The Tim50 subunit of the mitochondrial TIM23 complex contains a presequence-binding domain that is essential for viability and precursor transport across the inner membrane

Agnieszka Chacinska

Alder

Bauer

Bernard Guiard

Bernhard Schmidt

Brix

Bömer

Chacinska

Christian Schulz

de la Cruz

Dimova

Endo

Erdmann

Geissler

Gevorkyan-Airapetov

Jahn

Janke

Jonathan Melin

Junge

Kanamori

Kleywegt

Meinecke

Mick

Misura

Mokranjac

Mumberg

Neuhoff

Neupert

Olaf Jahn

Oleksandr Lytovchenko

Perry

Peter Rehling

Piotr Neumann

Qian

Ralf Ficner

Rapoport

Raveh

Schnell

Stojanovski

Söding

Tamura

Truscott

van der Laan

von Heijne

Vögtle

Wickner

Wittelsberger

Yamamoto

English

PubMed

Schulz, Christian

Lytovchenko, Oleksandr

Melin, Jonathan

Chacinska, Agnieszka

Guiard, Bernard

Neumann, Piotr

Ficner, Ralf

Jahn, Olaf

Schmidt, Bernhard

Rehling, Peter

Max Planck Society eDoc Server

Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex

Schulz, C.

Lytovchenko, O.

Melin, J.

Chacinska, A.

Guiard, B.

Neumann, P.

Ficner, R.

Jahn, O.

Schmidt, B.

Rehling, P.

