A novel intermembrane space-targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding.
Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step-specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.
Sideris
Dionisia P
DP
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece.
Petrakis
Nikos
N
Katrakili
Nitsa
N
Mikropoulou
Despina
D
Gallo
Angelo
A
Ciofi-Baffoni
Simone
S
Banci
Lucia
L
Bertini
Ivano
I
Tokatlidis
Kostas
K
D001665:Binding Sites; D002151:Calorimetry; D002352:Carrier Proteins / Q000378:metabolism; D027682:Cation Transport Proteins; D016384:Consensus Sequence; D000079922:Copper Transport Proteins; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones; D010084:Oxidation-Reduction; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
C507282:CHCHD4 protein, human; C100359:COX17 protein, S cerevisiae; C491006:COX17 protein, human; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; C495320:MIA40 protein, S cerevisiae; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine