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

Ahting U, Thieffry M, Engelhardt H, Hegerl R, Neupert W, Nussberger S - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Universität München, D-81377 München, Germany.

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.

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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.
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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.

Mentions: Tom40 and TOM core complex channels were further characterized in single channel records (Fig. 6). They were either directly integrated into the bilayer by adding detergent-solubilized protein to the bath solution or they were reconstituted into proteoliposomes, which were subsequently fused to lipid bilayers. The channels described previously for the core complex showed cation selectivity and were characterized by three main conductance levels separated from the fully open state (1,100 pS in 150 mM KCl) by two identical jumps of 440 pS (Fig. 6 A). At ∼0 mV, they were fully open, and they closed with slow kinetics at potentials of either polarity. In addition, a fast flicker between the three main conductance levels occurred only at voltages of one polarity around 70 mV (Fig. 6 A). These characteristics are similar to those of the dimeric peptide-sensitive channel (PSC) identified in outer membrane and holo complex fractions of Neurospora (Künkele et al. 1998b). The channels most often found in the Tom40 fraction had similar selectivity and voltage-dependence properties, but their maximum conductance (550 pS in 150 mM KCl) was half that of the channels described above. They exhibited only two main conductance levels separated by jumps of 440 pS and a fast flicker at voltages of one polarity (Fig. 6 B). They are thus similar to the monomeric form of the Neurospora PSC described previously (Künkele et al. 1998b). This form was also found in the core complex fraction but with a lower probability than the dimeric form. These results indicate that Tom40 can form the protein translocation channel of the mitochondrial outer membrane.


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

Ahting U, Thieffry M, Engelhardt H, Hegerl R, Neupert W, Nussberger S - J. Cell Biol. (2001)

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.
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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.
Mentions: Tom40 and TOM core complex channels were further characterized in single channel records (Fig. 6). They were either directly integrated into the bilayer by adding detergent-solubilized protein to the bath solution or they were reconstituted into proteoliposomes, which were subsequently fused to lipid bilayers. The channels described previously for the core complex showed cation selectivity and were characterized by three main conductance levels separated from the fully open state (1,100 pS in 150 mM KCl) by two identical jumps of 440 pS (Fig. 6 A). At ∼0 mV, they were fully open, and they closed with slow kinetics at potentials of either polarity. In addition, a fast flicker between the three main conductance levels occurred only at voltages of one polarity around 70 mV (Fig. 6 A). These characteristics are similar to those of the dimeric peptide-sensitive channel (PSC) identified in outer membrane and holo complex fractions of Neurospora (Künkele et al. 1998b). The channels most often found in the Tom40 fraction had similar selectivity and voltage-dependence properties, but their maximum conductance (550 pS in 150 mM KCl) was half that of the channels described above. They exhibited only two main conductance levels separated by jumps of 440 pS and a fast flicker at voltages of one polarity (Fig. 6 B). They are thus similar to the monomeric form of the Neurospora PSC described previously (Künkele et al. 1998b). This form was also found in the core complex fraction but with a lower probability than the dimeric form. These results indicate that Tom40 can form the protein translocation channel of the mitochondrial outer membrane.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie, Universität München, D-81377 München, Germany.

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.

Show MeSH
Related in: MedlinePlus