Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Abstract

Mitochondria import nuclear‐encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

Mia40 targets cysteines in a hydrophobic environment to direct oxidative protein folding in the mitochondria

“…To examine whether this modification influenced binding, we performed experiments in which the energy donor Trp was placed in Mia40 and the acceptor AEDANS in Cox17*. Four single-cysteine variants of Cox17* were produced and labelled with AEDANS (variants [8][9][10][11]. Their interaction with the wild-type form of Mia40 was then measured in equilibrium titrations, and its kinetics was followed in the stopped-flow instrument by the FRET between the single Trp (W294) of Mia40 and the AEDANS groups of the four Cox17* variants ( Supplementary Fig.…”

“…T he intermembrane space (IMS) of the mitochondria was long believed to provide a reducing environment, similar to the cytosol, but recently proteins with disulphide bonds and subsequently a thiol oxidase (Mia40, mitochondrial import and assembly) were discovered in the IMS [1][2][3][4][5][6][7][8][9][10][11][12] . Unlike the other known thiol oxidases, Mia40 does not belong to the thioredoxin family, and its catalytic disulphide is arranged in a unique Cys-Pro-Cys motif.…”

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

“…By a subsequent proteolytic step the mature protein is released into the IMS. On the other hand, there are many proteins of relatively small size that lack presequences but contain motifs of conserved cysteine residues, such as the twin CX 9 C motif in the copper chaperone Cox17p and in Cox19p, Mdm35p, Mic14p, and Mic17p or the twin CX 3 C motif in the family of small Tim proteins (6 -11).ProteinswithtwinCX n CmotifsusetheMia40p-dependent translocation pathway, which consists of at least two components in the IMS, Mia40p and Erv1p (8,(12)(13)(14)(15)(16)(17). Both proteins are essential for viability of yeast cells (12-14, 18 -20).…”

Mitochondrial ATP‐independent chaperones

“…After translocation through the TOM complex, small Tims are captured in a disulphide bondlinked intermediate with the intermembrane space oxidase Mia40. A ternary complex is formed between the substrate, Mia40 and the sulfhydryl oxidase Erv1, which is required for reoxidation of Mia40 (62)(63)(64). Although the small Tims contain no typical cleavable N-terminal signal sequence, a linear targeting signal within Tim9 and Tim10 has recently been shown to direct proteins across the outer membrane to the Mia40 translocase (65).…”