Signal recognition initiates reorganization of the presequence translocase during protein import

Lytovchenko, Oleksandr; Melin, Jonathan; Schulz, Christian; Kilisch, Markus; Hutu, Dana P.; Rehling, Peter

doi:10.1038/emboj.2013.23

Cited by 59 publications

(83 citation statements)

References 43 publications

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“…Then the core domain of Tim50 interacts with residues 417-421 of presequence-bound PBD to form a transient ternary complex. Shift of the core domain to the presequence binding domain (residues 409-415) on PBD will release the presequence from PBD for its transfer to the core domain following the affinity gradient [10]. Although presequence binding to PBD is conserved only among fungal Tim50, that to the core domain is conserved among Tim50 from yeast to human, suggesting the importance of the core domain for further transfer of the presequence to the Tim23 channel for translocation across the inner membrane.…”

Section: Discussionmentioning

confidence: 99%

“…The determined X-ray structure of residues 176-361 consists of five a-helices and nine b-strands, and a negatively charged groove near the protruding b-hairpin was proposed to bind to a positively charged presequence [8]. Interestingly, the presence of a presequence binding site was reported for not only the conserved core domain (residues 164-361) but also the C-terminal ''presequence binding'' domain (PBD: residues 395-476) in yeast Tim50 [9,10]. Abbreviations: IMS, intermembrane space; PBD, presequence binding domain; sPBD, a shorter variant of PBD; Tim50core, Tim50 core domain; pSu9N, the N-terminal half of the presequence of subunit 9 of F o -ATPase; pHsp60, the presequence of mitochondrial Hsp60…”

Section: Introductionmentioning

confidence: 99%

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NMR analyses on the interactions of the yeast Tim50 C‐terminal region with the presequence and Tim50 core domain

et al. 2014

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a b s t r a c tThe mitochondrial targeting signal in the presequence of mitochondrial precursor proteins is recognized by Tom20 and subsequently by Tim50 in mitochondria. Yeast Tim50 contains two presequence binding sites in the conserved core domain and in the fungi-specific C-terminal presequence binding domain (PBD). We report the NMR analyses on interactions of a shorter variant of PBD (sPBD), a shorter variant of PBD, with presequences. The presequence is recognized by sPBD in a similar manner to Tom20. sPBD can also bind to the core domain of Tim50 through the presequence binding region, which could promote transfer of the presequence from sPBD to the core domain in Tim50. Structured summary of protein interactions:Tim50 sPBD and Tim50core bind by nuclear magnetic resonance (View interaction) pSu9N and Tim50 sPBD bind by nuclear magnetic resonance (1, 2, 3, 4, 5) pSu9N and dTim20 bind by nuclear magnetic resonance (View interaction)

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

NMR analyses on the interactions of the yeast Tim50 C‐terminal region with the presequence and Tim50 core domain

et al. 2014

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“…Although import of matrix-targeted pSu9-DHFR was impaired with ssc1-3 mitochondria, import of Om45 was not affected (Supplementary Fig S5A), suggesting that mtHsp70 import motor is not required for Om45 import. However interestingly, depletion of Pam17, another subunit of the TIM23 complex that may link the TIM23 core part and mtHsp70 import motor [18], impaired import of Om45 like pSu9-DHFR (Supplementary Fig S5B).…”

Section: Embo Reportsmentioning

confidence: 99%

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

et al. 2014

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The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (DΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.

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“…1A). For photoaffinity labeling, the peptide probes contained the photoreactive amino acid derivative para-benzoylphenylalanine (BPA) either at the hydrophilic surface of the presequence helix (replacing serine 16; pS 16 B) or at the hydrophobic surface of the helix (replacing leucine 19; pL 19 B). These peptides are efficiently imported into isolated mitochondria, thus allowing for photoaffinity labeling during the transport process (15).…”

Section: Resultsmentioning

confidence: 99%

“…From the TOM complex, the precursor is passed to the presequence translocase of the inner mitochondrial membrane (TIM23 complex). The primary presequence receptor of TIM23 is Tim50, which is dedicated to presequence recognition and precursor transport to Tim23 (15)(16)(17)(18). Tim23 exposes a presequence-binding domain into the IMS and is the pore-forming subunit of the presequence translocase (13,15,19,20).…”

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confidence: 99%

Presequence Recognition by the Tom40 Channel Contributes to Precursor Translocation into the Mitochondrial Matrix

Melin

Schulz

Wróbel

et al. 2014

Molecular and Cellular Biology

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More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined. Using a photoaffinity-labeling approach with modified presequence peptides, we identified Tom40 as a presequence interactor of the TOM complex. Utilizing mass spectrometry, we mapped Tom40's presequence-interacting regions to both sides of the ␤-barrel. Analysis of a phosphorylation site within one of the presequence-interacting regions revealed altered translocation kinetics along the presequence pathway. Our analyses assess the relation between the identified presequence-binding region of Tom40 and the intermembrane space domain of Tom22. The identified presequence-interacting region of Tom40 is capable of functioning independently of the established trans-acting TOM presequence-binding domain during matrix import. Mitochondria receive the majority of their resident proteins from the cytoplasm. Therefore, mitochondria must mediate the influx of proteins, which have been translated on cytosolic ribosomes, in a spatially constrained manner, i.e., all substrates are required to be unfolded during membrane passage. This constraint is in part due to the static dimensions of the pore within the translocase of the outer mitochondrial membrane (TOM). The TOM complex is involved in the majority of mitochondrial import traffic and therefore serves as the main gateway into the mitochondrion (for reviews, see references 1 to 4).About two-thirds of mitochondrial proteins are targeted into mitochondria by N-terminal targeting signals (presequences) (5). These signals consist of amphipathic ␣-helices with a net positive charge and direct the precursors across the outer and inner mitochondrial membranes. Presequence recognition at the TOM complex is thought to commence with the interaction of the hydrophobic surface of the amphipathic presequence-helix with the Tom20 binding groove (6-8). Subsequently, the hydrophilic side is recognized by the cytosolic domain of the central receptor Tom22 that interacts with the presequence in a salt-sensitive manner (6). The exact series of initial presequence-binding events is still debated; however, there is growing support for a tertiary complex in which both Tom20 and Tom22 interact with the presequence simultaneously (9, 10). Following the positioning of the presequence via Tom22, the substrate is passed to the pore-forming subunit of the TOM complex, Tom40. This process involves Tom5, which serves as a TOM assembly and presequence-interacting subunit (11). As the presequence emerges from the Tom40 pore on the trans side, the intermembrane space (IMS) domain of Tom22 engages with the precursor (10,(12)(13)(14). From...

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Signal recognition initiates reorganization of the presequence translocase during protein import

Cited by 59 publications

References 43 publications

NMR analyses on the interactions of the yeast Tim50 C‐terminal region with the presequence and Tim50 core domain

NMR analyses on the interactions of the yeast Tim50 C‐terminal region with the presequence and Tim50 core domain

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

Presequence Recognition by the Tom40 Channel Contributes to Precursor Translocation into the Mitochondrial Matrix

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