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Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50

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Science Advances  01 Sep 2017:
Vol. 3, no. 9, e1700532
DOI: 10.1126/sciadv.1700532
  • Fig. 1 In vivo and in organello analysis of the CL dependence of the Tim23-Tim50 interaction.

    (A) Coimmunoprecipitation analysis of the Tim23-Tim50 interaction. Digitonin lysates of mitochondria from yeast strains containing full-length Tim50 (left) or Tim50IMS (right) were immunoprecipitated with affinity-purified antibodies to Tim23 (αTim23) or Tim50 (αTim50) or preimmune immunoglobulins (PI). Unbound (Sup.) and bound (Pellets) fractions were analyzed by SDS-PAGE and immunodecoration with antibodies against Tim50, Tim23, or Tim17, as indicated. Total and Sup. lanes indicate 20% of starting material; all other lanes represent undiluted samples. (B) Synthetic lethality of tim23yl and Tim50IMS. Yeast cells containing a chromosomal deletion of TIM23 complemented with a TIM23-containing URA3 plasmid were generated in a Tim50 or Tim50IMS background (delineated by dotted boxes). Cells were transformed with centromeric plasmids carrying either Tim23 or tim23yl, as indicated, and subjected to URA3 plasmid loss by plating on glucose-containing medium with 5-fluoroorotic acid (5-FOA). Cells were incubated at 30°C (upper plate) or 24°C (lower plate). (C) Genetic interaction of Δcrd1 and tim23yl. Tenfold serial dilutions were prepared from yeast cells containing WT Tim23 or the tim23yl mutant in a background of normal CL synthesis or CL synthase knockouts (Δcrd1). Cells were spotted onto YPD or YPLac medium as shown and grown at the indicated temperatures.

  • Fig. 2 In organello cross-linking–detected Tim23-Tim50 interactions.

    (A) Topology diagram of Tim23 and Tim50 in the mitochondrial IM. Tim23 (yellow) contains four predicted transmembrane segments (TMS1 to TMS4) and an intrinsically disordered IMS region with a heptad leucine repeat (thickened line). Sites in white depict residues changed to cysteine for thiol-based cross-linking. Sites in red depict the region of the Y70A,L71A mutations. Tim50 (cyan) contains a single native cysteine site (C268) in the IMS domain. (B) Cross-linking and immunoprecipitation (IP)–detected interaction of Tim50 with Tim23IMS. [35S]Tim23(S80C) (“23”) or [35S]Tim23(S80C) with the Y70A,L71A mutations (“yl”) was imported into mitochondria isolated from the strains indicated (WT, Δcrd1, Δtaz1, and Δcld1) and subjected to chemical cross-linking with BMOE. One subset of samples was retained for analysis of the cross-linking reaction (“Total”), and the other was subjected to immunoprecipitation with antibodies to Tim50 to identify Tim23-Tim50 adducts (“Immunoprecipitation”). The band corresponding to non–cross-linked [35S]Tim23(S80C) (or [35S]Tim23(S80C)yl) is indicated by an open arrowhead on the Total gel; cross-linked adducts between radiolabeled Tim23 constructs and Tim50 [used for quantitation in (C)] are indicated by the closed arrowheads on the Immunoprecipitation gel. (C) Quantitation of cross-linking efficiency between Tim50 and Tim23IMS. Means represent values from three independent experiments (normalized relative to [35S]Tim23 in WT mitochondria); error bars represent SDs. Dots indicate significant differences in comparison to results from control ([35S]Tim23(S80C) in WT mitochondria) by Student’s t test (P < 0.05; ••P < 0.01; •••P < 0.0001). (D) Interaction between Tim50 and Tim23 transmembrane segments. Left: Representative cross-linking and IP-detected interaction between Tim50 and the indicated sites on TMS1 and TMS2, showing the higher–molecular weight immunoprecipitated band. Right: Helical wheel projections of TMS1 and TMS2 indicating sites used for cross-linking. Sites in yellow indicate those with highest cross-linking to Tim50. The red arc on TMS2 indicates the aqueous channel–facing, substrate-interactive face of the helix. (E) IP-detected interaction of Tim50 with Tim23 TMS1. [35S]Tim23(F114C) (“23”) or [35S]Tim23(F114C)yl (“yl”) was subjected to chemical cross-linking and IP in mitochondria from the indicated strains exactly as in (B). Cross-linked adducts between radiolabeled Tim23 constructs and Tim50 [used for quantitation in (F)] are indicated by the closed arrowheads. (F) Quantitation of cross-linking efficiency between Tim50 and Tim23 TMS1. Means represent values from three independent experiments (normalized relative to [35S]Tim23 in WT mitochondria); error bars represent SDs. Dots indicate significant differences in comparison to results from control ([35S]Tim23(F114C) in WT mitochondria) by Student’s t test (••P < 0.01; •••P < 0.0001).

