The mitochondrial protein translocase (MPT), which brings nuclearly encoded preproteins into mitochondria, is very complex with numerous identified protein constituents that comprise at least five translocons, two in the outer membrane: Tom (Tom40/70/22/20) and Sam (Sam50/Sam35/Mas37) (SAM=sorting and assembly machinery], and three in the inner membrane, Tim23 (Tim23/17/44/50/Hsp70/Pam16/Pam18/Mge1) [PAM=presequence-associated import motor]; Tim22 (Tim22/54/18) and Oxa1p (TC #2.A.9). These systems function in conjunction with several chaperone proteins (Rehling et al., 2004). The outer membrane translocase (Tom) consists of import receptors and the proteins of the Tom channel complex. Non-identical integral outer membrane receptor proteins are called Tom70, Tom22 and Tom20. Of these receptor proteins, only Tom22 is essential for protein import. The receptor complex delivers the substrate proteins to the outer membrane channel consisting of 5 hydrophobic proteins, Tom40, Tom38, Tom7, Tom6 and Tom5. Tom40 is the core oligomeric subunit of this channel. It forms a β-stranded, cation-selective, high conductance pore that specifically binds to and transports mitochondrial-targeting peptides. The inner pore diameter has been estimated to be about 22 Å. The small Tom proteins may function in regulatory capacities and are not essential (Rehling et al., 2003, 2004).

The TOM complex can apparently function independently of the two TIM complexes, transporting proteins from the cytoplasm to the intermembrane space (Herrmann, 2003). Conserved, intraprotein, hydrophilic targeting sequences, different from matrix targeting sequences, are involved. Targeting translocation across the outer membrane may be independent of ATP and the pmf, but subsequent transport steps require ATP. About 30% of mitochondrial proteins lack matrix targeting sequences. They are present in the outer membrane, the intermembrane space and in the inner membrane. These proteins may be imported initially via the TOM complex, but their transport across or into the inner membrane is achieved by two independent systems, each with a different subset of targeted proteins.

The two inner membrane channel-forming complexes are both multicomponent. One, the Tim23 complex, consists of at least 2 integral membrane Tim proteins: Tim23 and Tim17 as well as peripheral membrane motor proteins, Tim44, Pam16 and Pam18. In this complex, other motor components (Tim14 and Mge1) are required (Mokranjac et al., 2003; Rehling et al., 2004; Truscott et al., 2003). Tim23, possibly together with Tim17, exhibits channel activity. Two ATP-dependent matrix chaperone proteins (mhsp70 and mGrpE) have been suggested to function together with Tim44 to drive active uptake. Tim44 (with or without the chaperone proteins) may function as an ATP-driven import motor that pulls the precursor polypeptide chain through the Tim23 channel into the matrix. The Tim23 complex imports presequence-containing matrix proteins as well as presequence-containing inner membrane monotonic (1TMS) proteins. The latter can laterally diffuse out of the channel into the lipid bilayer (Herrmann, 2003).

The second inner membrane channel-forming complex consists of at least three integral membrane proteins, Tim22, Tim54 and Tim18. The Tim22 complex has been purified and shown to insert inner membrane proteins by a twin-pore translocase that uses the pmf as the exclusive energy source and a half of sites mechanism (Rehling et al., 2003, 2004). The polytopic substrate proteins of the Tim22 complex (including Tim22 and Tim23) lack typical presequences and instead have internal targeting signals.

The Tom and Tim proteins have homologues in yeast, fungi and/or animals. Tim23, Tim17 and Tim22 are homologous to each other.

Some of the inner membrane proteins are translocated into the matrix via the Tim23 complex and then, following cleavage of its first signal sequence, they are targeted to the Oxa1 complex (TC #2.A.9). Proteins that are translated within the mitochondrial matrix are directly inserted into the inner membrane by the Oxa1 complex. Another group of inner membrane proteins is directly targeted to the inner membrane in a pathway requiring intermembrane protein complexes as well as the integral inner membrane Tim22 complex. While the proteins that use the Tim22 complex are of eukaryotic origin (e.g., MC family members (TC #2.A.29)), those that use the Oxa1 complex are usually of bacterial origin (Chacinska et al., 2002; Herrmann, 2003).

While the Tim23 import system depends on a single water-soluble intermembrane protein, Tim50 (Geissler et al., 2002; Yamamoto et al., 2002), the Tim22 import system depends on two distinct water-soluble protein complexes that are present in the intermembrane space. One contains the essential Tim9 and Tim10 proteins and mediates transport of cytosolically synthesized integral inner membrane proteins including the metabolite carrier proteins from the outer to the inner membrane. The other complex includes the Tim8 and Tim13 proteins and mediates import of a different subset of integral inner membrane proteins. These two intermembrane complexes deliver substrate proteins to the Tim22 complex. Thus, multiple pathways are proposed for transfer of proteins across the intermembrane space, for transfer across the inner membrane, and for insertion into the inner membrane.

The generalized transport reaction catalyzed by the MPT is:

protein (cell cytoplasm) protein (mitochondrial matrix or membrane)