A conserved R type Methionine Sulfoxide Reductase reverses oxidized GrpEL1/Mge1 to regulate Hsp70 chaperone cycle

Cells across evolution employ reversible oxidative modification of methionine and cysteine amino acids within proteins to regulate responses to redox stress. Previously we have shown that mitochondrial localized methionine sulfoxide reductase (Mxr2) reversibly regulates oxidized yeast Mge1 (yMge1), a co-chaperone of Hsp70/Ssc1 to maintain protein homeostasis during oxidative stress. However, the specificity and the conservation of the reversible methionine oxidation mechanism in higher eukaryotes is debatable as human GrpEL1 (hGrpEL1) unlike its homolog yMge1 harbors two methionine residues and multiple cysteines besides the mammalian mitochondria hosting R and S types of Mxrs/Msrs. In this study, using yeast as a surrogate system, we show that hGRPEL1 and R type MSRs but not the S type MSRs complement the deletion of yeast MGE1 or MXR2 respectively. Our investigations show that R type Msrs interact selectively with oxidized hGrpEL1/yMge1 in an oxidative stress dependent manner, reduce the conserved hGrpEL1-Met146-SO and rescue the Hsp70 ATPase activity. In addition, a single point mutation in hGrpEL1-M146L rescues the slow growth phenotype of yeast MXR2 deletion under oxidative duress. Our study illustrates the evolutionarily conserved formation of specific Met-R-SO in hGrpEL1/yMge1 and the essential and canonical role of R type Msrs/Mxrs in mitochondrial redox mechanism.

system can undergo early stress dependent structural transition. Oxidative and thermal stresses are known to change the ratio between active dimeric Mge1 to inactive monomeric form 14,15 . We have shown earlier that the conserved methionine at 155 th position in Mge1 responds to oxidative stress. In addition, mitochondrial localized methionine sulfoxide reductase 2 (Mxr2) reversibly regulates Mge1 by selectively reducing the Met155-SO to restore the activity of Mge1. Although, Mxr2 reduces the Met-SO of yeast Mge1 both in vitro and in vivo, our earlier study does not preclude the formation of only R type sulfoxide upon oxidation.
In contrast to yeast, mammalian mitochondria contain two isoforms of R type (MsrB2 and MsrB3) and one isoform of S type (MsrA) sulfoxide reductases 5,6 . In addition, two isoforms of GrpEL (GrpEL1 and GrpEL2) are present in mammalian mitochondria. GrpEL1 and GrpEL2 are differentially expressed across all tissues and high levels of GrpEL1 protein has been detected in many tissues 16,17 . To further increase the complexity, mammalian GrpEL1 contains multiple cysteines and two methionine residues (position 44 and 146) while its counterpart in yeast, Mge1 lacks cysteines and contains only one methionine (position 155). It is remains to be explored whether the MsrB mediated redox switch at conserved GrpEL1 methionine residue is required for GrpEL1 function and subsequent regulation of Hsp70/Ssc1p cycle.
In this study, we show that both the methionine residues in human GrpEL1 get oxidized upon exposure to H 2 O 2 . However, the conserved oxidized methionine at 146 is specifically reduced by R type of methionine sulfoxide reductase in vitro. Using an yeast heterologous system, we show that human hGRPEL1 and human R type MSR complement the deletion of yeast MGE1 and MXR2 respectively. Yeast cells expressing human GrpEL1-M146L mutant conferred better growth kinetics than yeast strain expressing wild type hGrpEL1 under oxidative stress. This study delineates the function of human GrpEL1 and R type Msrs in redox regulation besides the evolutionarily conserved role of Mge1/GrpEL1 in mitochondrial oxidative stress response pathway.

hGrpEL1 responds to oxidative stress and alters the ATPase stimulating activity of Hsp70/Ssc1 in vitro.