  • Fig. 3 Interactions between Tim50IMS and full-length Tim23 in nanodiscs.

    (A) Diagram of the experimental design. Full-length Tim23 (yellow) was reconstituted into nanodiscs (gray disc) containing an experimentally defined lipid composition and bound by MSP (black rings). Sites in white depict residues changed to cysteine for thiol-based cross-linking or fluorescence analysis. Sites in red depict the region of the Y70A,L71A mutation. Purified soluble Tim50IMS (cyan) contains a single cysteine site (C268). (B) Fluorescence analysis of NBD-Tim23-ND. NBD was cotranslationally incorporated into the Tim23 IMS region (Tim23 T24C) or TMS1 (Tim23 I111C) during in vitro translation, assembled into nanodiscs, and purified for spectral analyses. On the basis of emission scans, samples with the NBD probe in TMS1 [NBD-Tim23(I111C)-ND, cyan] had a 3.1-fold higher fluorescence yield and a 10-nm blue-shifted λmax relative to samples with the probe in the IMS domain [NBD-Tim23(T24C)-ND, red]. RU, relative units. (C) Quality control tests with the nanodisc-based cross-linking system. Purified nanodiscs containing [35S]Tim23(T94C) (lanes 1 to 3), [35S]Tim23ΔCys (lane 4), or [35S]Tim23yl(T94C) (lane 5) were subjected to reactions in the presence or absence of a cross-linking agent [bis-maleimidoethane (BMOE)] and Tim50IMS, as indicated, followed by IP with αTim50. The band corresponding to the non–cross-linked [35S]Tim23(T94C)-ND is indicated by the open arrowhead, and the cross-linked adduct between [35S]Tim23(T94C)-ND and Tim50IMS is indicated by the closed arrowhead. (D) Relative efficiency of nanodisc incorporation and Tim50IMS-[35S]Tim23-ND cross-linking. [35S]Tim23(F114C) was reconstituted into nanodiscs containing only POPC (“PC”) or a binary lipid composition containing 20 mole percent (mol %) TOCL (“PC + CL”), as indicated. Samples were resolved by SDS-PAGE in the absence or presence of cross-linking with Tim50IMS, as shown. The bands corresponding to the non–cross-linked [35S]Tim23(F114C)-ND are indicated by the open arrowhead, and the cross-linked adducts between [35S]Tim23(F114C)-ND and Tim50IMS are indicated by the closed arrowhead. The panel to the right shows the quantitation (means with SDs) of relative incorporation efficiencies of [35S]Tim23 into nanodiscs (“Nanodisc reconst.”) and Tim50IMS cross-linking (“X-link”) for both types of nanodisc lipid compositions (means of three independent experiments with SDs are shown). Dots indicate significant differences between PC + CL and the cognate and PC-only samples by Student’s t test (••P < 0.01; •••P < 0.0001). (E) Cross-linking and IP-detected interaction between Tim50IMS and [35S]Tim23-ND. [35S]Tim23(S80C) or [35S]Tim23(F114C) in the absence (“23”) or presence (“yl”) of the Y70A,L71A mutations was reconstituted into nanodiscs containing only POPC (“PC”) or with a binary lipid composition containing 20 mol % TOCL (“PC + CL”), as indicated. Samples from cross-linking reactions with Tim50IMS were resolved directly by SDS-PAGE (Total; top) or subjected to IP with αTim50 and resolved by SDS-PAGE (Immunoprecipitation; bottom). Cross-linked adducts between radiolabeled Tim23 constructs and Tim50 (used for quantitation) are indicated by the closed arrowheads. Numbers above each “yl” lane indicate the percent cross-linking efficiency relative to the corresponding experiment with WT Tim23 (lane to the immediate left), shown graphically in (F). (F) Quantitation of cross-linking efficiency between Tim50IMS and [35S]Tim23-ND. Means represent values from three independent experiments; error bars represent SDs. Dots indicate significant differences between PC + CL and the cognate and PC-only samples by Student’s t test (•••P < 0.0001).

