Human, yeast and other eukaryotic cells contain compartments called mitochondria. These compartments are surrounded by two membranes and are most famous for their essential role in supplying the cell with energy. While mitochondria can make a few of their own proteins, the vast majority of mitochondrial proteins are produced elsewhere in the cell and are subsequently imported into mitochondria. During the import process, most proteins need to cross both mitochondrial membranes.

Many mitochondrial proteins are transported across the inner mitochondrial membrane by a molecular machine called the TIM23 complex. The complex forms a channel in the inner membrane and contains an import motor that drives the movement of mitochondrial proteins across the membrane. However, it is not clear how the channel and import motor are coupled together. There is some evidence that a protein within the TIM23 complex called Tim44 – which is made of two sections called the N-terminal domain and the C-terminal domain – is responsible for this coupling. It has been suggested that mainly the N-terminal domain of Tim44 is required for this role.

Banerjee et al. used biochemical techniques to study the role of Tim44 in yeast. The experiments show that both the N-terminal and C-terminal domains are essential for its role in transporting mitochondrial proteins. The N-terminal domain interacts with the import motor, whereas the C-terminal domain interacts with the channel and the mitochondrial proteins that are being moved.

Banerjee et al. propose a model of how the TIM23 complex works, in which the import of proteins into mitochondria is driven by rearrangements in the two domains of Tim44. A future challenge is to understand the nature of these rearrangements and how they are influenced by other components of the TIM23 complex.

Mitochondria perform a number of essential cellular functions ranging from production of ATP and diverse other metabolic intermediates to initiation of apoptosis. It is thus not very surprising that disturbances in mitochondrial function are associated with a number of human diseases, including neurodegenerative disorders, diabetes, and various forms of cancer (Nunnari and Suomalainen, 2012; Quirós et al., 2015; Youle and van der Bliek, 2012). An essential prerequisite for correctly functioning mitochondria is import of about 1000 different proteins synthesized as precursor proteins in the cytosol. Recent studies revealed that mitochondrial protein import machineries are sensitive indicators of functionality of mitochondria (Harbauer et al., 2014; Nargund et al., 2012; Yano et al., 2014), demonstrating that a deep understanding of mitochondrial protein import pathways and their regulation will be essential for understanding the role mitochondria have under physiological and pathophysiological conditions. Over half of mitochondrial proteins are synthesized with cleavable, N-terminal extensions called presequences. Import of such precursor proteins requires a coordinated action of the TOM complex in the outer membrane and the TIM23 complex in the inner membrane and is driven by membrane potential across the inner membrane and ATP in the matrix (Dolezal et al., 2006; Endo et al., 2011; Koehler, 2004; Mokranjac and Neupert, 2009; Neupert and Herrmann, 2007; Schulz et al., 2015; Stojanovski et al., 2012).

The TIM23 complex mediates translocation of presequence-containing precursor proteins into the matrix as well as their lateral insertion into the inner membrane. The latter process requires the presence of an additional, lateral insertion signal. After initial recognition on the intermembrane space side of the inner membrane by the receptors of the TIM23 complex, Tim50 and Tim23, precursor proteins are transferred to the translocation channel in the inner membrane in a membrane-potential dependent step (Bajaj et al., 2014; Lytovchenko et al., 2013; Mokranjac et al., 2009; Shiota et al., 2011; Tamura et al., 2009). The translocation channel is formed by membrane-integrated segments of Tim23, together with Tim17 and possibly also Mgr2 (Alder et al., 2008; Demishtein-Zohary et al., 2015; leva et al., 2014; Malhotra et al., 2013). At the matrix-face of the inner membrane, precursor proteins are captured by the components of the import motor of the TIM23 complex, also referred to as PAM (presequence translocase-associated motor). Its central component is mtHsp70 whose ATP hydrolysis-driven action fuels translocation of precursor proteins into the matrix (De Los Rios et al., 2006; Liu et al., 2003; Neupert and Brunner, 2002; Schulz and Rehling, 2014). Multiple cycles of mtHsp70 binding to and release from translocating proteins are required for complete translocation across the inner membrane. The ATP hydrolysis-driven cycling of mtHsp70 and thereby its binding to proteins is regulated by the J- and J-like proteins Tim14(Pam18) and Tim16(Pam16) as well as by the nucleotide-exchange factor Mge1 (D'Silva et al., 2003; Kozany et al., 2004; Mapa et al., 2010; Mokranjac et al., 2006; 2003b; Truscott et al., 2003). Tim21 and Pam17 are two nonessential components that bind to Tim17-Tim23 core of the TIM23 complex and appear to modulate its activity in a mutually antagonistic manner (Chacinska et al., 2005; Popov-Celeketic et al., 2008; van der Laan et al., 2005).

The translocation channel and the import motor of the TIM23 complex are thought to be coupled by Tim44, a peripheral inner membrane protein exposed to the matrix (D'Silva et al., 2004; Kozany et al., 2004; Schulz and Rehling, 2014). Like other components of the TIM23 complex, Tim44 is a highly evolutionary conserved protein and is encoded by an essential gene. In mammals, Tim44 has been implicated in diabetes-associated metabolic and cellular abnormalities (Wada and Kanwar, 1998; Wang et al., 2015). A novel therapeutic approach using gene delivery of Tim44 has recently shown promising results in mouse models of diabetic nephropathy (Zhang et al., 2006). In addition, mutations in Tim44 were identified that predispose carriers to oncocytic thyroid carcinoma (Bonora et al., 2006). Understanding the function of Tim44 and its interactions within the TIM23 complex will therefore be essential for understanding how the energy of ATP hydrolysis is converted into unidirectional transport of proteins into mitochondria and may provide clues for therapeutic treatment of human diseases.

