The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly

Wróbel, Lidia; Sokòl, Anna M.; Chojnacka, Magdalena; Chacińska, Agnieszka

doi:10.1038/srep27484

Cited by 39 publications

(38 citation statements)

References 31 publications

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“…Affinity purification revealed the interaction between His TIM22 and TIM29 (Fig. A), in addition to native TIM22 . Other mitochondrial proteins were not found in the eluate, demonstrating assay specificity.…”

Section: Resultsmentioning

confidence: 95%

TIM29 is a subunit of the human carrier translocase required for protein transport

et al. 2016

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Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.

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Section: Resultsmentioning

confidence: 95%

TIM29 is a subunit of the human carrier translocase required for protein transport

et al. 2016

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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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“…Tim22 is supposed to have four transmembrane helices 19 . The cryo-EM density of the membrane region is relatively lower than the soluble region, ranging from 5 Å to 3.2 Å. Luckily, the local resolution of the loop between TM2 and TM3 is high enough, around 3.5 Å, which allow us identify three bulky residues (Y146, R147, and W152) in the linker.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM Structure of the Human Mitochondrial Translocase TIM22 Complex

Wang

Guan

et al. 2019

Preprint

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21Mitochondria play vital functions in cellular metabolism, homeostasis, and apoptosis 1-3 . 22 Most of the mitochondrial proteins are synthesized as precursors in the cytosol and 23 imported into mitochondria for folding or maturation 4,5 . The translocase TIM22 24 complex is responsible for the import of multiple hydrophobic carrier proteins that are 25 then folded in the inner membrane of mitochondria 6-8 . In mammalian cells, the TIM22 26 complex consists of at least six components, Tim22, Tim29, AGK, and three Tim 27 chaperones (Tim9, Tim10a and Tim10b) 9-14 . Here, we report the cryo-EM structure of 28 the human translocase TIM22 complex at an overall resolution of 3.7 angstrom. The 29 core subunit, Tim22, contains four transmembrane helices, forming a partial pore that is 30 open to the lipid bilayer. Tim29 is a single transmembrane protein that provides an 31 N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space (IMS) 32 domain to connect AGK and two TIM chaperone hexamers to maintain complex 33 integrity. One TIM hexamer comprises Tim9 and Tim10a in a 3:3 molar ratio, and the 34 other consists of two Tim9 units, three Tim10a units, and one Tim10b unit. The latter 35 hexamer faces the intramembrane region of Tim22, likely providing the dock to load the 36 precursors to the partial pore of Tim22. Our structure serves as a molecular basis for 37 the mechanistic understanding of TIM22 complex function. 38 39 40 3Mitochondria are essential eukaryotic cellular organelles with multiple vital functions, 41 such as energetics, metabolism, and cellular signaling 1,2 . These functions are performed by 42 more than 1,000 proteins 3,5 . However, only a small set of these proteins are synthesized in the 43 mitochondria; most of the mitochondria functioning proteins (~99%) are encoded by nuclear 44 genes, and imported into the correct mitochondrial compartment by specific preprotein 45 translocase complexes 3,5,15,16 . These complexes include the translocase of outer membrane 46 (the TOM complex) 17-20 , the carrier translocase of inner membrane complex (the TIM22 47 7 toward the intermembrane space and interact with the Tim9/10a/10b hexamer. Helix 1 and 129 loop 1, similar to a hook, sinuously wind around the groove between the inner helices and the 130 outer helices of Tim9 and Tim10a. Helix 1 was nestled in a greasy pocket formed by Tim9 131 and Tim10a (Fig. 2b), and likely functioned as a plug to obstruct the hydrophobic carrier 132 precursor from wedging within the hexamer chaperone from this side (Extended Data Fig. 6a). 133The recently identified disease-related mutation (Val33Leu) of Tim22 was located in the helix 134 1 51 . 135 136 Specific interactions comprise van der Waals contacts and one hydrogen bond. 137

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The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly

Cited by 39 publications

References 31 publications

TIM29 is a subunit of the human carrier translocase required for protein transport

TIM29 is a subunit of the human carrier translocase required for protein transport

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

Cryo-EM Structure of the Human Mitochondrial Translocase TIM22 Complex

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