A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.
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.
Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.Show MeSH
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Mentions: To study in vivo interactions with OM components, we generated yeast that produced the fusion model protein Mix17FLAG (Böttinger et al., 2012; Bragoszewski et al., 2013). The assembled TOM complex (Supplemental Figure S2C), as well as the abundance of its components and other mitochondrial OM and IMS proteins, was largely unaffected upon the overexpression of Mix17FLAG (Supplemental Figure S2D). The mitochondrial presence of Mix17FLAG inhibited the import of radiolabeled Mix17, which was expected for proteins that use the same native MIA biogenesis pathway (Supplemental Figure S2E). In contrast, Mix17FLAG did not affect the import of presequence-containing protein (Supplemental Figure S2F). We checked which components are involved in the translocation of Mix17FLAG. Affinity purification from the cellular protein extract (Figure 4A) revealed that Mix17FLAG interacted with Mia40, which has been previously demonstrated (Böttinger et al., 2012; Figure 4B). Of interest, Tom40 protein was also found as a Mix17FLAG interaction partner (Figure 4B). The interaction with Tom40 was less efficient than the interaction with Mia40, suggesting more transient complex formation. Mix17FLAG did not interact with newly imported radiolabeled Tom40, demonstrating that only mature Tom40 can be engaged in the interaction with Mix17 (Supplemental Figure S2G). In contrast to Tom40, peripherally attached TOM receptors, such as Tom20 and Tom70, did not interact with Mix17FLAG (Figure 4B). Consistent with this finding, the import of radiolabeled Mix17 into mitochondria that lacked Tom70 was not significantly affected (Figure 4C). The import of another MIA substrate, Tim9, did not depend on Tom70 (Figure 4D, lanes 1–6) or Tom20 (Figure 4D, lanes 7–12).
Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.