A presequence-binding groove in Tom70 supports import of Mdl1 into mitochondria

• The receptor of carrier family protein Tom70 can recognize presequences.
• Presequences bind to the preprotein binding groove in Tom70.
• Presequence binding requires Tom70 in the semi-open or open conformation.
• M551R mutation decreases presequence binding and specifically impairs Mdl1 import.

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry gate into mitochondria for almost all imported proteins. A variety of specific receptors allow the TOM complex to recognize targeting signals of various precursor proteins that are transported along different import pathways. Aside from the well-characterized presequence receptors Tom20 and Tom22 a third TOM receptor, Tom70, binds proteins of the carrier family containing multiple transmembrane segments. Here we demonstrate that Tom70 directly binds to presequence peptides using a dedicated groove. A single point mutation in the cavity of this pocket (M551R) reduces the presequence binding affinity of Tom70 ten-fold and selectively impairs import of the presequence-containing precursor Mdl1 but not the ADP/ATP carrier (AAC). Hence Tom70 contributes to the presequence import pathway by recognition of the targeting signal of the Mdl1 precursor.