  • Fig. 4 Ab initio SAXS reconstruction of Tim50IMS.

    (A) Tim50IMS SAXS molecular envelope aligned with the homology model. The ab initio envelope generated by DAMAVER analysis of SAXS data from Tim50IMS (0.5 mg/ml; gray) is shown in comparison with the Tim50IMS homology model (ribbon structure), aligned using SITUS. The domains of the Tim50IMS homology model are color-coded with respect to the linear domain representation shown above. (B) Analysis of the fit between the envelope and the homology model. The experimental SAXS scattering data for Tim50IMS (0.5 mg/ml; black) and theoretical scattering data for the Tim50IMS homology model (red) were fit using the CRYSOL package, yielding a χ2 value of 1.12.

  • Fig. 5 Analysis of Tim50IMS-bilayer interactions from CG MD simulations.

    (A and B) Time course profiles of the Tim50IMS-bilayer distance. The minimum distance between Tim50IMS and the bilayers composed of pure POPC (A) or POPC with 20 mol % TOCL (B) is shown for five MD simulations each. (C) Four unique modes of Tim50IMS binding to TOCL-containing bilayers. N-terminal region, green; Tim50CORE, blue; β-hairpin, purple; C-terminal region, red; lipid headgroups, yellow; lipid hydrocarbon chains, gray. (D) Lipid headgroup phosphate contacts with Tim50IMS in binding mode 4. Tim50IMS is shown in space-filling representation with protein coloring the same as that in (C). POPC phosphates, cyan; TOCL phosphates, orange. (E) Tim50IMS β-hairpin–bilayer interactions in binding mode 4. Detailed image of key β-hairpin side chains shown to stabilize the Tim50IMS-bilayer interaction (W207, H211, W213, and R214) and the site of extrinsic fluorescent probe attachment (S208; see Fig. 7). POPC headgroup beads, cyan; TOCL headgroup beads, orange; lipid hydrocarbon tails, gray. (F) Free energy binding curves. Umbrella sampling results are shown for the four unique binding modes as well as for the four-point mutant of mode 4. The abscissa is a normalized coordinate such that x = 0 when Tim50IMS is bound and x = 1 when Tim50IMS is fully dissociated. This coordinate was used for clarity because the different modes required different absolute displacements from the bilayer to become fully dissociated.

  • Fig. 6 Cosedimentation analysis of the CL-dependent association of Tim50IMS with lipid bilayers.

