Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria

Abstract

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

Figure 2

EM and projection map of purified Tom40. (A) Survey view of negatively stained Tom40 particles. The image was filtered to the first zero of the electron microscope transfer function. (B and C) Statistical analysis of Tom40 particles. From electron micrographs a total of 1,550 Tom40 particles were extracted and subjected to multireference alignment. Based on eigenimage analysis, the data sets of the two most prominent groups were split into 20 classes. (B) Class averages of one pore particle images. (C) Group averages of the two pore classes. The numbers given for a specific class average represent the number of merged particle images. Group averages containing <10 particle images were omitted. Bars: (A) 20 nm; (B and C) 10 nm.

Figure 1

Purification of Tom40. (A) Purified TOM core complex carrying Tom22 with a hexahistidinyl tag was solubilized in 0.1% DDM and bound to an Ni-NTA affinity column. Tom40 and Tom7 were eluted with 3% (wt/vol) OG; Tom22 and Tom6 were eluted with 300 mM imidazole. Aliquots of the resulting column fractions were analyzed by high Tris/urea SDS-PAGE and staining with Coomassie blue. (Lane 1) TOM core complex; (lanes 2 and 3) column fractions 3 and 5 of the OG eluate; (lane 4) column fraction 3 of the imidazole eluate. (B) Fractions containing Tom40 were pooled and further purified by anion exchange chromatography on a Resource Q column equilibrated with 0.5% DDM. The peak fraction of the column was analyzed by high Tris/urea SDS-PAGE and Coomassie staining. Tom7 and residual amounts of Tom6 were removed completely from Tom40 after passage over the anion exchange column. (C) SDS-PAGE of VDAC isolated from N. crassa mitochondria. The gel was stained with Coomassie blue. (D) Size exclusion chromatography of isolated Tom40. Tom40 purified by anion exchange chromatography was subjected to gel filtration on a Superose 6 column equilibrated with 0.1% DDM. Column fractions (16–33) were analyzed by SDS-PAGE and staining with Coomassie (top). For comparison, gel filtration analysis of purified TOM core complex is shown at the bottom. Protein M _r standards: ApoF, apoferritin (M _r 443,000); ADH, alcohol dehydrogenase (M _r 155,000); CA, carboanhydrase (M _r 29,000).

Figure 3

Far ultraviolet CD spectra of Tom40, TOM core complex, and mitochondrial porin isolated from N. crassa mitochondria. (A) Tom40; (B) TOM core complex; and (C) porin. Protein was solubilized in 0.1% DDM, 50 mM potassium-acetate/MOPS, pH 7.0, 10% glycerol. 10 scans were accumulated at 4°C. The protein concentrations of Tom40 (2.5 μM) and mitochondrial porin (6.9 μM) relevant for computing the molar ellipiticities Θ_R were determined by UV absorbance spectroscopy. The protein concentration of the TOM core complex was determined using a colorimetric assay.

Figure 4

Original and deconvoluted FTIR spectra of Tom40, TOM core complex, and mitochondrial porin. The spectra of Tom40 (A), TOM core complex (C), and porin (E) were recorded on thin films on Ge crystals applying the ATR approach. After the end of measurements, the films were exposed to D₂O-saturated nitrogen gas for 120 min, and spectra of deuterated protein were recorded (B, D, and F) in order to separate contributions of α-helix from random coil components. Spectral bands assigned to α-helix structure are centered at ∼1,650 cm⁻¹, random components at 1,645–1,640 cm⁻¹, and β-sheet at 1,630–1,625 cm⁻¹. The shoulder at 1,695 cm⁻¹ indicates antiparallel β-sheet with particularly short turns. For all spectra, the baseline and residual water vapor components were subtracted if necessary.

Figure 5

Single-channel recording of isolated Tom40. (A) Purified Tom40 (∼4 μg/ml final protein concentration) was added to both sides of a black lipid membrane formed by diphytanoyl phosphatidyl choline/n-decane/butanol, and single channel conductances were measured in the presence of a membrane potential of +20 mV. (B) Histogram of channel conductances. P(G) is the probability that a given conductance increment G is observed. A total of n = 114 conductance increments were analyzed. The aqueous phase contained 1 M KCl, 5 mM Hepes, pH 7.0.

Figure 6

Properties of single channels of Tom40 and purified TOM core complex. Samples of current traces of channels of TOM core complex (A) and purified Tom40 (B). Currents were recorded after voltage jumps from 0 mV to the voltages indicated on the left of the traces. The main conductance levels are indicated on the right of the traces recorded at +80 and −80 mV. (C) Blockade of a channel from purified Tom40 by a mitochondrial presequence peptide. Currents were recorded after voltage jumps from 0 mV to the voltages indicated on the left of the traces. Left, control, before peptide addition; middle, after addition to the cis (cytosolic) side of a peptide corresponding to the first 32 residues of yeast pF₁β (final concentration 0.5 μM); right, after further addition of the same peptide to the trans (intermembrane space) side (final concentration 1 μM). The orientation of the channels in the bilayer was determined from the polarity of the membrane potential at which the characteristic flicker occurred (Künkele et al. 1998b). (D) Properties of nontypical channels formed by purified Tom40. Samples of current traces recorded after voltage jumps from 0 mV to the voltages indicated on the left of the traces. The first type (right) is voltage dependent and exhibits rectification. The second type (right) does not rectify and is not voltage dependent. Both types are devoid of the characteristic flicker. The dashed lines represent the 0 pA levels. For all records, the cis and trans compartments contained 150 mM KCl, 10 mM Hepes, pH 7.0. Data were sampled at 400 Hz and filtered at 200 Hz.