The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.
Mia40 is a recently identified oxidoreductase in the intermembrane space (IMS) of mitochondria that mediates protein import in an oxidation-dependent reaction. Substrates of Mia40 that were identified so far are of simple structure and receive one or two disulphide bonds. Here we identified the protease Atp23 as a novel substrate of Mia40. Atp23 contains ten cysteine residues which are oxidized during several rounds of interaction with Mia40. In contrast to other Mia40 substrates, oxidation of Atp23 is not essential for its import; an Atp23 variant in which all ten cysteine residues were replaced by serine residues still accumulates in mitochondria in a Mia40-dependent manner. In vitro Mia40 can mediate the folding of wild-type Atp23 and prevents its aggregation. In these reactions, the hydrophobic substrate-binding pocket of Mia40 was found to be essential for its chaperone-like activity. Thus, Mia40 plays a much broader role in import and folding of polypeptides than previously expected and can serve as folding factor for proteins with complex disulphide patterns as well as for cysteine-free polypeptides.
H2S is an important signalling molecule involved in diverse biological
processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect
the activity of target proteins. Like thiols, persulfides show reactivity towards
electrophiles and behave similarly to other cysteine modifications in a biotin
switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based
method allowing the isolation of persulfide containing peptides in the mammalian
proteome. With this method, we demonstrated that H2S donors differ in
their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis
revealed that persulfide formation affects all subcellular compartments and various
cellular processes. Negatively charged amino acids appeared more frequently adjacent
to cysteines forming persulfides. We confirmed our proteomic data using pyruvate
kinase M2 as a model protein and showed that several cysteine residues are prone to
persulfide formation finally leading to its inactivation. Taken together, the
site-specific identification of persulfides on a proteome scale can help to identify
target proteins involved in H2S signalling and enlightens the biology of
H2S and its releasing agents.
Most mitochondrial proteins are synthesized in the cytosol and directed into the organelle; matrix proteins contain presequences that guide them through translocases in contact sites of the outer and inner membrane. In contrast, the import of many intermembrane space proteins depends on cysteine residues and the oxidoreductase Mia40. Here, we show that both import machineries can cooperate in the biogenesis of matrix proteins. Mrp10, a conserved protein of the mitochondrial ribosome, interacts with Mia40 during passage into the matrix. Mrp10 contains an unconventional proline-rich matrix-targeting sequence that renders import intermediates accessible to Mia40. Although oxidation of Mrp10 is not essential for its function in mitochondrial translation, the disulfide bonds prevent proteolytic degradation of Mrp10 and thereby counteract instability of the mitochondrial genome. The unconventional import pathway of Mrp10 is presumably part of a quality-control circle that connects mitochondrial ribosome biogenesis to the functionality of the mitochondrial disulfide relay.
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