Import of p34 into isolated rat liver mitochondria.

<p>(<b>A</b>) Proteolysis of p34. Radiolabelled p34 was synthesized in reticulocyte lysate in the presence of ³⁵S-methionine and incubated with increasing concentrations of proteinase K (PK) as indicated. The samples were incubated for 10 min at 0°C, proteolysis was stopped by addition of PMSF. Aliquots were analyzed by SDS-PAGE. The radiolabelled protein was visualized and the relative amounts were determined using a phosphoimager. At 10 µg PK/ml, about 1% of p34 was retained, no p34 was detected at 20 µg PK/ml. (<b>B</b>) Import of p34 into mitochondria and digitonin fractionation. ³⁵S-labelled p34 and Tom70 (a subunit of 70 kDa of the protein translocase of the mitochondrial outer membrane) were synthesized in reticulocyte lysate and incubated with freshly isolated rat liver mitochondria for 10 min at 25°C. The mitochondria were reisolated by centrifugation and resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS KOH, pH 7.2. As indicated, digitonin was added at increasing concentrations (up to 1% w/v), or proteinase K (PK) at a final concentration of 50 µg/ml, respectively. Proteolysis was stopped by addition of PMSF, and the mitochondria were collected by centrifugation. The proteins were separated by SDS-PAGE for subsequent analysis by digital autoradiography using a phosphoimager. (<b>C</b>) Import after exchange of residues in the p34 N-terminus. Reticulocyte lysate containing ³⁵S-labelled p34 was incubated with mitochondria for 10 min at 25°C (upper panel). Parallel samples contained mutant versions of p34 (P9A, G14A, K33A). Samples 3 and 4 contained valinomycin to dissipate the membrane potential, samples 2 and 4 were subsequently incubated with proteinase K (PK, final conc. 25 µg/ml). The mitochondria were reisolated and the proteins were separated by SDS-PAGE. (<b>D</b>) Proteolysis of p34(1–35)-DHFR. The experiment was carried out as in (A). (<b>E</b>) Function of the p34 N-terminus. Left panel (lanes 1 and 2), incubation of N-terminally truncated p34 (residues 37-319) with mitochondria. Reticulocyte lysate containing the radiolabelled protein was incubated with isolated rat liver mitochondria as in (C). 25 µg/ml proteinase K were subsequently added to sample 2 to degrade the protein outside the mitochondria. Right panel (lanes 3–5), import of a hybrid protein comprising the 35 N-terminal residues of p34 fused to the entire dihydrofolate (DHFR) of the mouse. Lane 3, sample of reticulocyte lysate containing the radiolabelled hybrid protein p34(1–35)-DHFR and DHFR. Lanes 4 and 5, incubation of the lysate with mitochondria and reisolation of the mitochondria. Proteinase K was added to sample 5 (+PK). Asterix (*), degradation product of the hybrid protein. (<b>F</b>) Import of p34 into trypsin-pretreated mitochondria. Rat liver mitochondria were pretreated with trypsin (20 µg/ml) for 10 min at 0°C, proteolysis was stopped by addition of soybean trypsin inhibitor and the mitochondria were reisolated. ³⁵S-labelled p34 was synthesized in reticulocyte lysate and incubated with the mitochondria at 25°C for different times as indicated. The trypsin pretreatment was omitted in parallel samples. The mitochondria were again reisolated and the proteins were separated by SDS-PAGE. The relative amounts of ³⁵S-labelled p34 were determined using a phosphoimager. The highest value was set to 100% (control). (<b>G</b>) Import of p34(1–35)-DHFR into trypsin-pretreated mitochondria. The experiment was carried out as in (F).</p

Distribution of p34 in intact cells.

<p>(<b>A</b>) Hybrid proteins containing an EGFP domain (enhanced green fluorescent protein) fused to the complete p34 (residues 1–319), or parts of p34 comprising residues 37–319 or 1–36, respectively, were expressed in HeLa cells (green fluorescence) and their distribution was monitored by confocal microscopy. About 10% of the cells were found to be transfected. Mitochondria were visualized with a Tom20-specific labelling (red colour; Tom20 is a subunit of the protein translocase of the mitochondrial outer membrane), the DNA of the cells was stained with DAPI (blue). The overlay (right panel) shows the co-localization between the EGFP-labelled proteins and Tom20 (yellow). (<b>B</b>) Hydrophobicity plot of p34(1–319) and sequence of the 32 N-terminal residues.</p

Import of p34 into isolated yeast mitochondria.