Mge1 is known to be oxidized at conserved Met155 amino acid residue both in vitro and in vivo upon exposure to oxidative stress. Human GrpEL1 contains methionine residue at 44 th and 146 th position and the latter one is analogous to yMge1 Met155. To test whether hGrpEL1 is oxidized at conserved methionine residue like yMge1, purified recombinant hGrpEL1 was treated with or without H 2 O 2 , separated on SDS-PAGE, Coomassie stained and trypsin digested fragments were analyzed by MALDI-TOF-MS/MS (Supplementary Figure S2A-D). In the absence of H 2 O 2 , Met146 containing peptides were resolved as a major un-oxidized 1764 Da mass and as a minor oxidized 1780 Da mass with a difference of 16 Da (Fig. 1A). In contrast, H 2 O 2 treated hGrpEL1 displayed a relatively higher form of oxidized Met146 peptide compared to the un-oxidized Met146 peptide (Fig. 1B). MS/ MS analysis of the aforementioned peptides confirmed that Met also exists as Met-SO by attaining a mass of 16 Da with H 2 O 2 treatment ( Fig. E and F). Examination of the Met44 containing peptides revealed that Met44 also exists in two forms, the minor un-oxidized 1794 Da form and the major oxidized 1810 form, the latter becoming the more dominant form in the presence of H 2 O 2 ( Fig. 1C and D). Interestingly, Met44 amino acid is located in un-structured N-terminal region whereas Met146 residue is present in two helix bundle domain of Mge1 (Fig. 1G).
To test the significance of oxidized hGrpEL1, we performed an ATPase activity of purified yeast mitochondrial His-yHsp70/Ssc1in the presence of Mge1 or hGrpEL1 with or without H 2 O 2 treatment as described in the methods. In the absence of GrpEL1/Mge1 proteins, the total yHsp70/Ssc1 mediated ATP hydrolysis in the reaction is minimal (Fig. 2). In the presence of yMge1 or hGrpEL1, the ATPase activity of yHsp70/Ssc1 is enhanced by 3-4 folds. However, the ATPase activity of yHsp70/Ssc1 is reduced when yMge1 or hGrpEL1 was treated with H 2 O 2 prior to its addition in the assay (Fig. 2). This result suggests that oxidation of GrpEL1 protein might have physiological consequences on the chaperone activity of yHsp70/Ssc1. hGRPEL1 complements the deletion of yeast MGE1. Mge1 is an essential protein and it has been shown that human GRPEL1 does not complement the deletion of MGE1 deletion 18 . To investigate if the function of MGE1 is conserved across evolution, we initially examined if over-expression of hGRPEL1 could complement yMGE1 chromosomal deletion. Using plasmid shuffling, we created yeast strains deleted for chromosomal MGE1 but over-expressing yMGE1 or hGRPEL1 from a high copy plasmid as described in the methods 19 . The growth phenotypes of strains expressing yMGE1 and hGRPEL1 were compared on YPD and YPGE plates. Strain expressing hGRPEL1 had growth that was comparable to the strain expressing yMGE1 on YPD and YPGE plates at 30 °C ( Fig. 3A and B). To ensure the protein expression of hGRPEL1, we carried out immunoblot analysis of cell lysates obtained from strains expressing yMGE1 and hGRPEL1 as described in the Methods section. Immunoblotting studies using antibodies specific against Mge1 and hGrpEL1 confirmed the presence of Mge1 and GrpEL1 (Fig. 3C). Mge1 was not detected in the strain expressing hGRPEL1 confirming that the strain indeed had chromosomal deletion of MGE1 (Fig. 3C). Our results clearly demonstrate that over expression of hGRPEL1 can efficiently complement yMGE1 deletion.
Human Methionine R sulfoxide Reductase complements yeast MXR2 deletion and interacts with oxidized hGrpEL1. Oxidation of methionine can result in two forms of enantiomers, Met-R-SO or Met-S-SO. The protein bound Met-SO enantiomers are known to be specifically reduced by either MsrA or MsrB enzymes 20,21 . Mammalian mitochondria contain three methionine sulfoxide reductases that include two R type (MsrB2 and MsrB3) and one S type (MsrA) sulfoxide reductases. As hGrpEL1 is sensitive to oxidative stress in vitro, we wished to examine if the Met SO modification is reversible, akin to Mge1.
