The Tim50 subunit of the mitochondrial TIM23 complex contains a presequence-binding domain that is essential for viability and precursor transport across the inner membrane.

The mitochondrial presequence translocase interacts with presequence-containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix-targeting signals activate the translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence translocase initiates reorganization among Tim-proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal-sensitive manner in a process that involves the IMS-domain of the Tim23 channel. The signal-driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor-associated form of the TIM23 complex required for matrix transport.

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.

More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined. Using a photoaffinity-labeling approach with modified presequence peptides, we identified Tom40 as a presequence interactor of the TOM complex. Utilizing mass spectrometry, we mapped Tom40's presequence-interacting regions to both sides of the ␤-barrel. Analysis of a phosphorylation site within one of the presequence-interacting regions revealed altered translocation kinetics along the presequence pathway. Our analyses assess the relation between the identified presequence-binding region of Tom40 and the intermembrane space domain of Tom22. The identified presequence-interacting region of Tom40 is capable of functioning independently of the established trans-acting TOM presequence-binding domain during matrix import. Mitochondria receive the majority of their resident proteins from the cytoplasm. Therefore, mitochondria must mediate the influx of proteins, which have been translated on cytosolic ribosomes, in a spatially constrained manner, i.e., all substrates are required to be unfolded during membrane passage. This constraint is in part due to the static dimensions of the pore within the translocase of the outer mitochondrial membrane (TOM). The TOM complex is involved in the majority of mitochondrial import traffic and therefore serves as the main gateway into the mitochondrion (for reviews, see references 1 to 4).About two-thirds of mitochondrial proteins are targeted into mitochondria by N-terminal targeting signals (presequences) (5). These signals consist of amphipathic ␣-helices with a net positive charge and direct the precursors across the outer and inner mitochondrial membranes. Presequence recognition at the TOM complex is thought to commence with the interaction of the hydrophobic surface of the amphipathic presequence-helix with the Tom20 binding groove (6-8). Subsequently, the hydrophilic side is recognized by the cytosolic domain of the central receptor Tom22 that interacts with the presequence in a salt-sensitive manner (6). The exact series of initial presequence-binding events is still debated; however, there is growing support for a tertiary complex in which both Tom20 and Tom22 interact with the presequence simultaneously (9, 10). Following the positioning of the presequence via Tom22, the substrate is passed to the pore-forming subunit of the TOM complex, Tom40. This process involves Tom5, which serves as a TOM assembly and presequence-interacting subunit (11). As the presequence emerges from the Tom40 pore on the trans side, the intermembrane space (IMS) domain of Tom22 engages with the precursor (10,(12)(13)(14). From...