<p>(<b>A</b>) Protease-sensitivity of acid-pretreated p34. Reticulocyte lysate containg ³⁵S-labelled p34 was pretreated with HCl at pH 5 for 5 min, diluted twentyfold into 1 mM EDTA, 10 mM MOPS/KOH pH 7.4, and incubated with proteinase K at increasing concentrations. (<b>B</b>) Import of p34 into mitochondria. p34 was imported as described in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000878#ppat-1000878-g002" target="_blank">Fig. 2</a>, using yeast mitochondria instead of mammalian mitochondria. The organelles were isolated from the wild type strain PK82 (lanes 1–7) or from the mutant strain <i>ssc1-3</i> (lane 8). No mitochondria were added in the sample of lane 4. (<b>C</b>) Import of p34 in protease-pretreated yeast mitochondria. The mitochondria were incubated with trypsin (20 µg/ml) for 10 min at 0°C, proteolysis was stopped by addition of soybean trypsin inhibitor and the mitochondria were reisolated. ³⁵S-labelled p34 was incubated with the mitochondria at 25°C for different times as indicated. (<b>D</b>) Import of p34 into mitochondria isolated from a strain lacking the outer membrane import receptor Tom20 (<i>tom20Δ</i>). Parallel samples contained mitochondria from the corresponding wildtype (WT). The standard deviation was calculated from 3 experiments, the highest value of each series was set to 100% (control). (<b>E</b>) Relative import efficiencies of p34(1–319), p34(1–35)-DHFR, and porin with mitochondria isolated from yeast strains lacking Tom20 or Tom70, respectively. The radiolabelled proteins were incubated with the mitochondria for 5 min at 25°C, reisolated, and treated with proteinase K. In parallel samples, the proteins were incubated with mitochondria isolated from the corresponding wildtype strains. The proteins were analyzed by SDS-PAGE and the relative amounts of the radiolabelled proteins were determined using the phosphoimager. The amounts detected in wildtype mitochondria were set to 100%. The standard deviations were calculated from 5 independent experiments. (<b>F</b>) Import of ³⁵S-labelled p34 into mitochondria isolated from a yeast strain containing a defect in Tom40 (<i>tom40-4</i>). The highest value of each experiment was set to 100% (control), n = 3. (<b>G</b>) Import of radiolabelled p34 into mitochondria and preparation of outer and inner membrane vesicles. ³⁵S-labelled p34 was imported into isolated yeast mitochondria, the mitochondria were reisolated and sonified. The membrane vesicles were layered on top of a sucrose step gradient and centrifuged for 16 h at 100.000 g. Fractions were collected for TCA-precipitation of proteins and subsequent analysis by SDS-PAGE, immuno blotting and visualization of radiolabelled p34 by digital autoradiography. Polyclonal antisera were used for labelling of Tom40 (outer membrane) and Tim23 (inner membrane), respectively. Fraction 1, upper part of the gradient (0.85 M sucrose); fraction 8, lower part of the gradient (1.6 M sucrose). Upper panel, radiolabelled p34 as visualized by digital autoradiography; lower panel, immuno blotting to visualize Tom40 and Tim23. (<b>H</b>) Quantification of the proteins shown in (G), the highest values were set to 100%.</p

Electrophysiology of p34.

<p>(<b>A</b>) Current recordings of a bilayer containing purified p34 at indicated holding potential. Buffer conditions were symmetrical with 1.5 M KCl, 10 mM MOPS/Tris, pH 7.0 in <i>cis</i> and <i>trans</i> chamber. (<b>B</b>) Current-voltage relationship of p34. The conductance was calculated from the slope of the graph. (<b>C</b>) Current recordings of a bilayer containing p34(37–319) at holding potential as indicated. Buffer conditions were symmetrical with 1.5 M KCl, 10 mM Na-Acetat, pH 4.0 in <i>cis</i> and <i>trans</i> chamber. (<b>D</b>) Current-voltage relationship of p34(37–319). (<b>E</b>) Current recordings of a bilayer containing p34 at holding potential as indicated. The buffer conditions were the same as in (A). (<b>F</b>) Current recordings in the presence of 100 µM NPPB.</p

Complex formation of purified p34.

<p>(<b>A</b>) p34 was expressed in <i>Escherichia coli</i> and purified using conventional methods. Lane 1, inclusion bodies collected by centrifugation; lane 2, proteins dissolved in presence of 8 M urea and precipitated by ammonium sulfate (30% saturation); lane 3, eluate from Phenyl-Sepharose; lane 4, flow through from DEAE-Sephacel column. The proteins were separated by SDS-PAGE and the gel was stained by Coomassie. (<b>B</b>) CD spectra of p34. The purified protein was dissolved in 8 mM N-Decyl-β-D-Maltopyranosid, 10 mM KCl, 20 mM K₂HPO₄/KH₂PO₄, pH 7.0 and analyzed using a Jasco J-810 spectrapolarimeter. (<b>C</b>) Chemical cross-linking. Purified p34 was incubated with 50 µM DSS (lane 2) or 0.5 mM Sulfo-MBS (lanes 3 and 4) for 30 min at 0°C. The reaction was stopped by addition of 0.5 M Tris-HCl pH 7.4, the proteins were precipitated by TCA, separated by SDS-PAGE, transferred on nitrocellulose, and labelled using a polyclonal antiserum. (<b>D</b>) Analysis of p34 in BN-PAGE. Purified p34 was dissolved in 0.5% Triton X-100, 10% Glycerol, 50 mM NaCl, 0.1 mM EDTA, PMSF 1 mM, 20 mM Tris-HCl pH 7.0, and applied on a gel for BN-PAGE in the presence of 500 mM ε-aminocaproic acid (first dimension). A lane from the gel was excised and layered on top of a conventional SDS-PAGE for separation of the proteins under denaturing conditions (second dimension). The proteins were transferred on nitrocellulose and labelled using a polyclonal antiserum directed against p34 (upper panel). Truncated p34 comprising residues 37–319 was similarly analyzed (middle panel), or dissolved in the presence of 8 M urea and 1% dodecylmaltoside for separation in the first dimension (lower panel).</p