Article Figures & Data
Figures
- FIG. 1.
Interaction of the targeting signal of BSC1 with the TOM complex. (A) BCS1(1-126)-DHFR and pSu9(1-69)-DHFR were incubated with N. crassa OMV for 20 min at 0°C in the presence of MTX/NADPH. OMV were then treated with buffer containing either 20 or 200 mM KCl. The OMV were reisolated and resuspended inbuffer, and the suspensions were halved. One half was treated with trypsin at 0°C (folded material), while the second half was kept at 0°C (bound). Proteins were then analyzed by SDS-PAGE and phosphorimaging. The amount of protein bound at 20 mM salt was set to 100%. (B) Preprotein bound to OMV can be coimmunoprecipitated with components of the TOM complex. Radiolabeled BCS1 and BCS1 (1-126)-DHFR were incubated with OMV at 0°C in the presence of MTX/NADPH or at 25°C in the absence of MTX/NADPH. The reaction mixtures were adjusted to 20 or 200 mM KCl at 0°C, and OMV were reisolated and resuspended in SEM buffer. Immunoprecipitation was performed with antibodies raised against Tom22 or Tom40 or with preimmune serum. To control for binding, an aliquot was removed before the coimmunoprecipitation and precipitated with trichloroacetic acid (TCA) (Total). (C) Precursor of BCS1 interacts with purified TOM complex. Radiolabeled precursor of BCS1 was incubated for 20 min at 25°C with purified TOM core complex. Immunoprecipitation was performed with antibodies or preimmune serum, as described for panel B. To exclude unspecific interactions, immunoprecipitation was also performed in the absence of the TOM complex (−TOM). (D) A matrix-destined precursor can outcompete the precursor of BCS1(1-126)-DHFR. Radiolabeled precursor of BCS1(1-126)-DHFR was incubated for 20 min at 25°C with either mitochondria alone (−) or with mitochondria preincubated with the indicated amounts of proteins for 2 min on ice. The mitochondria were either intact (upper panel) or pretreated with trypsin before incubation with proteins (lower panel). At the end of the import reactions, mitochondria were treated with proteinase K, washed, reisolated, and analyzed by SDS-PAGE.
- FIG. 2.
Precursor of BCS1 interacts with receptor components of the TOM complex. (A) BCS1 is in the vicinity of Tom20, Tom22, and Tom40 on its insertion pathway. Radiolabeled BCS1(1-126)-DHFR precursor was incubated with isolated OMV for 30 min at 0°C. OMV were reisolated and resuspended in SEM buffer. One aliquot was left on ice (−DFDNB), while the chemical cross-linker DFDNB was added to the others for 40 min on ice. Aliquots were subjected to immunoprecipitation with antibodies against Tom20, Tom22, or Tom40 or with preimmune serum (PIS). Asterisks, adducts consisting of BCS1 cross-linked to Tom proteins. (B) BCS1 is in the vicinity of Tom70 on its insertion pathway. Radiolabeled BCS1(1-126)-DHFR precursor was incubated with isolated OMV for 2 min at 0°C. The sample was split; one aliquot was left on ice (−DSS), and the chemical cross-linker DSS was added to the other for 40 min on ice. Aliquots were subjected to immunoprecipitation with antibodies against Tom70 or with PIS. Asterisk, adduct consisting of BCS1 cross-linked to Tom70. A longer exposure of the immunoprecipitation with Tom70 is presented for clarity. (C) The tom70 null mutation affects import of BCS1. Radiolabeled precursors of BCS1 and pSu9-DHFR were incubated at 15°C for the indicated time periods with mitochondria from either tom70 null mutant (tom70) or its wild type parent (WT). At the end of the import, proteinase K was added, and mitochondria were reisolated and analyzed by SDS-PAGE. The protease-protected bands of BCS1 and mature Su9-DHFR were quantified. (D) Binding of mitochondrial preproteins to purified Tom70 cytosolic domain. The purified cytosolic domain of Tom70 was bound to an Ni-NTA column. Then radiolabeled preproteins were incubated with the bound protein for 30 min at 4°C. After a washing step, bound proteins were eluted with sample buffer and analyzed by SDS-PAGE. The total amount of each preprotein added was set to 100%.