It has been shown that mammalian MSRB can complement the deletion of yeast MXR2 22 . Initially, we analyzed the importance of mitochondrial localized mammalian Msrs during oxidative stress by specifically targeting them to yeast mitochondria. We employed mitochondrial pre-sequence, N-terminal Su-9 MTS, upstream   of MSRB3 and MSRA gene sequence to target them to mitochondria. We constructed yeast strains as described in the methods for the expression of human mitochondrial methionine sulfoxide reductases, Su9-MTS-MsrA, Su9-MTS-MsrB3, MsrB2 and yeast Mxr2 with Flag epitopes in a mxr2Δ background. The parent strain, WT was transformed with an empty vector and used as a control strain. We compared the growth of Flag-Msr expressing strains with the control strain on SC-URA in the presence and absence of H 2 O 2 . All the strains had comparable growth on SC-URA plates in the absence of H 2 O 2 . However, in the presence of H 2 O 2 , strains expressing human MsrB2 or MsrB3 or yeast Mxr2 had growth phenotype similar to control strain (Fig. 4A). However, growth of strain expressing mammalian MsrA is severely hampered in the presence of H 2 O 2 . These results suggest that only Met-R-SO reducing enzymes can efficiently complement the MXR2 deletion. To confirm the mitochondrial  localization of the human mitochondrial Msrs in yeast, subcellular fractionation of yeast strains expressing different Flag-Msr was carried out and the fractions were resolved on SDS-PAGE, blotted and probed with antibodies specific to the mitochondria fraction (Aconitase), cytosolic faction (Glycerol kinase) and to the Flag epitope. As shown in the Fig. 4, Flag-Msr proteins are enriched in the mitochondrial fraction like Aconitase ( Fig. 4B-D). These results suggest that human Flag-Msr proteins are efficiently targeted to the yeast mitochondria and only the R type of human Msrs are able to complement the deletion of yeast MXR2.
To determine the specificity of methionine sulfoxide enantiomers, we performed interaction studies between oxidized GrpEL1 and Msr proteins by in vitro Ni-NTA pull down assay as described 19 . The cell lysates expressing mammalian and yeast Flag-Msr proteins were passed through Ni-NTA beads pre-bound with recombinant His-GrpEL1 pre-treated with increasing concentrations of H 2 O 2 . To ascertain specificity, only Ni-NTA beads without His-GrpEL1 was used as control. The beads were washed and the proteins bound to Ni-NTA beads were resolved on SDS-PAGE and blotted as described in the Methods section (Fig. 5). Immunoblotting was carried out using antibodies specific to Flag, GrpEL1 and His tag. Our results show that binding of MsrB2, MsrB3 and Mxr2 depends on the oxidation of GrpEL1 (Fig. 5A,B and D). Most importantly, in the absence of H 2 O 2 , the binding of GrpEL1 to the Msrs or Mxr2 is very low. In addition, human mitochondrial MsrA and yeast cytosolic Mxr1 fail to bind to GrpEL1 irrespective of H 2 O 2 treatment ( Fig. 5C and E). To ascertain the binding activity of MsrA, we repeated the Ni-NTA pull down assay using MRP6, a known substrate of Msrs instead of GrpEL1. We observe a specific interaction of MsrA with MRP6 pre-treated with H 2 O 2 (Fig. 5F). Our results provide evidence that Met R specific reductases either from yeast or human are able to specifically interact with GrpEL1 in an oxidation dependent manner potentially indicating a physiological role for the binding.
