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A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

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Tom40HA verifies the specificity of the in vivo interaction with Mix17FLAG. (A) Schematic representation (left) of the immunoaffinity purification of Tom40HA upon solubilization with digitonin of isolated mitochondria with Tom40 or Tom40HA (right). (B) Schematic representation (left) of the immunoaffinity purification of Mix17FLAG upon disruption and solubilization with digitonin of yeast cell extracts with Tom40 or Tom40HA (right). (A, B) The samples were analyzed by reducing SDS–PAGE, followed by immunodecoration with specific antisera. Load, 2%; eluate, 100%.
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Figure 5: Tom40HA verifies the specificity of the in vivo interaction with Mix17FLAG. (A) Schematic representation (left) of the immunoaffinity purification of Tom40HA upon solubilization with digitonin of isolated mitochondria with Tom40 or Tom40HA (right). (B) Schematic representation (left) of the immunoaffinity purification of Mix17FLAG upon disruption and solubilization with digitonin of yeast cell extracts with Tom40 or Tom40HA (right). (A, B) The samples were analyzed by reducing SDS–PAGE, followed by immunodecoration with specific antisera. Load, 2%; eluate, 100%.

Mentions: We next verified the specificity of the interaction between Mix17FLAG and Tom40 using a yeast strain that carried Tom40HA, a tagged version of Tom40. Tom40HA met the control requirements, in which affinity chromatography via anti-hemagglutinin (HA) agarose allowed the efficient and specific purification of other TOM components, such as Tom22, Tom5, and the peripheral receptors Tom70 and Tom20 (Figure 5A). Affinity chromatography from yeast cells via FLAG tag showed that both Tom40 and Tom40HA were able to interact with Mix17FLAG, thus demonstrating that the Mix17-Tom40 interaction is specific (Figure 5B).


A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Tom40HA verifies the specificity of the in vivo interaction with Mix17FLAG. (A) Schematic representation (left) of the immunoaffinity purification of Tom40HA upon solubilization with digitonin of isolated mitochondria with Tom40 or Tom40HA (right). (B) Schematic representation (left) of the immunoaffinity purification of Mix17FLAG upon disruption and solubilization with digitonin of yeast cell extracts with Tom40 or Tom40HA (right). (A, B) The samples were analyzed by reducing SDS–PAGE, followed by immunodecoration with specific antisera. Load, 2%; eluate, 100%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263444&req=5

Figure 5: Tom40HA verifies the specificity of the in vivo interaction with Mix17FLAG. (A) Schematic representation (left) of the immunoaffinity purification of Tom40HA upon solubilization with digitonin of isolated mitochondria with Tom40 or Tom40HA (right). (B) Schematic representation (left) of the immunoaffinity purification of Mix17FLAG upon disruption and solubilization with digitonin of yeast cell extracts with Tom40 or Tom40HA (right). (A, B) The samples were analyzed by reducing SDS–PAGE, followed by immunodecoration with specific antisera. Load, 2%; eluate, 100%.
Mentions: We next verified the specificity of the interaction between Mix17FLAG and Tom40 using a yeast strain that carried Tom40HA, a tagged version of Tom40. Tom40HA met the control requirements, in which affinity chromatography via anti-hemagglutinin (HA) agarose allowed the efficient and specific purification of other TOM components, such as Tom22, Tom5, and the peripheral receptors Tom70 and Tom20 (Figure 5A). Affinity chromatography from yeast cells via FLAG tag showed that both Tom40 and Tom40HA were able to interact with Mix17FLAG, thus demonstrating that the Mix17-Tom40 interaction is specific (Figure 5B).

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

Show MeSH