MPG.PuRe

Presequence receptor function of Tim50 • Schulz et al.THE JOURNAL OF CELL BIOLOGYS1JCBSupplemental materialSchulz et al., http://www.jcb.org/cgi/content/full/jcb.201105098/DC1JCB S2 Figure S1.  Validation of the approach using Tom20CD. (A) Isolated Tom20CD and GST were incubated with presequence probes under UV irradiation, sam-ples analyzed by SDS-PAGE, and proteins subjected to in-gel digest before LC MALDI MS/MS analysis. PA, photo-adduct. (B) Fragment ion mass spectrum of peptide Tom2053-58 cross-linked to pL19B18-24 (precursor mass 1573.75). Photo-adduct of pL19B with Tom20CD (A) was digested with trypsin and subjected to LC MALDI MS/MS analysis. (C) Fragment ion mass spectrum of peptide Tom20103-109 cross-linked to pL19B18-24 (precursor mass 1629.84). Sample was analyzed as in B. (D) Representative model of the NMR structure of rat Tom20 in complex with pALDH presequence peptide (Protein Data Base accession no. 1OM2; Abe et al., 2000), left, and modeled yeast Tom20 in complex with pALDH (blue), right. Leu19 of pALDH and Ala82 or Met105 are colored in or-ange or magenta, respectively. N and C denote the N and C termini. Presequence receptor function of Tim50 • Schulz et al. S3Figure S2.  Characterization of the photo-peptides. (A) Presequence-probes pL19B (top panels) or pS16B (bottom panels) were imported into isolated mito-chondria in the presence of  for indicated times at 25°C. After import, mitochondria were converted into mitoplasts or resuspended in Triton X-100 con-taining buffer and subjected to proteinase K digestion. Samples were analyzed by Western blotting using streptavidin-HRP (horseradish peroxidase) or indicated antibodies. Input, 25% of peptide used for each time point. M, mitochondria. MP, mitoplasts. Tx-100, Triton X-100. (B) Mitochondria were incu-bated for 10 min with 2 µM presequence peptide in the presence or absence of . After re-isolation of mitochondria, protein import of a radiolabeled pre-cursor protein (F1 and Su9-DHFR) was assessed. Therefore, the radiolabeled precursor was added to the treated or untreated mitochondria and import performed for 15 min. Subsequent to proteinase K treatment, samples were analyzed by SDS-PAGE and digital autoradiography. Imported precursor was quantified relative to untreated mitochondria (100%). (C) Radiolabeled AAC was imported into isolated mitochondria in the presence of 2 µM of the indi-cated peptides for 45 min. After proteinase K treatment, mitochondria were solubilized and samples analyzed by BN-PAGE and digital autoradiography. Asterisk denotes unspecific band. (D) Isolated mitochondria were preincubated with 2 µM pL19B for the indicated time on ice before subjecting the samples to UV irradiation for 10 min. Samples were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. PA, photo-adduct. (E) Tim50 immu-noprecipitation from mitochondria after photo cross-linking with 2 µM photo-peptides for 30 min. Samples were analyzed by Western blotting with indi-cated antibodies or streptavidin-HRP, SA-HRP. Asterisk denotes cross-reactive protein. Total, 5%; Eluate, 100%. PA, photo-adduct.JCB S4 Figure S3.  Identification of the presequence-binding site in Tim50. (A) After UV-induced cross-linking of Tim50IMS with peptide pL19B or pS16B and purifica-tion by SDS-PAGE, the lower bands containing photo-adducts (Fig. 2 B) were digested with trypsin and the peptides subjected to LC MALDI MS/MS analy-sis. The a-, b-, and y-ion series are indicated. Peptide Tim50412-419 cross-linked to peptide pL19B18-24 (precursor mass 1873.90). (B) Peptide Tim50412-419 cross-linked to peptide pL19B18-24 (precursor mass 1573.78). Sample was analyzed as in A. (C–J) After UV-induced cross-linking of Tim50PBD with peptide pL19B or pS16B and purification by SDS-PAGE, bands corresponding to photo-adducts (Fig. 2 F) were digested with trypsin and the peptides subjected to LC MALDI MS/MS analysis. Where needed, lower and upper indicate the band excised from the gel and analyzed. The a-, b-, and y-ion series are indi-cated. (C) Peptide Tim50405-411 cross-linked to peptide pL19B18-24 (precursor mass 1800.86); lower. (D) Peptide Tim50405-411 cross-linked to peptide pL19B18-24 (precursor mass 1800.89); upper. (E) Peptide Tim50412-419 cross-linked to peptide pL19B18-24 (precursor mass 1873.91); lower. (F) Peptide Tim50412-419 cross-linked to peptide pL19B18-24 (precursor mass 1889.92); upper. (G) Peptide Tim50412-419 cross-linked to peptide pS16B15-17 (precursor mass 1573.86); lower. (H) Peptide Tim50412-419 cross-linked to peptide pS16B15-17 (precursor mass 1573.81); lower. (I) Peptide Tim50422-435 cross-linked to peptide pL19B18-24 (precursor mass 2447.25). (J) Peptide Tim50436-450 cross-linked to peptide pL19B18-24 (precursor mass 2574.23); lower.Presequence receptor function of Tim50 • Schulz et al. S5Figure S4.  Photo-adducts to Tim50PBD contain mainly one presequence peptide. (A) After UV-induced cross-linking of purified Tim50PBD with pL19B or pS16B, for 30 min as in Fig. 2 F, the sample was analyzed by Western blotting using anti-Tim50 antibodies. PA, photo-adduct. (B) Samples from A were analyzed by linear MALDI-TOF-MS. The calculated molecular masses are as follows: Mcalc(Tim50PBD) = 10428 Da; Mcalc(Tim50PBD+1pL19B) = 14338 Da; Mcalc(Tim50PBD+1pS16B) = 14364 Da; Mcalc(Tim50PBD+2pL19B) = 18247 Da; Mcalc(Tim50PBD+2pS16B) = 18299 Da. Observed shifts toward higher masses are most likely due to methionine oxidation. The relative areas of the relevant signals are 80.8, 17.8, and 1.4% for pL19B; and 83.4, 14.6, and 2.0% for pS16B.JCB S6 Figure S5.  Properties of different mutant mitochondria. (A) Steady-state protein analysis of isolated Tim50↓ mitochondria expressing either Tim50HA1 or Tim50PBD HA1 analyzed by Western blotting with the indicated antibodies. (B) Radiolabeled F1 was imported into wild-type and Tim23↓ mitochondria for the indicated times at 25°C. Subsequent to proteinase K digestion samples were analyzed by SDS-PAGE and digital autoradiography. m, mature protein. (C) Isolated mitochondria were incubated with 20 µM of the indicated peptides and subjected to chemical cross-linking using 1 mM DFDNB for 30 min. Samples were analyzed as in A. (D) Isolated Tim50↓ mitochondria expressing either Tim50HA1 or Tim50PBD HA1 were converted into mitoplasts and sub-jected to proteinase K treatment. Samples were analyzed as in A.ReferenceAbe, Y., T. Shodai, T. Muto, K. Mihara, H. Torii, S. Nishikawa, T. Endo, and D. Kohda. 2000. Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell. 100:551–560. http://dx.doi.org/10.1016/S0092-8674(00)80691-1

Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex.

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.</jats:p

Crossref

The Journal of Cell Biology

Tim50’s presequence receptor domain is essential for signal driven transport across the TIM23 complex

Import of proteins into mitochondria. Methods Cell Biol. 80:783–806. http://dx.doi .org/10.1016/S0091-679X(06)80036-1

Motor-free mitochondrial presequence translocase drives membrane integration of preproteins.

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Protein translocons: multifunctional mediators of protein translocation across membranes.

Structural basis for the function of Tim50 in the mitochondrial presequence translocase.

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

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Tim50’s presequence receptor domain is essential for signal driven transport across the TIM23 complex

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