**2. Protein Translocation**

All but 13 of the estimated >1000 human mitochondrial proteins [6] required to perform key mitochondrial functions are encoded by the nucleus and synthesised on cytoplasmic ribosomes and thus must be imported into mitochondria through highly conserved protein translocation pathways (Figure 1). Owing to the double membrane bound structure of mitochondria, these multistep protein translocation pathways involve numerous protein complexes (Figure 1 and Table 1). Moreover, their proteome consists of soluble, membrane-bound, and transmembrane proteins with different mitochondrial sub-localisations. Therefore, specialised import machinery has evolved to successfully import all classes of proteins.

usually do not cross the Tom40 channel. Instead, OMM α **Figure 1.** Overview of human mitochondrial protein import pathways. The TOM complex acts as the central entry gate for precursor proteins to enter the IMS, where they are diverted into one of five pathways, depending on their structure, function, and target destination. The MIM pathway (only currently understood in yeast) is an exception in that proteins usually do not cross the Tom40 channel. Instead, OMM α-helical proteins are recognised by Tom70 and transferred through MIM to be inserted into the OMM. The five major pathways proteins take after crossing the TOM channel are the following. The presequence pathway: Presequences containing precursor proteins are transported via the presequence pathway. Of these proteins, proteins with a hydrophobic sorting sequence are inserted into the IMM by the TIM23 SORT complex, whereas hydrophilic matrix proteins are pulled through the TIM23MOTOR complex, with the help of the PAM complex and

ATP hydrolysis cycles. The presequences of both these groups of proteins are cleaved by MPP on the matrix side. The OXA1 pathway: N-terminally inserted multispanning membrane proteins, once passed through TIM23MOTOR and cleaved by MPP, are passed to OXA1L, which inserts them into the IMM in the N-terminal formation. OXA1L is also responsible for the insertion of mtDNA encoded proteins into the IMM. The SAM pathway: β-barrel proteins are transported to the TOM complex by cytoplasmic chaperones. They are then passed through the TOM complex and received by small TIM chaperones on the other side for insertion into the OMM by the SAM complex. The MIA pathway: Cysteine-rich proteins in an unfolded, reduced state are passed via the TOM complex to the MIA complex, which inserts disulphide bonds in them, allowing them to reside in a folded, oxidised state in the IMS. Carrier pathway: Proteins with internal targeting signals are protected in the cytosol by cytosolic chaperones (Stage I), which pass them to the TOM complex (Stage II). They are received on the IMS side by small TIM chaperones (Stage III), which transfer them through the IMS to the TIM22 complex (Stage IV) for insertion into the IMM (Stage IV).

**Table 1.** Structure and function of subunits of the mitochondrial translocase complexes in humans and their yeast counterparts.



**Table 1.** *Cont*.

#### *2.1. Crossing the Outer Membrane*

All proteins destined to the mitochondria must first cross the outer mitochondrial membrane (OMM), which they gain access to via the translocase of the outer membrane (TOM) complex (Figure 1). The TOM core complex (TOM-CC) consists of five components: TOM40, TOM22, TOM7, TOM6, and TOM5. The TOM holo-complex is formed following weak association of the TOM-CC with an additional two subunits: TOM20 and TOM70 [2,7]. These subunits are highly conserved between humans and yeast (Table 1); however, we refer to the yeast translocases in the following section, since this was the first organism it was discovered in. Precursor proteins are recognised by the receptor proteins Tom20, which recognises proteins with a mitochondrial targeting sequence (MTS), i.e., presequence proteins [8,9], and Tom70, which specifically recognises precursors with internal targeting signals, such as those belonging to the solute carrier family (SLC25) [10,11]. Proteins are then passed to the Tom40 pore via another receptor component, Tom22, which has also been shown to assist in the assembly of the TOM complex [12–14]. Tom22 physically interacts with Tom40 via its transmembrane segment, whilst its cytosolic domain has been suggested to act as a docking site for the other receptor proteins, Tom70 and Tom20. Recently, the OMM porin metabolite channel (also known as the voltage-dependent anion channel) has been reported to regulate Tom22 integration into the TOM complex in yeast, thus regulating the assembly and stability of the TOM complex [15,16]. Por1, the major yeast isoform of Porin, binds newly imported Tom22 and integrates it into the TOM complex, promoting formation of the mature trimeric form of the TOM complex required for import of precursor proteins [15]. Por1 also sequesters dissociated Tom22, stabilising the dimeric TOM complex under situations where this is preferable, i.e., for the import of proteins destined for the mitochondrial intermembrane space (IMS) assembly (MIA) pathway [15]. Porin is also thought to cooperate with Tom6 in regulating trimeric TOM assembly and stability and thereby modulating protein import during the cell cycle [15,17].

The different oligomeric states of the TOM complex and the nature of these different states remains unclear. Whilst it had generally been accepted that the mature form of TOM complex exists as a trimer [18–21], a cryogenic electron microscopy (cryo-EM) study in *Neurosposa crassa* showed the TOM complex in a dimeric form [22]. More recently, high resolution cryo-EM studies in *Saccharomyces cerevisiae* showed that the TOM-CC exists as dimers and tetramers. The latter is essentially a dimer of the dimeric form of TOM-CC, achieved by lateral stacking of the dimeric TOM complex [7]. Due to the dynamic properties of the TOM complex, it may be proposed that the trimeric complex is formed by dissociation of a monomer from the tetrameric form.

Of note, the only protein of the TOM complex with a significant IMS domain is Tom22, which is important for its role in directing emerging precursor proteins to the Tim50 receptor of the translocase of the inner membrane 23 (TIM23) complex for further translocation [23]. Structural analysis of the interactions between these differing structural subunits showed that association is mainly mediated by hydrophobic interactions, along with high surface complementarity between the transmembrane domains [7].