- FIG. 3.
Screening of a peptide library with soluble receptor domains. Cytosolic domains of the indicated Tom components (150 nM) were incubated with a peptide library on a cellulose membrane covering amino acid residues 1 to 126 of BCS1 (length of peptides, 13 residues; overlap, 10 residues). The bound proteins were blotted to PVDF membranes and decorated with the corresponding antibody.The labeling indicates the numbers of the peptides in the beginning and the end of each row. Binding was quantified by scanning densitometry from three independent experiments. The various domains of BCS1 are displayed below the corresponding peptides. TM, transmembrane domain; H, putative presequence-like helix.
- FIG. 4.
BCS1 can bind to the TOM complex in the absence of the transmembrane domain. (A) The indicated radiolabeled precursors were incubated with OMV for 20 min at 0°C. Samples were thenadjusted to the indicated KCl concentrations. OMV were reisolated, dissolved in sample buffer, and analyzed for bound precursor proteins by SDS-PAGE and phosphorimaging. The amount of preprotein bound at 20 mM salt was set as 100%. (B) pBCS1(1-126)ΔTM-DHFR was incubated with OMV for 20 min at 25°C in the presence or absence of excess amounts of pSu9(1-69)-DHFR. The reaction mixtures were adjusted to 200 mM KCl at 0°C, and OMV were reisolated and resuspended in SEM buffer. Immunoprecipitation was performed with antibodies raised against Tom22 or Tom40 or with preimmune serum. To control for binding, an aliquot was removed before the coimmunoprecipitation and precipitated with TCA (Total). (C) pBCS1(1-126)-DHFR and pBCS1(1-126)ΔTM-DHFR were incubated with uncoupled mitochondria for 20 min at 25°C. The mitochondria were washed with a buffer containing 50 mM KCl, reisolated, and solubilized in 0.75% digitonin buffer. Further treatment and coimmunoprecipitation were as described in the legend to Fig. 1B. (D) The bands corresponding to immunoprecipitated proteins from three experiments as described in the legend for panel C were quantified. The average values are presented as percentages of the bound material.
- FIG. 5.
The sequence comprising residues 66 to 86 of BCS1 behaves like a typical mitochondrial presequence and interacts with Tomcomponents. (A) Radiolabeled pBCS1Δ1-65 and, as a control, pSu9 (1-69)-DHFR were incubated for 20 min at 15°C in SI buffer with either mitochondria (−) or mitochondria preincubated with the indicated amounts of competitor proteins for 2 min on ice. At the end of the import reactions, mitochondria were washed, reisolated, and analyzed by SDS-PAGE. p, precursor; i, intermediate-size form; m, mature form. (B) Residues 66 to 86 can direct a cytosolic protein into the mitochondrial matrix. Radiolabeled BCS1(66-86)-DHFR was incubated at 25°C for the indicated time periods with mitochondria in SI buffer in the absence or presence of valinomycin (1 μM). Mitochondria were reisolated, resuspended in SEM, and divided into two halves. One half was left on ice (−PK) while the other was treated with proteinase K (+PK). The import reactions were analyzed by SDS-PAGE. p, precursor form; m, mature form. (C) Residues 66 to 86 can promote interaction with the TOM complex. Radiolabeled pBCS1 (66-86)-DHFR was incubated with OMV for 20 min at 25°C. The OMV were washed with a buffer containing 100 mM KCl, reisolated, and solubilized in 0.75% β-dodecyl maltoside-containing buffer. Further treatment and immunoprecipitation (IP) were as described in the legend to Fig. 1B. (D) Radiolabeled BCS1(66-86)-DHFR precursor was incubated in the presence of MTX/NADPH with isolated OMV for 2 min at 0°C, followed by incubation for 5 min at 25°C. The chemical cross-linker N-succinimidyl[4-iodoacetyl]aminobenzoate (SIAB) was then added for a further 40 min at 10°C. Cross-linked samples were immunoprecipitated with antibodies against Tom70 or Tom40. Asterisks, adducts consisting of BCS1 cross-linked to Tom proteins.
