Abstract
Mitochondria are central organelles to cellular metabolism. Their function relies largely on nuclear-encoded proteins that must be imported from the cytosol, and thus the protein import pathways are important for the maintenance of mitochondrial proteostasis. Mitochondrial HSP70 (mtHsp70) is a key component in facilitating the translocation of proteins through the inner membrane into the mitochondrial matrix. Its protein folding cycle is regulated by the nucleotide-exchange factor GrpE, which triggers the release of folded proteins by ATP rebinding. Vertebrates have two mitochondrial GrpE paralogs, GRPEL1 and 2, but without clearly defined roles. Using BioID proximity labeling to identify potential binding partners of the GRPELs in the mitochondrial matrix, we obtained results supporting a model where both GRPELs regulate mtHsp70 as homodimers. We show that GRPEL2 is not essential in human cultured cells, and its absence does not prevent mitochondrial protein import. Instead we find that GRPEL2 is redox regulated in oxidative stress. In the presence of hydrogen peroxide, GRPEL2 forms dimers through intermolecular disulfide bonds in which Cys87 is the thiol switch. We propose that the dimerization of GRPEL2 may activate the folding machinery responsible for protein import into mitochondrial matrix or enhance the chaperone activity of mtHSP70, thus protecting mitochondrial proteostasis in oxidative stress.