 amino acids of the enzyme were sufficient for the targeting but dispensable for enzymatic activity. The full-length recombinant hMSRA and a deletion mutant lacking the first 22 amino acids were active.

Mitochondrial signal peptides tend to form amphiphilic helices. Elements important for the targeting were recently identified in these helices. Arginine residues located on the hydrophilic side of such a helix may interact with corresponding negative charges on the surface of TOM22 of the mitochondrial protein import machinery. Substitution of each of the three Arg residues consequently hampered localization of hMSRA. Hydrophobic Leu residues on the other side of the amphiphilic helix were shown to be crucial for the interaction with TOM20, another constituent of the outer membrane part of the mitochondrial protein import machinery. Removal of three of the four consecutive Leu residues of hMSRA impaired localization in most cells examined.

The mitochondrial localization may be typical for the family of mammalian MSRA enzymes characterized. The recently cloned rat MSRA was also localized in mitochondria when expressed as EGFP fusion protein. With an antibody raised against the recombinant hMSRA lacking its NH₂ terminus, we localized the enzyme directly in mouse and rat liver slices using fluorescence microscopy. The localization could be verified in colocalization experiments using an antibody against the chaperone protein HSP60 that was shown to be located in mitochondria. Staining was also visible in the cytoplasm. Immunoperoxidase staining and electron microscopy demonstrated that the enzyme is located in the mitochondria matrix.

In mitochondria, the site of intensive ROS production, hMSRA may exert a function to prevent or repair oxidative damage (Fig. 2 ). Cyclic oxidation/reduction of Met residues may reduce the ROS release from mitochondria. Met oxidation at specific targets and its reduction by MSRA may modulate mitochondrial functions such as Ca²⁺ signaling.

View larger version (21K): [in this window] [in a new window]   Figure 2. Putative function of MSRA in mitochondria. a) Oxygen radicals (O2•) are generated inside mitochondria during respiration; resulting ROS will locally oxidize methionines and may be released into the cytosol. b) By reduction of free or protein-bound methionine sulfoxide (MO), MSRA may take part in a cyclic oxidation/reduction process leading to a decrease in ROS release from mitochondria.

It remains elusive how the mitochondria-localized MSRA enzyme can reduce methionine sulfoxide in other subcellular locations. In Arabidopsis thaliana, two different MSRA variants were detected with 70% homology to each other. One of these enzymes is targeted to the plastids; the other is located in the cytoplasm. Such functional variants have not yet been characterized in mammalian species. The staining seen with hMSRA-specific antibodies may indicate the presence of such MSR isoforms in the cytoplasm.

In conclusion, we demonstrate that MSRA is targeted to mitochondria, where it may function in reducing the amount of ROS released from these organelles.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0737fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0737fje.

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