Reduction of hGrpEL1-Met SO at Met146 by R type enzymes. In vitro pull down assays described above clearly demonstrate that methionine sulfoxide R reductases bind with relatively greater affinity to the oxidized form of GrpEL1/Mge1 than to its reduced form. In contrast, the S type of methionine sulfoxide reductases are unable to interact with either form of GrpEL1/Mge1. To understand if the interaction between the R type Msrs and the oxidized form of GrpEL1 have functional consequences, we utilized MALDI/TOF analysis to examine the oxidized state of GrpEL1 in the presence of Msrs. As observed earlier, majority of the recombinant GrpEL1 protein is oxidized with H 2 O 2 treatment. The oxidation state is reflected by the appearance of peptides with masses 1780 Da and 1810 Da that correspond to oxidized Met146 and Met44 peptides respectively ( Fig. 6A and B). Strikingly, when oxidized GrpEL1 was further incubated with the R type of reductases (MsrB2 or MsrB3), we observe a selective reduction in the percentage of oxidized 1780 Da peptide with a concomitant increase in the reduced Met1764 Da peptide ( Fig. 6C and D). However, there is no efficient reduction in the 1810 Da peptide that harbors the oxidized Met44 amino acid ( Fig. 6E and F). MsrA had no effect on the MALDI spectrum of oxidized GrpEL1 (Fig. 6G and H). Consistent with mammalian R type Msrs, yeast Mxr2 is able to efficiently reduce oxidized GrpEL1 at Met146 and not Met44 ( Fig. 6I and J), a site that corresponds to Met155 in its canonical Next, we wished to test the functional relevance of oxidized GrpEL1 reduced by MsrB at Met146 in an ATPase stimulating activity of yHsp70/Ssc1. The results from this assay will also allow us to differentiate the effect of Met44/Met146 oxidation in the influence of ATPase stimulating activity by GrpEL1. We performed ATPase stimulating activity of GrpEL1 on yHsp70/Ssc1 as described in the Methods section. We pre-treated recombinant GrpEL1 with or without H 2 O 2 prior to incubating it with yeast or mammalian Msrs. After the incubation step with Msrs, the ATPase stimulating activity was measured as described in Methods. yHsp70 alone exhibits a low intrinsic ATPase activity (Fig. 6K). Addition of recombinant GrpEL1 stimulates ATPase activity of yHsp70 by four folds while oxidized GrpEL1 does not have any significant effect. Prior incubation of oxidized GrpEL1 with R type of Msr's (MsrB2, MsrB3 and yMxr2) restores the ATPase stimulating activity of GrpEL1 (Fig. 6K). However, pretreatment of oxidized GrpEL1 with S epimer reducing enzyme, MsrA has no effect in augmenting the ATPase activity of yHsp70 by GrpEL1. Our studies thus far, strongly suggest that the observed binding between oxidized GrpEL1 and the R type Msrs irrespective of their origin have a functional consequence as evident from the restoration of the ATPase stimulating activity of a 'reduced' GrpEL1 by Msr enzymes. hGRPEL1 M146L rescues the oxidative sensitive phenotype of yeast MXR2 deletion. Ectopically expressed Mge1 is able to complement the chromosomal deletion of MGE1. We have shown previously that an oxidative resistant mutant, MGE1 M155L is able to complement both MGE1 and MXR2 deletions under oxidative stress 15,19 . To probe if GrpEL1 M146L can functionally mimic Mge1 M155L mutant, we generated yeast strains that ectopically express either wild type hGRPEL1 or hGRPEL1 M146L in a mxr2Δ and mge1Δ background as described in the Methods section. The growth phenotype of strains expressing either hGRPEL1 or h GRPEL1 M146L were compared to their parent wild type strain with vector alone as control in the presence and absence of H 2 O 2 on SC-Leu plates. Yeast strains expressing wild type and mutant GrpEL1 grew normally in the absence of H 2 O 2 while GrpEL1 M146L mutant displayed better growth on minimal plates in presence of H 2 O 2 (Fig. 7A). Similar growth resistant phenotype of hGrpEL1 M146L was observed in liquid cultures with H 2 O 2 (Fig. 7B). These results indicate GrpEL1 is sensitive to oxidation in vivo under normal physiological conditions. We have shown earlier that mxr2Δ strain fails to grow on non-fermentable carbon sources while MGE1 M155L can complement the growth defect. Similarly, we find that GRPEL1 M146L is able to grow on non-fermentable carbon source when compared to the strain expressing WT GRPEL1 in mxr2Δ strain background (Fig. 7A right panel  and 7C). Further, we have observed no change in the steady state levels of WT GrpEL1, GrpEL1 M146L and other mitochondrial proteins in yeast strains expressing either WT hGrpEL1 or hGrpELl M146L mutant in mxr2Δ background (Fig. 7D, Supplementary Figure S3). Together, our results provide compelling evidence to the existence of an evolutionarily conserved mechanism that is orchestrated by mitochondrial proteins Mxr2-Mge1-Ssc1 to regulate cellular redox homeostasis.

