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A selectivity filter in Tim17 mediates intramitochondrial protein sorting - Dataset II

ABSTRACT: Mitochondria import hundreds of proteins into the matrix and the inner membrane. The translocase of the inner membrane of mitochondria (TIM23 complex) sorts these proteins using topogenic signals in their structure and directs them into the matrix or inserts them into the inner membrane. How these signals are deciphered is unknown. Here we show that Tim17, the protein-conducting subunit of the TIM23 complex, contains a highly conserved selection filter for incoming proteins. Using a fluorescence-based sorting reporter, we identified the novel mitochondrial oxidoreductase Dmo2 (akin to DMAC1 in humans) as critical factor for intramitochondrial protein sorting. Molecular dynamics simulations show that a Dmo2-dependent disulfide bond into Tim17 constitutes a negatively charged selection filter at the vestibule of the TIM23 complex that arrests membrane proteins and facilitates the lateral insertion. Our work provides a first molecular rationale as to how the TIM23 translocase differentiates between soluble and membrane proteins.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Markus Räschle

LAB HEAD: Markus Räschle

PROVIDER: PXD078122 | Pride | 2026-08-03

REPOSITORIES: Pride

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			Action	DRS
	20260205_HF1_MaRa_SA_P0312_Dmo2_01.raw	Raw
	20260205_HF1_MaRa_SA_P0312_Dmo2_02.raw	Raw
	20260205_HF1_MaRa_SA_P0312_Dmo2_03.raw	Raw
	20260205_HF1_MaRa_SA_P0312_Dmo2_04.raw	Raw
	20260205_HF1_MaRa_SA_P0312_EV_01.raw	Raw

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A selectivity filter in Tim17 mediates intramitochondrial protein sorting - Data set I

Project description:The translocase of the inner membrane of mitochondria (TIM23 complex) imports proteins into the matrix and, in the case of membrane proteins, releases precursors laterally into the inner membrane. The sorting of precursor proteins relies on topogenic signals in their structure. How these signals are deciphered is unknown. Using a fluorescence-based sorting reporter, we systematically screened yeast depletion libraries and identified Dmo2 (akin to DMAC1 in humans) as a novel protein critical for the membrane insertion of inner membrane proteins. Dmo2 serves as oxidoreductase that introduces a disulfide bond into Tim17, the protein-conducting subunit of the TIM23 complex. Molecular dynamics simulations show that the disulfide bond in Tim17 positions the negatively charged N terminus of Tim17 to the protein-conducting vestibule of the TIM23 translocase to constitute a negatively charged selection filter for incoming precursor proteins. Our paper provides the first molecular rationale as to how the TIM23 translocase differentiates between soluble and membrane proteins.

2026-08-03 | PXD068062 | Pride

A selectivity filter in Tim17 mediates intramitochondrial protein sorting - Dataset III

Project description:Mitochondria import hundreds of proteins into the matrix and the inner membrane. The translocase of the inner membrane of mitochondria (TIM23 complex) sorts these proteins using topogenic signals in their structure and directs them into the matrix or inserts them into the inner membrane. How these signals are deciphered is unknown. Here we show that Tim17, the protein-conducting subunit of the TIM23 complex, contains a highly conserved selection filter for incoming proteins. Using a fluorescence-based sorting reporter, we identified the novel mitochondrial oxidoreductase Dmo2 (akin to DMAC1 in humans) as critical factor for intramitochondrial protein sorting. Molecular dynamics simulations show that a Dmo2-dependent disulfide bond into Tim17 constitutes a negatively charged selection filter at the vestibule of the TIM23 complex that arrests membrane proteins and facilitates the lateral insertion. Our work provides a first molecular rationale as to how the TIM23 translocase differentiates between soluble and membrane proteins.

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Expression data of WT and Attim17-1 Arabidopsis seeds during stratification and germination

Project description:The translocase of the inner membrane 17-1 (Tim17-1) plays a defined role in germination in Arabidopsis thaliana A transcriptomic analysis of Attim17-1 during seed germination

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Project description:Damaged mitochondria can be cleared from the cell by mitophagy, using a pathway formed by the recessive Parkinson’s disease genes PINK1 and Parkin. Whether the pathway senses diverse forms of mitochondrial damage by a common mechanism, however, remains uncertain. Here, using a novel Parkin reporter in genome-wide screens, we identified that diverse forms of mitochondrial damage converge on loss of mitochondrial membrane potential (MMP) to activate PINK1. Loss of MMP, but not the PAM import motor, blocked progression of PINK1 import through the translocase of the inner membrane (TIM23), causing it to remain bound to the translocase of the outer membrane (TOM). Ablation of TIM23 was sufficient to arrest PINK1 in TOM, irrespective of MMP. Meanwhile, TOM (including subunit TOMM5) was required for PINK1 retention on the mitochondrial surface. The energy-state outside of the mitochondria further modulated the pathway by controlling the rate of new PINK1 synthesis. Together, our findings point to a convergent mechanism of PINK1-Parkin activation by mitochondrial damage: loss of MMP stalls PINK1 import during its transfer from TOM to TIM23.

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A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

Project description:Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane- associated respiratory chain, and consequently in the generation of minimal inner membrane potential (), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on and ATP hydrolysis, while TIM22 requires only . The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown.

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Project description:Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase (DHFR) fused to the N-terminus of the inner membrane protein MIC60. While stabilization of the DHFR domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, surprisingly, plugging the channel of translocase of the outer membrane (TOM) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 appears to play a crucial role in mitochondrial quality control to counteract precursor stalling in the TOM complex and unoccupied TIM23 channels.

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The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

Project description:Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.

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Impaired protein import drives mitochondrial dysfunction upon mutation of TIMM50 in mitochondrial disease

Project description:TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of presequence-containing precursors into the matrix or inner membrane. Here we describe a mitochondrial disease patient that is homozygous for a novel variant in TIMM50 and establish the first proteomic profile of mitochondrial disease associated with TIMM50 mutation. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient cells and a TIMM50 HEK293T cell model of disease, we reveal that laterally inserted substrates imported via the TIM23SORT complex are more sensitive to loss of TIMM50. Indeed, proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in laterally-released TIM23SORT substrates, providing a biochemical mechanism for the specific defects in TIMM50 patients. These results highlight the power of using proteomics for elucidating molecular mechanisms of disease and uncovering novel features of fundamental biology.

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Succinate and its carrier Sfc1 mediate metabolic control of mitochondrial protein import by the TIM23 translocase

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Project description:Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early onset Parkinson disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane (OMM) forming a high molecular weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates ubiquitin, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved, remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress, such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 NT-CTE module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

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