A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import

Ramesh, Ajay; Peleh, Valentina; Martínez-Caballero, Sonia; Wollweber, Florian; Sommer, Frederik; Laan, Martin van der; Schroda, Michael; Alexander, R. Todd; Campo, María Luisa; Herrmann, J. M.

doi:10.1083/jcb.201602074

Cited by 51 publications

(51 citation statements)

References 58 publications

(105 reference statements)

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“…A recent report highlights that the Tim17 disulfide bond is critical for protein translocation and channel gating (79). Additionally, mutants lacking the Tim17 disulfide bond showed larger Tim21-bound TIM23 complexes and depleted TIM23 core complex, suggesting "extensive remodelling of TIM23-complex" (79). On the other hand, our findings highlight that the mutations in G/AXXXG/A motifs result in complete destabilization of the TIM23 complex due to defective recruitment of Pam18, Pam16, and Tim21.…”

Section: Tim17 Maintains Mtdna and Tim23 Complex Integritycontrasting

confidence: 48%

“…Presumably, the loss of membrane polarity due to mutation in G/AXXXG/A motifs can arise as a consequence of perturbed helix-helix packing between Tim23 and Tim17. A recent report highlights that the Tim17 disulfide bond is critical for protein translocation and channel gating (79). Additionally, mutants lacking the Tim17 disulfide bond showed larger Tim21-bound TIM23 complexes and depleted TIM23 core complex, suggesting "extensive remodelling of TIM23-complex" (79).…”

Section: Tim17 Maintains Mtdna and Tim23 Complex Integritymentioning

confidence: 99%

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Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

Matta

Pareek

Bankapalli

et al. 2017

Molecular and Cellular Biology

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Mitochondrial life cycle and protein import are intricate cellular processes, which require precise coordination between the transport machineries of outer and inner mitochondrial membranes. Presequence translocase performs the indispensable function of translocating preproteins having N-terminal targeting sequences across the inner membrane. Tim23 forms the core of the voltage-gated import channel, while Tim17 is presumed to maintain the stoichiometry of the translocase. However, mechanistic insights into how Tim17 coordinates these regulatory events within the complex remained elusive. We demonstrate that Tim17 harbors conserved G/AXXXG/A motifs within its transmembrane regions and plays an imperative role in the translocase assembly through interaction with Tim23. Tandem motifs are highly essential, as most of the amino acid substitutions lead to nonviability due to the complete destabilization of the TIM23 channel. Importantly, Tim17 transmembrane regions regulate the dynamic assembly of translocase to form either the TIM23 (PAM)-complex or TIM23 (SORT)-complex by recruiting the presequence translocase-associated motor (PAM) machinery or Tim21, respectively. To a greater significance, tim17 mutants displayed mitochondrial DNA (mtDNA) instability, membrane potential loss, and defective import, resulting in organellar dysfunction. We conclude that the integrity of Tim17 transmembrane regions is critical for mitochondrial function and protein turnover.KEYWORDS mitochondria, presequence translocase, mtDNA stability, preprotein import, membrane potential T he vast majority of mitochondrial proteins are encoded by nuclear genes and synthesized on cytosolic ribosomes with an amino-terminal positively charged cleavable signal sequence. The efficient import of mitochondrial proteins requires precise coordination between the translocases of the outer membrane (TOM complex) and the inner membrane (TIM23 complex) (1-9). After initial recognition and transport through TOM complex, these preproteins are actively sorted based on their destination. The preproteins directed toward the matrix compartment possess an N-terminal positively charged presequence and are imported into the matrix compartment with the aid of presequence translocase (TIM23 complex) (1-10). The core of presequence translocase consists of a membrane-bound channel component formed by . Tim23 and Tim17 are phylogenetically related integral membrane proteins and have similar transmembrane topology. Tim23 contributes to the formation of voltage-gated protein-conducting pores, while the evidence suggests that Tim17 is majorly involved in regulating the channel activity (16)(17)(18)(19)(20)(21)(22)(23). Other components such as Tim50 and Tim21 act as receptors and coordinate with the intermembrane space (IMS) domain of Tim23 to facilitate preprotein recognition and transfer from the TOM complex to presequence translocase (16, 17, 22, 24-32). Mgr2 has been recently