Discussion
Molecular Chaperones are essential components of cellular protein homeostasis and belong to a highly conserved protein family. Most of the chaperones assist in protein folding by utilizing ATP as an energy source 10,23 . Certain chaperones display stress dependent expression and 24 are also regulated by post translational modifications. Very few chaperones have been reported to be regulated through reversible oxidative modifications at cysteine or methionine residues. It has been shown that thermal stress induces the expression of Hsp33 gene and additionally regulates its activity through a redox mechanism by forming disulfide bond at its zinc binding site 25,26 . Bacterial Hsp70 undergoes reversible glutathionylation in response to thermal and oxidative stress 27 . In eukaryotes, oxidation of Cys residues within cytosolic Hsp70 and Hsp90 chaperones result in their inactivation and induction of Unfolded Protein Response (UPR) 24,28 . In addition, Met and Cys oxidation in the eye lens chaperone, α-Crystallins is thought to be one of the major causes of eye defects 29 .
Most of the mitochondrial matrix targeted proteins are nuclear encoded, translated in cytosol and imported into mitochondria as pre-proteins 30 . Hsp70/Ssc1 chaperone is a component of the import motor that plays an essential role in pulling the proteins across inner membrane into mitochondrial matrix. Hsp70/Ssc1 chaperone system is also required for mitochondrial proteostasis, iron sulfur cluster biogenesis and in mtDNA maintenance 31 . Mge1/GrpEL1 is a conserved essential subunit of Hsp70/Ssc1 machinery and is majorly required for the efficiency of ATPase cycle. Dimeric GrpEL1 structure has two long N-terminal α-helix regions, a small four-helix bundle region with antiparallel topological arrangement followed by a C-terminal β-sheet domain 32,33 . Several point mutations in the four helix bundle of E.coli GrpE and yeast Mge1 are known to cause severe growth defects and loss of Ssc1 dependent functions 15,34 . Methionine 155 in yMge1 and the corresponding 146 in hGrpEL1, both resident in the four helix bundle region, are sensitive to oxidative stress and are conserved across eukaryotes. Yeast Mxr2 interacts with Mge1 in an oxidative stress dependent manner and restores Mge1 dependent Hsp70/ Ssc1 functions both in vitro and in vivo 15,19 . In this report, we show that hGrpEL1 modulates the ATPase activity of yHsp70/Ssc1 during oxidative stress (Fig. 2). In mammals, two isoforms of GrpEL (GrpEL1 and GrpEL2) are present in the mitochondria and their presence is ubiquitous. Our results show that hGrpEL1 but not hGrpEL2 is able to complement yMGE1 deletion indicating that it is probably playing a role similar to Mge1 in regulating the activity of Hsp70/Ssc1 chaperone ( Fig. 3 and unpublished results). Interestingly, hGrpEL2 does not contain methionine corresponding to the conserved Met155 position of yeast Mge1. Oxidation resistant mutant, hGr-pEL1 M146L is able to rescue the growth defect associated with mxr2Δ strain in presence of H 2 O 2 indicating the conserved role of GrpEL1 during oxidative stress (Fig. 7).