    (A) Liposome cosedimentation analysis of Tim50IMS. Left: SDS-PAGE/SYPRO Orange stain profiles of Tim50IMS (1 μM) cosedimented with sucrose-loaded LUVs of defined lipid composition and increasing lipid concentration (up to 1.0 mg lipid/ml). Lipid compositions include PC (100 mol % POPC), PC + CL (80 mol % POPC and 20 mol % TOCL), PC + PG (80 mol % POPC and 20 mol % POPG), and PC + PE (50 mol % POPC and 50 mol % POPE). Bands used for quantitation are shown by the closed arrowheads. Right: Quantitation of relative Tim50IMS liposome cosedimentation for each lipid composition. Means represent values from three independent experiments (normalized with respect to band intensity in the absence of added liposomes); error bars represent SDs. (B) Cosedimentation analysis of Tim50IMS variants. Cosedimentation experiments conducted as above for 1 μM Tim50IMS, Tim50IMS W207A H211A W213A R214A (Tim50IMS WHWR), Tim50CORE (residues 165 to 361), and Tim50IMSΔN (residues 165 to 476) in the presence of PC + CL LUVs (final concentration, 0.5 mg lipid/ml). Means are normalized relative to Tim50IMS binding for three independent experiments; error bars represent SDs.

  • Fig. 7 Analysis of CL-mediated changes in Tim50IMS proteolysis, labeling, and bilayer exposure.

    (A) Limited proteolysis of Tim50IMS. Tim50IMS (1 μM) was preincubated without lipid vesicles (lanes 1 to 5) or with vesicles composed of POPC only (“PC”; lanes 6 to 10) or POPC with 20 mol % TOCL (“PC + CL”; lanes 11 to 15). Samples were incubated in the absence of protease (lanes 1, 6, and 11) or in the presence of increasing proteinase K (“PK”; 0.67 nM, lanes 2, 7, and 12; 2.0 nM, lanes 3, 8, and 13; 5.0 nM, lanes 4, 9, and 14; 100 nM, lanes 5, 10, and 15) and resolved by SDS-PAGE. Intact Tim50IMS is indicated by the arrowhead, the smallest product detected with Tim50IMS alone or with POPC vesicles is indicated by “*,” and the predominant band protected in the presence of POPC + TOCL vesicles is indicated by “**.” (B) Lipid-dependent cysteine accessibility of Tim50IMS. Tim50IMS (1 μM) was incubated in the presence of vesicles composed of POPC only (“PC”) or POPC with 20 mol % TOCL (“PC + CL”) at the indicated lipid concentrations and subjected to labeling with the thiol-reactive reagent TMM(PEG)12. Representative gels (below) show unlabeled (open arrowhead) and labeled (closed arrowhead) Tim50IMS. Quantification of the labeling efficiency (percent labeled relative to total labeled and unlabeled; means of three independent samples with SDs) is shown for each lipid concentration in plots above. Dots indicate significant differences in labeling compared to results from control (no liposomes) by Student’s t test (P < 0.05; ••P < 0.01; •••P < 0.0001). (C) Fluorescence-detected interaction between Tim50IMS and lipid bilayers. NBD-Tim50IMS(S208C) (500 nM) was incubated in the absence of lipid vesicles or in the presence of liposomes (1 mg lipid/ml) with differing lipid composition [POPC only (“PC”) or POPC with 20 mol % TOCL (“PC + CL”), dCL (“PC + dCL”), or MLCL (“PC + MLCL”), as indicated] and samples were subjected to fluorescence emission scans. (D) Tim50IMS-bilayer interactions with different CL variants. NBD-Tim50IMS(S208C) was incubated with varying concentrations of vesicles containing the indicated lipid compositions [described in (C)] and used for spectral analysis. The extent of interaction is reported as the fractional increase in emission intensity at the λmax (F/F0). (E) Tim50IMS-bilayer interaction with increasing TOCL concentration. NBD-Tim50IMS(S208C) was incubated with liposomes containing up to 40 mol % TOCL, and the fractional increase in emission intensity was measured.

  • Fig. 8 Substrate-dependent changes in the Tim50-Tim23 interaction with and without CL.

