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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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Purification of the TOM core complex. A, Isolation of the TOM core complex from mitochondria. Mitochondria from a Neurospora strain containing a Tom22 with a 6×His tag were solubilized in DDM and bound to Ni-NTA. Bound complex was eluted with imidazole. Fractions containing Tom40 were pooled and further purified by anion-exchange chromatography on a MonoQ column. Fractions were analyzed by SDS-PAGE and proteins were stained with Coomassie brilliant blue. Lane 1, Total mitochondrial protein solubilized in DDM; lane 2, eluant from Ni-NTA; lane 3, peak fraction of MonoQ chromatography. B, Size-exclusion chromatography on a TSK G4000 PWXL column of purified TOM core complex. The elution profile was revealed by monitoring absorption at 280 nm. The peak fraction corresponds to ∼410 kD. C, The peak fraction of the TSK sizing column analyzed by high Tris/urea SDS-PAGE and Coomassie staining. The asterisk denotes the band that possibly represents Tom5. D, Analysis of peak column fractions by SDS-PAGE and immunoblotting using antibodies against Tom40 and Tom20. Tom20 was completely removed from the core complex after passage over a Ni-NTA affinity, MonoQ, and TSK G4000 sizing column.
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Figure 2: Purification of the TOM core complex. A, Isolation of the TOM core complex from mitochondria. Mitochondria from a Neurospora strain containing a Tom22 with a 6×His tag were solubilized in DDM and bound to Ni-NTA. Bound complex was eluted with imidazole. Fractions containing Tom40 were pooled and further purified by anion-exchange chromatography on a MonoQ column. Fractions were analyzed by SDS-PAGE and proteins were stained with Coomassie brilliant blue. Lane 1, Total mitochondrial protein solubilized in DDM; lane 2, eluant from Ni-NTA; lane 3, peak fraction of MonoQ chromatography. B, Size-exclusion chromatography on a TSK G4000 PWXL column of purified TOM core complex. The elution profile was revealed by monitoring absorption at 280 nm. The peak fraction corresponds to ∼410 kD. C, The peak fraction of the TSK sizing column analyzed by high Tris/urea SDS-PAGE and Coomassie staining. The asterisk denotes the band that possibly represents Tom5. D, Analysis of peak column fractions by SDS-PAGE and immunoblotting using antibodies against Tom40 and Tom20. Tom20 was completely removed from the core complex after passage over a Ni-NTA affinity, MonoQ, and TSK G4000 sizing column.

Mentions: To isolate the TOM core complex directly from mitochondria in high amounts, mitochondria from a strain with a hexahistidinyl-tag on Tom22 were solubilized in DDM at a concentration of 1% (wt/vol). The extract was loaded onto a Ni-NTA affinity column and, after extensive washing, bound material was eluted with an imidazole gradient. Tom40, Tom22, and the smaller Tom components all coeluted within five major fractions that accounted for ∼0.2% of protein loaded onto the column (Fig. 2 A, lane 2). Further anion-exchange chromatography resulted in a virtually pure TOM core complex (Fig. 2 A, lane 3). The yield of purified core complex was ∼2 mg complex per 1 g isolated mitochondrial protein.


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)

Purification of the TOM core complex. A, Isolation of the TOM core complex from mitochondria. Mitochondria from a Neurospora strain containing a Tom22 with a 6×His tag were solubilized in DDM and bound to Ni-NTA. Bound complex was eluted with imidazole. Fractions containing Tom40 were pooled and further purified by anion-exchange chromatography on a MonoQ column. Fractions were analyzed by SDS-PAGE and proteins were stained with Coomassie brilliant blue. Lane 1, Total mitochondrial protein solubilized in DDM; lane 2, eluant from Ni-NTA; lane 3, peak fraction of MonoQ chromatography. B, Size-exclusion chromatography on a TSK G4000 PWXL column of purified TOM core complex. The elution profile was revealed by monitoring absorption at 280 nm. The peak fraction corresponds to ∼410 kD. C, The peak fraction of the TSK sizing column analyzed by high Tris/urea SDS-PAGE and Coomassie staining. The asterisk denotes the band that possibly represents Tom5. D, Analysis of peak column fractions by SDS-PAGE and immunoblotting using antibodies against Tom40 and Tom20. Tom20 was completely removed from the core complex after passage over a Ni-NTA affinity, MonoQ, and TSK G4000 sizing column.
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Related In: Results  -  Collection

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

Figure 2: Purification of the TOM core complex. A, Isolation of the TOM core complex from mitochondria. Mitochondria from a Neurospora strain containing a Tom22 with a 6×His tag were solubilized in DDM and bound to Ni-NTA. Bound complex was eluted with imidazole. Fractions containing Tom40 were pooled and further purified by anion-exchange chromatography on a MonoQ column. Fractions were analyzed by SDS-PAGE and proteins were stained with Coomassie brilliant blue. Lane 1, Total mitochondrial protein solubilized in DDM; lane 2, eluant from Ni-NTA; lane 3, peak fraction of MonoQ chromatography. B, Size-exclusion chromatography on a TSK G4000 PWXL column of purified TOM core complex. The elution profile was revealed by monitoring absorption at 280 nm. The peak fraction corresponds to ∼410 kD. C, The peak fraction of the TSK sizing column analyzed by high Tris/urea SDS-PAGE and Coomassie staining. The asterisk denotes the band that possibly represents Tom5. D, Analysis of peak column fractions by SDS-PAGE and immunoblotting using antibodies against Tom40 and Tom20. Tom20 was completely removed from the core complex after passage over a Ni-NTA affinity, MonoQ, and TSK G4000 sizing column.
Mentions: To isolate the TOM core complex directly from mitochondria in high amounts, mitochondria from a strain with a hexahistidinyl-tag on Tom22 were solubilized in DDM at a concentration of 1% (wt/vol). The extract was loaded onto a Ni-NTA affinity column and, after extensive washing, bound material was eluted with an imidazole gradient. Tom40, Tom22, and the smaller Tom components all coeluted within five major fractions that accounted for ∼0.2% of protein loaded onto the column (Fig. 2 A, lane 2). Further anion-exchange chromatography resulted in a virtually pure TOM core complex (Fig. 2 A, lane 3). The yield of purified core complex was ∼2 mg complex per 1 g isolated mitochondrial protein.

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