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
Mentions: We determined whether other core components of the TOM complex interact with Mix17FLAG. However, during the isolation of mitochondria, we observed the partial degradation of Mix17FLAG to lower–molecular weight products that were also recognized via anti-FLAG antibodies. The addition of the metalloprotease inhibitor 1,10-phenanthroline during solubilization partially inhibited Mix17FLAG degradation and also improved the interaction with Mia40 (Supplemental Figure S2H). These conditions were used for further affinity purification experiments that were performed with isolated mitochondria. Although Mix17FLAG efficiently interacted with Mia40 and Tom40, it is surprising that the central TOM receptors, Tom22 and Tom5, were not found in the eluate fraction (Figure 6A). We then investigated the function of the central receptors. The import of MIA-dependent proteins showed variable dependence on Tom5. Consistent with previous studies (Kurz et al., 1999; Vögtle et al., 2012), Tim9 was imported less efficiently into mitochondria that lacked Tom5 (Figure 6B). Cox12 and Pet191 were also affected (Figure 6B). However, half of the tested precursors, including Cox17, Cox19, and Mix17, did not depend on Tom5 for mitochondrial localization (Figure 6B). The import of all of the tested MIA-dependent precursor proteins into mitochondria that lacked Tom22 was unaffected (Figure 6C). Similarly, the translocation of Tim9 and Mix17 did not depend on Tom6 and Tom7 (Supplemental Figure S3, A and B). Thus only Tom40 was found to be universally involved in OM translocation of IMS proteins. A recent study reported that the TOM and SAM complexes are linked to form a supercomplex (Qiu et al., 2013). Thus we investigated whether the SAM components play a role in the translocation of MIA substrates. However, neither Sam50 nor Sam37 was pulled down via Mix17FLAG (Figure 6D).
Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.