Journal of Molecular Biology
Volume 424, Issue 5, 14 December 2012, Pages 227-239
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Impaired Folding of the Mitochondrial Small TIM Chaperones Induces Clearance by the i-AAA Protease

https://doi.org/10.1016/j.jmb.2012.09.019Get rights and content
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Abstract

The intermembrane space of mitochondria contains a dedicated chaperone network—the small translocase of the inner membrane (TIM) family—for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX3C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1).

Highlights

► Individual cysteine residues of the CX3C motif in Tim9 and Tim10 are not essential for viability. ► A single disulfide bond in Tim9 and Tim10 is essential for function. ► Misfolded small TIM subunits are targeted for proteolytic degradation within mitochondria. ► Misfolded and unassembled small TIM subunits are degraded by Yme1.

Abbreviations

TOM
translocase of the outer membrane
TIM
translocase of the inner membrane
IMS
intermembrane space
MIA
mitochondrial IMS import and assembly machinery
AAA
ATPases associated with diverse cellular activities
BN-PAGE
blue native polyacrylamide gel electrophoresis
Yme1
yeast mitochondrial escape 1
5-FOA
5-fluoroorotic acid
AAC
ADP/ATP carrier
DHFR
dihydrofolate reductase
SAM
sorting and assembly machinery
TCA
trichloroacetic acid
i-AAA
IMS-AAA

Keywords

disulfide bond
intermembrane space
MIA
mitochondria
Yme1

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