Methionine sulfoxide reductases are important regulators of oxidative stress, as they reduce oxidized methionine in proteins. Mge1, a cochaperone of mtHsp70, is a physiological substrate of Mxr2 and regulates reversibly to maintain mitochondrial protein homeostasis and oxidative stress.
Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.
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.
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