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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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Preprotein binding to TOM core complex. Recombinant pSu9-DHFR (100 μg) was incubated with purified mitochondrial outer membrane vesicles (200 μg). They were incubated with DDM and subjected to gel filtration on TSK G-4000 PWXL. TOM complex and pSu9-DHFR in the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing Tom22 and DHFR. As a control, the same protocol was performed with DHFR instead of pSu9-DHFR.
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Figure 6: Preprotein binding to TOM core complex. Recombinant pSu9-DHFR (100 μg) was incubated with purified mitochondrial outer membrane vesicles (200 μg). They were incubated with DDM and subjected to gel filtration on TSK G-4000 PWXL. TOM complex and pSu9-DHFR in the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing Tom22 and DHFR. As a control, the same protocol was performed with DHFR instead of pSu9-DHFR.

Mentions: Does the TOM core complex retain its ability to bind a chemically pure preprotein in the presence of detergent? To address this question, we incubated chemical amounts of pure preprotein (pSu9-DHFR) with mitochondrial outer membrane vesicles. Membranes were solubilized with DDM at a concentration identical to that used for the isolation of the core complex directly from mitochondria, and the lysate was subjected to size exclusion chromatography. All column fractions were analyzed by SDS-PAGE and immunoblotting. A large fraction of pSu9-DHFR coeluted with Tom22 (Fig. 6) and Tom40 (not shown) in a high molecular weight complex. Only background binding was observed when DHFR lacking a mitochondrial presequence was analyzed, excluding the possibility that formation of the TOM-pSu9-DHFR complex was the result of unspecific binding. Thus, pSu9-DHFR remained firmly bound to the TOM core complex in a signal-sequence dependent manner, even at high levels of nonionic detergent.


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)

Preprotein binding to TOM core complex. Recombinant pSu9-DHFR (100 μg) was incubated with purified mitochondrial outer membrane vesicles (200 μg). They were incubated with DDM and subjected to gel filtration on TSK G-4000 PWXL. TOM complex and pSu9-DHFR in the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing Tom22 and DHFR. As a control, the same protocol was performed with DHFR instead of pSu9-DHFR.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169338&req=5

Figure 6: Preprotein binding to TOM core complex. Recombinant pSu9-DHFR (100 μg) was incubated with purified mitochondrial outer membrane vesicles (200 μg). They were incubated with DDM and subjected to gel filtration on TSK G-4000 PWXL. TOM complex and pSu9-DHFR in the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing Tom22 and DHFR. As a control, the same protocol was performed with DHFR instead of pSu9-DHFR.
Mentions: Does the TOM core complex retain its ability to bind a chemically pure preprotein in the presence of detergent? To address this question, we incubated chemical amounts of pure preprotein (pSu9-DHFR) with mitochondrial outer membrane vesicles. Membranes were solubilized with DDM at a concentration identical to that used for the isolation of the core complex directly from mitochondria, and the lysate was subjected to size exclusion chromatography. All column fractions were analyzed by SDS-PAGE and immunoblotting. A large fraction of pSu9-DHFR coeluted with Tom22 (Fig. 6) and Tom40 (not shown) in a high molecular weight complex. Only background binding was observed when DHFR lacking a mitochondrial presequence was analyzed, excluding the possibility that formation of the TOM-pSu9-DHFR complex was the result of unspecific binding. Thus, pSu9-DHFR remained firmly bound to the TOM core complex in a signal-sequence dependent manner, even at high levels of nonionic detergent.

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