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The TOM core complex: the general protein import pore of the outer membrane of mitochondria.

Ahting U, Thun C, Hegerl R, Typke D, Nargang FE, Neupert W, Nussberger S - J. Cell Biol. (1999)

Bottom Line: It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles.Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm.Tom40 is the key structural element of the TOM core complex.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie der Universität München, D-80336 München, Germany.

ABSTRACT
Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.

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Channel conductance of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Purified TOM core complex (2 μg ml−1 final concentration) was added to both sides of a black lipid bilayer formed of diphytanoyl phosphatidyl choline/n-decane. Single channel conductances were measured at a membrane potential of ΔV = +20 mV. Histograms of channel conductances in polymer-free solution (A), in the presence of PEG1000 (B), and in the presence of PEG8000 (C). P(G) is the probability that a given conductance increment G is observed. D, Dependence of PEG-induced channel conductance change on the polymer weight. The electrolytes contained 1 M KCl, 5 mM Hepes, pH 7.0; the PEG concentrations were 20% (wt/vol), respectively. The data represent the mean conductances of n = 84, 56, 59, 22, 97, 53, 54, and 31 measurements in the absence and the presence of PEG 200, PEG 1000, PEG 3,350, PEG 6000, PEG 8,000, PEG 12,000, and PEG 20,000, respectively. Note that addition of PEG to a polymer-free solution decreases the single channel conductance due to the reduced bulk electrolyte conductance.
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Figure 5: Channel conductance of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Purified TOM core complex (2 μg ml−1 final concentration) was added to both sides of a black lipid bilayer formed of diphytanoyl phosphatidyl choline/n-decane. Single channel conductances were measured at a membrane potential of ΔV = +20 mV. Histograms of channel conductances in polymer-free solution (A), in the presence of PEG1000 (B), and in the presence of PEG8000 (C). P(G) is the probability that a given conductance increment G is observed. D, Dependence of PEG-induced channel conductance change on the polymer weight. The electrolytes contained 1 M KCl, 5 mM Hepes, pH 7.0; the PEG concentrations were 20% (wt/vol), respectively. The data represent the mean conductances of n = 84, 56, 59, 22, 97, 53, 54, and 31 measurements in the absence and the presence of PEG 200, PEG 1000, PEG 3,350, PEG 6000, PEG 8,000, PEG 12,000, and PEG 20,000, respectively. Note that addition of PEG to a polymer-free solution decreases the single channel conductance due to the reduced bulk electrolyte conductance.

Mentions: To test whether the isolated core complex contains pores, we analyzed its channel forming activity after reconstitution into lipid membranes. Purified core complex was added to both sides of a black lipid membrane bilayer. Current recordings showed characteristic steps of conductance increase that reflect insertion of the core complex into the lipid bilayer. An average conductance of ∼2.3 nS in the presence of 1 M KCl was observed (Fig. 5 A). The trypsin-treated TOM holo complex had an average conductance of 2.7 nS (data not shown). These average conductances were similar to that of the holo complex (2.3 nS in 1 M KCl; Künkele et al. 1998). The hydrophilic import receptor domains of Tom70, Tom22, and Tom20 apparently play only a minor role in the channel properties of the TOM complex.


The TOM core complex: the general protein import pore of the outer membrane of mitochondria.

Ahting U, Thun C, Hegerl R, Typke D, Nargang FE, Neupert W, Nussberger S - J. Cell Biol. (1999)

Channel conductance of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Purified TOM core complex (2 μg ml−1 final concentration) was added to both sides of a black lipid bilayer formed of diphytanoyl phosphatidyl choline/n-decane. Single channel conductances were measured at a membrane potential of ΔV = +20 mV. Histograms of channel conductances in polymer-free solution (A), in the presence of PEG1000 (B), and in the presence of PEG8000 (C). P(G) is the probability that a given conductance increment G is observed. D, Dependence of PEG-induced channel conductance change on the polymer weight. The electrolytes contained 1 M KCl, 5 mM Hepes, pH 7.0; the PEG concentrations were 20% (wt/vol), respectively. The data represent the mean conductances of n = 84, 56, 59, 22, 97, 53, 54, and 31 measurements in the absence and the presence of PEG 200, PEG 1000, PEG 3,350, PEG 6000, PEG 8,000, PEG 12,000, and PEG 20,000, respectively. Note that addition of PEG to a polymer-free solution decreases the single channel conductance due to the reduced bulk electrolyte conductance.
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Related In: Results  -  Collection

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Figure 5: Channel conductance of the TOM core complex in the presence of differently sized nonelectrolyte polymers. Purified TOM core complex (2 μg ml−1 final concentration) was added to both sides of a black lipid bilayer formed of diphytanoyl phosphatidyl choline/n-decane. Single channel conductances were measured at a membrane potential of ΔV = +20 mV. Histograms of channel conductances in polymer-free solution (A), in the presence of PEG1000 (B), and in the presence of PEG8000 (C). P(G) is the probability that a given conductance increment G is observed. D, Dependence of PEG-induced channel conductance change on the polymer weight. The electrolytes contained 1 M KCl, 5 mM Hepes, pH 7.0; the PEG concentrations were 20% (wt/vol), respectively. The data represent the mean conductances of n = 84, 56, 59, 22, 97, 53, 54, and 31 measurements in the absence and the presence of PEG 200, PEG 1000, PEG 3,350, PEG 6000, PEG 8,000, PEG 12,000, and PEG 20,000, respectively. Note that addition of PEG to a polymer-free solution decreases the single channel conductance due to the reduced bulk electrolyte conductance.
Mentions: To test whether the isolated core complex contains pores, we analyzed its channel forming activity after reconstitution into lipid membranes. Purified core complex was added to both sides of a black lipid membrane bilayer. Current recordings showed characteristic steps of conductance increase that reflect insertion of the core complex into the lipid bilayer. An average conductance of ∼2.3 nS in the presence of 1 M KCl was observed (Fig. 5 A). The trypsin-treated TOM holo complex had an average conductance of 2.7 nS (data not shown). These average conductances were similar to that of the holo complex (2.3 nS in 1 M KCl; Künkele et al. 1998). The hydrophilic import receptor domains of Tom70, Tom22, and Tom20 apparently play only a minor role in the channel properties of the TOM complex.

Bottom Line: It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles.Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm.Tom40 is the key structural element of the TOM core complex.

View Article: PubMed Central - PubMed

Affiliation: Institut für Physiologische Chemie der Universität München, D-80336 München, Germany.

ABSTRACT
Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.

Show MeSH
Related in: MedlinePlus