- FIG. 6.
The sequence comprising residues 84 to 126 of BCS1 can promote binding to the TOM complex. (A) Precursor lacking the first 82 amino acid residues of BCS1 can bind to OMV in a receptor-dependent manner. Radiolabeled BCS1 and BCS1(Δ1-82) were incubated for 20 min at 25°C with either intact OMV or OMV pretreated with trypsin. OMV were then washed with buffer containing 200 mM KCl, reisolated, and analyzed by SDS-PAGE and phosphorimaging. The average of results of three experiments is presented. (B) Residues 84 to 126 of BCS1 promote binding to the TOM complex. Radiolabeled precursors of BCS1(84-126)-DHFR or pSu9(1-69)-DHFR for comparison were incubated for 20 min at 0°C with OMV in the absence (−Comp. precursor) or presence (+Comp. precursor) of excess amounts of pSu9(1-69)-DHFR. Further treatment was as described in the legend for panel A. The amount of protein bound to untreated OMV was set to 100%. (C) BCS1(84-126)-DHFR bound to OMV can be coimmunoprecipitated with components of the TOM complex. Radiolabeled precursor was incubated for 20 min at 25°C with OMV. The OMV were then treated with buffer containing 100 mM KCl. OMV were reisolated, pelleted, and resuspended in SEM buffer. Immunoprecipitation (IP) was performed with antibodies raised against Tom20 or Tom40 or with preimmune serum. To control for binding, an aliquot was removed before coimmunoprecipitation and precipitated with TCA (Total). (D) Residues 87 to 126 of BCS1 increase binding to OMV. BCS1(1-86)-DHFR and BCS1(1-126)-DHFR were incubated with OMV in the presence of MTX/NADPH at 15°C for the indicated time periods. OMV were then washed with buffer containing 20 mM KCl, reisolated, dissolved in sample buffer, and analyzed by SDS-PAGE and phosphorimaging. For each protein, the amount of radiolabeled precursor added to the reaction was set to 100%.
- FIG. 7.
BCS1 passes the TOM complex in a loop structure. (A) Radiolabeled DHFR-BCS1(1-250)-DHFR was incubated in a MTX/NADPH-containing buffer with mitochondria for 20 min at 25°C. The mitochondria were washed in a buffer containing 20 mM salt, resuspended in SEM buffer, and treated with the indicated concentration of trypsin for 15 min on ice. After inhibition of the protease by PMSF, samples were precipitated with TCA and analyzed by SDS-PAGE. The bands corresponding to the DHFR domain, the precursor protein (p), and the proteolytic fragments (f) are indicated. (B) Radiolabeled DHFR-BCS1(1-250)-DHFR was incubated with mitochondria as described in the legend for panel A. After treatment with trypsin (5 μg/ml), the mitochondria were spun down. Pellets were dissolved directly in sample buffer, while the supernatants were first precipitated with TCA and then dissolved in sample buffer. All samples were analyzed by SDS-PAGE. The bands corresponding to the DHFR domain and the proteolytic fragments (f) are indicated. (C) Radiolabeled DHFR-BCS1(1-250)-DHFR was incubated in a MTX/NADPH-containing SI buffer with mitochondria for 20 min at 25°C. The samples were halved; one aliquot was treated with proteinase K (20 μg/ml), and the other was left untreated. The mitochondria were sedimented, dissolved in buffer containing 0.4% digitonin, and analyzed by BNGE. The left panel shows the autoradiography, while the right panel represents immunodecoration of the same membrane with antibody against the cytosolic domain of Tom22. The radiolabeled precursor migrating with the TOM complex is indicated with an asterisk.