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Section: Tim17 Maintains Mtdna and Tim23 Complex Integritycontrasting

confidence: 48%

Section: Tim17 Maintains Mtdna and Tim23 Complex Integritymentioning

confidence: 99%

Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

Matta

Pareek

Bankapalli

et al. 2017

Molecular and Cellular Biology

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“…Therefore, interaction of the N-terminal domain of ScTim17 with the presequence is necessary for import of the precursor protein through the TIM23-17 channel. Recent data also showed that cysteine residue at position 10 is involved in disulfide bond formation with cysteine at position 77 [42,43]. Mutation of any of these two cysteines is detrimental for Tim17 function, particularly the gating of the TIM23 channel.…”

Section: Discussionmentioning

confidence: 99%

The divergent N-terminal domain of Tim17 is critical for its assembly in the TIM complex in Trypanosoma brucei

Weems

Singha

Smith

et al. 2017

Molecular and Biochemical Parasitology

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Trypanosoma brucei Tim17 (TbTim17), the single member of the Tim17/23/22 protein family, is an essential component of the translocase of the mitochondrial inner membrane (TIM). In spite of the conserved secondary structure, the primary sequence of TbTim17, particularly the N-terminal hydrophilic region, is significantly divergent. In order to understand the function of this region we expressed two N-terminal deletion mutants (Δ20 and Δ30) of TbTim17 in T. brucei. Both of these mutants of TbTim17 were targeted to mitochondria, however, they failed to complement the growth defect of TbTim17 RNAi cells. In addition, the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria were not restored by expression of the N-terminal deletion mutants but complemented by knock-in of the full-length protein. Further analysis revealed that Δ20-TbTim17 and Δ30-TbTim17 mutants were not localized in the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by two-dimensional Blue-native/SDS-PAGE revealed that none of these mutants are assembled into the TbTim17 protein complex. However, FL-TbTim17 was integrated into mitochondrial inner membrane and assembled into TbTim17 complex. Co-immunoprecipitation analysis showed that unlike the FL-TbTim17, mutant proteins are not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N-terminal domain of TbTim17 plays unique and essential roles for its sorting and assembly into the TbTim17 protein complex.

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“…Strains used to compare the effect of Djp1, Tom70 and Mim1 (ISY7475, djp1, tom70/71tom70/71djp1, mim1) were already described in (6). The tim17 ts mutant is described in (21).…”

Section: Yeast Strainsmentioning

confidence: 99%

An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast

Hansen

Aviram

Laborenz

et al. 2018

Science

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142

173

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The majority of organellar proteins are translated on cytosolic ribosomes and must be sorted correctly to function. Targeting routes have been identified for organelles such as peroxisomes and the endoplasmic reticulum (ER). However, little is known about the initial steps of targeting of mitochondrial proteins. In this study, we used a genome-wide screen in yeast and identified factors critical for the intracellular sorting of the mitochondrial inner membrane protein Oxa1. The screen uncovered an unexpected path, termed ER-SURF, for targeting of mitochondrial membrane proteins. This pathway retrieves mitochondrial proteins from the ER surface and reroutes them to mitochondria with the aid of the ER-localized chaperone Djp1. Hence, cells use the expanse of the ER surfaces as a fail-safe to maximize productive mitochondrial protein targeting.

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A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import

Abstract: Here, Ramesh et al. show that import and oxidation of Tim17, a membrane-embedded subunit of the mitochondrial protein import machinery, are mediated by the mitochondrial disulfide relay, although its disulfide bond is formed differently than soluble intermembrane space proteins.

Cited by 51 publications

References 58 publications

Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

The divergent N-terminal domain of Tim17 is critical for its assembly in the TIM complex in Trypanosoma brucei

An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast

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