Msrs act as antioxidants to regulate various biological processes. Msrs reduce Met-R-SO and Met-S-SO oxidized derivatives of methionine. They are evolutionarily conserved, and human mitochondria contain MsrA, MsrB2 and MsrB3 isoforms. MsrBs display a preference to proteins that have Met R SO oxidized form rather than Met-S-SO 5 . Our previous and current in vitro data suggest that oxidized hGrpEL1/Mge1is specifically reduced by R epimer reducing enzymes. Although both methionines in GrpE1 are oxidized in vitro, we find Met44 to be pre-oxidized during most of our experimental conditions. This may be due to auto-oxidation that has been reported earlier 35 . Intriguingly, we have not observed a reduction of oxidized Met44. Met44 is present in the un-structured tails of GrpEL1 that is part of the non-conserved region and not essential for GrpEL1 dimerization nor for stimulating Hsp70/Ssc1 ATPase activity. MsrB isoform shows preference towards M146-SO over Met44-SO (Figs 5 and 6) in reducing the sulfoxide. Additionally, only R type of human MSR isoforms can complement the loss of yMXR2 under oxidative conditions. Based on the above results, we believe that the conserved oxidized methionine in Mge1 and GrpEL1 is most likely to be the R epimer form. Besides mitochondria, MsrB3 is also localized to the ER in mammals 36 . Interestingly, we find that MSRB3 lacking the upstream Su9-MTS sequence fails to complement MXR2 deletion under oxidative stress (unpublished results). This shows that ER targeted MxrB3 has no apparent role in mitochondria.
Further, we find that hGRPEL1 M146L mutant complements the slow growth phenotype of MXR2 deleted cells (Fig. 7). Curiously, we find that wild type strain grows significantly much faster than cells devoid of MXR2 but harboring hGrpEL1 M146L in liquid cultures when compared to plate based assay. It has been shown that good aeration exacerbates the growth and viability defect caused by deletion of superoxide dismutase (SOD), while this difference from wild type strain is mitigated under low aeration 37 . Good aeration increases the release of ROS in mitochondria. We believe that this same effect is seen in the case of absence of MXR2. In liquid cultures where there is good aeration, the effect of MXR2 deletion is exacerbated. In contrast, in a plate assay that causes lower aeration, hGrpEL1 M146 mutant even in absence of MXR2 exhibits better growth characteristics. Additionally, cells harboring WT hGrpEL1 exhibit a growth phenotype that is akin to cells harboring hGrpEL1 M146L at later time points in liquid culture. This may due to the diminishing amount of H 2 O 2 with time.
Mitochondria accounts for 90% of total cellular ROS. Physiological levels of ROS regulate protein functions, however, increased ROS can damage various biomolecules. Excessive buildup of oxidized (reversible and irreversible) macromolecule aggregates promote aging 38  decreased antioxidant capacity of cell can contribute to imbalances in ROS production 1,39,40 . Methionine oxidation, loss of Msrs and increased mitochondrial ROS have been implicated in several neurological disorders like Parkinson's and Alzheimer's diseases including aging. We hypothesize that GrpEL1 oxidation might be playing a key role in Msr associated pathologies. Age dependent accumulation of ROS or decreased antioxidant property or altered Msr activities can lead to increased Met SO of GrpEL1. The cascading effects of Met oxidation of Mge1/ GrpEL1 include defective chaperone system and altered protein homeostasis that further aggravate several mitochondrial associated disease conditions. Mitochondrial hGrpEL1 perhaps acts like a redox sensor that transduces appropriate signals for mitochondrial protein homeostasis. The amplified products were cloned into pNB543 as BamHI -HindIII fragments to generate pNB639 and pNB643 containing MSRA and MSRB3 respectively. Since MSRB2 contains internal BamHI site, we amplified full length gene with primers MSRB2-Fwd2 and MSRB2-Rev2. Amplified gene was digested with NcoI and HindIII and cloned into SpeI and HindIII predigested plasmid pNB475 19 to express MSRB2 full length protein with FLAG epitope. Similarly, GRPEL1 WT was sub-cloned into pTEF LEU plasmid to generate plasmid pNB598 with Su9 MTS for yeast expression. Pet28a + carrying GRPEL1-M146L (pNB555) was created by site-directed mutagenesis of GRPEL1 present in pNB243 using primers GRPEL1_Fwd (5′GGG CTG GTC CTG ACT GAA GTC 3′) and GRPEL1 Rev (5′-GAC TTC AGT CAG GAC CAG CCC-3′). The mutant GRPEL1 was further amplified using pNB555 as template and sub-cloned into pTEF LEU plasmid to generate pNB605 with Su9-MTS.