    Analysis of in organello cross-linking between Tim50 and [35S]Tim23(S80C) (left) or between Tim50 and [35S]Tim23(F114C) (right) was conducted as above (Fig. 2), except in the presence of increasing pSu9-DHFR concentration. Panels above each graph show a representative cross-linked adduct between Tim50 and the relevant [35S]Tim23 construct (closed arrowhead). Relative cross-linking efficiency means represent relative efficiencies for each substrate titration type (relative to the highest efficiency), taken from a minimum of four independent experiments, and error bars represent SDs. Dots indicate significant differences in comparison to results from the respective no-substrate control by Student’s t test (P < 0.05; ••P < 0.01). (A) Cross-linking in the presence of CL. Experiments were conducted using mitochondria isolated from WT yeast strains. (B) Cross-linking in the absence of CL. Experiments were conducted using mitochondria isolated from the Δcrd1 yeast strain.

  • Fig. 9 Working models for Tim50IMS-bilayer and Tim50-Tim23 interactions.

    (A) Interaction between Tim50IMS and CL-containing bilayers. In solution, Tim50IMS is initially attracted to the negatively charged bilayer by long-range Coulombic forces. The electric field orients a basic face of the protein, including the β-hairpin, toward the bilayer. Upon electrostatic docking, hydrophobic interactions between the nonpolar residues at the binding interface and the nonpolar bilayer core stabilize the interaction in a manner stimulated by the HII propensity of CL. Coupled to membrane association are changes in Tim50IMS conformation relative to its water-solvated state. Membrane association of Tim50IMS will be promoted if these Coulombic and hydrophobic interactions are favorable enough to offset the energetic penalty of desolvation. Note that the left panel displays the Tim50IMS homology model of this study; subsequent panels show hypothetical outlines of the Tim50IMS structure in cyan, either in the membrane bound state or in the presence of the TIM23 complex. (B) CL- and substrate-dependent Tim50 dynamics in the context of the TIM23 complex. The presence of CL (left) favors the membrane-associated state of Tim50IMS, which facilitates interaction with the channel domain of Tim23. In the presence of substrate, Tim50IMS retains its interaction with both the Tim23IMS and Tim23 channel regions. The absence of CL (right) favors the membrane-dissociated state of Tim50IMS, which blocks its interaction with the Tim23 channel region. The presence of substrate further promotes the membrane-dissociated state of Tim50IMS. Note that the stippled region of Tim50 depicts the β-hairpin.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1700532/DC1

    fig. S1. The TIM23 complex and CL biogenesis.

    fig. S2. The effect of CL synthase knockout on Tim50IMS functionality and association with the TIM23 complex.

    fig. S3. Import and integration of [35S]Tim23 into isolated mitochondria.

    fig. S4. Response of [35S]Tim23 cross-linking to decreased Δψm.

    fig. S5. Incorporation of [35S]Tim23 into nanodiscs: control tests.

    fig. S6. Tim50IMS structural predictions and SAXS analyses.

    fig. S7. SAXS analysis of Tim50CORE.

    fig. S8. Effect of ionic strength on the Tim50IMS-bilayer interaction.

    fig. S9. Quantitative analysis of Tim50IMS binding.

    fig. S10. Structural features of the Tim50IMS homology model.

    table S1. SAXS structural parameters.

    movie S1. Tim50IMS-bilayer no CL.

    movie S2. Tim50IMS-bilayer with CL.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The TIM23 complex and CL biogenesis.
    • fig. S2. The effect of CL synthase knockout on Tim50IMS functionality and
      association with the TIM23 complex.
    • fig. S3. Import and integration of 35STim23 into isolated mitochondria.
    • fig. S4. Response of 35STim23 cross-linking to decreased Δψm.
    • fig. S5. Incorporation of 35STim23 into nanodiscs: control tests.
    • fig. S6. Tim50IMS structural predictions and SAXS analyses.
    • fig. S7. SAXS analysis of Tim50CORE.
    • fig. S8. Effect of ionic strength on the Tim50IMS-bilayer interaction.
    • fig. S9. Quantitative analysis of Tim50IMS binding.
    • fig. S10. Structural features of the Tim50IMS homology model.
    • table S1. SAXS structural parameters.

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Tim50IMS-bilayer no CL.
    • movie S2 (.avi format). Tim50IMS-bilayer with CL.

    Files in this Data Supplement: