Abstract
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.
Highlights
Chaperones play an important role in mitochondrial biogenesis through an efficient protein translocation, assembly, iron-sulfur cluster formation, mtDNA maintenance and protein homeostasis[10]
Using an yeast heterologous system, we show that human hGRPEL1 and human R type MSR complement the deletion of yeast MGE1 and MXR2 respectively
To test whether human GrpEL1 (hGrpEL1) is oxidized at conserved methionine residue like yMge[1], purified recombinant hGrpEL1 was treated with or without H2O2, separated on SDS-PAGE, Coomassie stained and trypsin digested fragments were analyzed by MALDI-TOF-MS/MS (Supplementary Figure S2A–D)
Summary
Chaperones play an important role in mitochondrial biogenesis through an efficient protein translocation, assembly, iron-sulfur cluster formation, mtDNA maintenance and protein homeostasis[10]. The indispensable Hsp[70] chaperone system consists of several conserved components that include DnaK/DnaJ or J-complex and Mge1/GrpE proteins. We have shown earlier that the conserved methionine at 155th position in Mge[1] responds to oxidative stress. Mitochondrial localized methionine sulfoxide reductase 2 (Mxr2) reversibly regulates Mge[1] by selectively reducing the Met155-SO to restore the activity of Mge[1]. Mxr[2] reduces the Met-SO of yeast Mge[1] both in vitro and in vivo, our earlier study does not preclude the formation of only R type sulfoxide upon oxidation. 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
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