Protein expression and purification. Plasmids containing His-MSRA, His-MSRB2, His-MSRB3 and
His-MRP6 genes were transformed into E. coli BL21 (DE3) Codon Plus (RIL) cells. Protein expression was induced with 1 mM IPTG and soluble proteins were further purified by using Ni-NTA talon affinity resin (GE Healthcare). Purified proteins were dialyzed in phosphate buffered saline (PBS) pH 7.2 with 5 mM β-ME. Similarly, hGrpEL1 wild type protein was expressed, purified and dialyzed in PBS. yMxr2, yMge1 and Hsp70/ Ssc1 proteins were expressed and purified as described earlier 15,19 . All the purified proteins were separated on SDS-PAGE to assess the purity (Supplementary Figure S4).

Yeast strain construction.
Yeast strain yNB65 is deleted for chromosomal MGE1 but has MGE1 expressed ectopically from a high copy URA3 plasmid (14). yNB158 and yNB159 were created by plasmid shuffling pNB598 (hGRPEL1 in a high copy LEU2 plasmid) and pNB605 (hGRPEL1 M146L high copy LEU2) respectively into yNB65 and evicting the URA3 plasmid by growing the transformants on 5 FOA. The transformants were further confirmed by checking the loss of viability on 5-FOA and growth on SC-URA plates ( Supplementary Fig. 1), by PCR for MGE1 deletion and by immunoblot analysis for hGrpEL1. yNB126 is deleted for chromosomal copies of MXR2 and MGE1 while ectopically expressing MGE1 from a high copy URA3 plasmid 15 . The URA3 plasmid in yNB126 was evicted by growing the PNB598 and PNB605 transformants on 5 FOA to generate yNB138 and yNB139 expressing hGRPEL1 and hGRPEL1 M146L respectively. yNB117 is deleted for chromosomal MXR2 19 and was used to generate yNB145, yNB146 and yNB147 by transforming it with URA3 high copy plasmids pNB639 (MSRA), pNB641 (MSRB3) and pNB645 (MSRB2) respectively. The strains were selected by growth on SC-URA plates and confirmed by immunoblot analysis with anti-FLAG antibody.
Yeast media and Growth assay. Standard conditions were used for culturing and maintaining of yeast strains. To evict URA3 plasmid, yeast strains were plated on SC medium containing 5 FOA (0.67% SC-URA, 2% dextrose, 50 μg/ml uracil, 2% agar and 0.1% 5-FOA). For performing growth assays, yeast strains were freshly streaked onto a YPD plates and the resultant colonies were grown in YPD medium overnight. These cultures were normalized to OD 600 1.0 and subjected to a ten-fold serial dilution. 5 µl from each dilution was spotted on SD-Leu plates with or without 1 mM H 2 O 2 and on YPGE plates (1% yeast extract. 2% peptone, 3% glycerol and 2% ethanol). Images were taken after 2 days of incubation in case of SD plates or 3-4 days in case of YPGE plates at 30 °C.
