A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N‐terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence‐containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex. Synopsis This study reveals a novel pathway for the insertion of an N‐anchor mitochondrial protein Om45 into the outer membrane from the intermembrane space side, which requires the TIM23 complex in the inner membrane, a translocator for presequence‐containing proteins. Om45 uses the TOM40 complex to cross the outer mitochondrial membrane and the TIM23 complex and ΔΨ to reach the intermembrane space. Om45 is inserted into the outer membrane from the intermembrane space side. Graphical Abstract This study reveals a novel pathway for the insertion of an N‐anchor mitochondrial protein Om45 into the outer membrane from the intermembrane space side, which requires the TIM23 complex in the inner membrane, a translocator for presequence‐containing proteins.