In vitro interaction assay. 100 μg Mitochondrial lysate from the strains expressing FLAG-MsrA, FLAG-MsrB2, FLAG-MsrB3, FLAG-Mxr2 or 100 μg cytosol lysate from the strain expressing FLAG-Mxr1 were used for in vitro interaction studies with or without H 2 O 2 treated 10 μg of His-hGrpEL1 or His-Mrp6 proteins. His-hGrpEL1 and His-Mrp6 were allowed to bind to Ni-NTA beads and the lysates were passed through. Ni-NTA beads were washed 3 times with 10 mM imidazole buffer. The beads were collected, suspending in SDS sample buffer and separated on SDS-PAGE. The gels were transferred for immunoblot analysis. In vitro reduction of oxidized hGrpEL1 by Msrs. Enzymatic activities of Msrs were initially checked on methionine rich Mrp6 protein pre-treated with H 2 O 2 followed by reduction in a Trx coupled system as described 41 . Oxidized GrpEL1 was incubated with Msr proteins in a reaction buffer containing 50 mM sodium phosphate, 50 mM sodium chloride and 10 mM DTT and separated on SDS-PAGE. The gel was Coomassie stained and the GrpEL1 bands were excised and subjected to trypsin digestion prior to mass spectrometric analysis.
Hsp70/Ssc1 single turnover ATPase assay. Hsp70/Ssc1 ATPase activity assay was performed as described earlier 15 with minor modifications. yMge1 and hGrpEL1 proteins were pre-treated with or without 5 mM H 2 O 2 and dialyzed. 5 μg of yMge1 or hGrpEL1 were added to the reaction buffer (50 mM HEPES/ KOH, pH 7.2, 5 mM MgCl 2 , and 100 mM KCl) containing 2 µg of yHsp70/Ssc1 and 0.05 mM [γ-32 P] ATP (3000 Ci/mmol). The effect of Msr on Hsp70/Ssc1 ATPase activity via GrpEL1 was also monitored by incubating oxidized hGr-pEL1 with 5 μg of Msr protein in presence of 10 mM DTT for 30 min prior to the Hsp70/Ssc1 assay. The amount of radioactive inorganic phosphate (pi) released after 5 min was measured in a scintillation counter as described 15 . Data was analyzed using Jandel scientific sigma software by one way ANOVA followed by post hoc Duncan's test for multiple comparion.
Mitochondria Isolation. Isolation of mitochondria was performed as described earlier 42,43 . Briefly, yeast strains expressing Flag-MsrB2, Flag-Su9-MsrB3 and Flag-Su9-MsrA were grown overnight in 2% lactate medium. Cells were centrifuged at 5000 rpm and washed with 100 mM Tris-SO 4 pH 9.4 buffer followed by lysis with lyticase (Sigma-L2524) in 1.2 M sorbitol and 20 mM phosphate buffer pH 7.0. The lysed cells were homogenized in SEM buffer (250 mM Sucrose, 1 mM EDTA, 10 mM MOPS/KOH pH 7.2) and centrifuged at 3500 rpm. The supernatant was collected and centrifuged at 10000 rpm. The resultant mitochondrial pellet was washed twice and suspended in SEM buffer and stored at −80 °C. The cytosolic fraction from yeast strain expressing Flag-Mxr1 was obtained by following the above procedure except that in the last step, instead of mitochondrial pellet, the post-mitochondrial fraction/supernatant is taken and stored at −20 °C. MALDI studies. Mass spectrometry of hGrpEL1 proteins was performed as described earlier 19 . Briefly, 20 mM H 2 O 2 treated 20 µg of hGrpEL1 was incubated with individual 10 µg of Msr proteins in a reaction buffer. The sample was incubated for 2 hours followed by separation of proteins on SDS-PAGE. The hGrpEL1 protein bands were excised from gel and digested with trypsin followed by mass spectrometric analysis. Relative levels of peptides of interest were quantified and plotted. The intensity of the peaks are taken into account while plotting the graph of oxidized and un-oxidized peptides.