Tim44 binds to the Tim17-Tim23 core of the translocation channel (Kozany et al., 2004; Mokranjac et al., 2003b). Tim44 also binds to mtHsp70, recruiting it to the translocation channel. The interaction between Tim44 and mtHsp70 is regulated both by nucleotides bound to mtHsp70 as well as by translocating proteins (D'Silva et al., 2004; Liu et al., 2003; Slutsky-Leiderman et al., 2007). Tim44 is likewise the major site of recruitment of the Tim14-Tim16 subcomplex, recruiting them both to the translocation channel as well as to mtHsp70 (Kozany et al., 2004; Mokranjac et al., 2003b). In this way, Tim44 likely ensures that binding of mtHsp70 to the translocating polypeptides, regulated by the action of Tim14 and Tim16, takes place right at the outlet of the translocation channel in the inner membrane.

Tim44 is composed of two domains, depicted as N- and C-terminal domains (Figure 1A). Recent studies suggested that the N-terminal domain is responsible for the majority of known functions of Tim44. Segments of the N-terminal domain were identified that are important for interaction of Tim44 with Tim16 and with mtHsp70 (Schilke et al., 2012; Schiller et al., 2008). Furthermore, using site-specific crosslinking, residues in the N-terminal domain were crosslinked to the matrix-exposed loop of Tim23 (Ting et al., 2014). However, the C-terminal domain of Tim44 shows higher evolutionary conservation. Still, the only function that has so far been attributed to the C-terminal domain is its role in recruitment of Tim44 to cardiolipin-containing membranes (Weiss et al., 1999). Based on the crystal structure of the C-terminal domain, a surface-exposed hydrophobic cavity was initially suggested to be important for membrane recruitment (Josyula et al., 2006). However, subsequent biochemical studies combined with molecular dynamics simulations, demonstrated that the helices A1 and A2 (residues 235–262 in yeast Tim44), present in the beginning of the C-terminal domain, are important for membrane recruitment (Marom et al., 2009). Deletion of helices A1 and A2 abolished membrane association of the C-terminal domain. Interestingly, attachment of helices A1 and A2 to a soluble protein was sufficient to recruit it to a model membrane (Marom et al., 2009).

Figure 1 with 1 supplement see all

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We report here that the function of the full-length Tim44 cannot be rescued by its N-terminal domain extended to include membrane-recruitment helices of the C-terminal domain, demonstrating an unexpected essential function of the core of the C-terminal domain. Surprisingly, we observed that the two domains of Tim44, when expressed in trans, can support, although poorly, growth of yeast cells, giving us a tool to dissect the role of the C-terminal domain in vivo. We identify the C-terminal domain of Tim44 as the domain of Tim44 that is in contact with translocating proteins and that directly interacts with Tim17, a component of the translocation channel. Our data suggest that intricate rearrangements of the two domains of Tim44 are required during transfer of translocating precursor proteins from the channel in the inner membrane to the ATP-dependent motor at the matrix face.

The major question of protein import into mitochondria that has remained unresolved is how translocation of precursor proteins through the channel in the inner membrane is coupled to the ATP-dependent activity of the Hsp70-based import motor at the matrix face of the inner membrane.

Results presented here demonstrate that the two domain structure of Tim44 is essential during this process. We show here that the two domains of Tim44 have different interaction partners within the TIM23 complex. In this way, Tim44 holds the TIM23 complex together. Our data revealed a direct, previously unexpected interaction between the C-terminal domain of Tim44 with the channel component Tim17. This result not only assigned a novel function to the C-terminal domain of Tim44 but also shed new light on Tim17, the component of the TIM23 complex that has been notoriously difficult to analyze. Recent mutational analysis of the matrix exposed loop between transmembrane segments 1 and 2 of Tim17 revealed no interaction site for Tim44 (Ting et al., 2014), suggesting its presence in another segment of the protein. Our data also confirmed the previously observed interactions of the N-terminal domain of Tim44 with the components of the import motor (Schilke et al., 2012; Schiller et al., 2008). We did, however, not observe any direct interaction between Tim23 and the N-terminal domain of Tim44 that has previously been seen by crosslinking in intact mitochondria (Ting et al., 2014). It is possible that this crosslinking requires a specific conformation of Tim23 only adopted when Tim23 is bound to Tim17 in the inner membrane. This notion is supported by our previous observation that the stable binding of Tim44 to the translocation channel requires assembled Tim17-Tim23 core of the TIM23 complex (Mokranjac et al., 2003b). We observed a direct Tim17-Tim44 interaction here probably because of a high local concentration of the C-terminal domain when bound to the beads.

The core of the C-terminal domain is preceded by a segment that contains two amphipathic, membrane-recruitment helices. This central segment connects the two domains of Tim44. Intriguingly, the two currently available crystal structures of the C-terminal domains of yeast and human Tim44s showed different orientations of the two helices relative to the core domains (Handa et al., 2007; Josyula et al., 2006). The conformational change was likely induced upon PEG binding to this region of human Tim44 during crystallization (Handa et al., 2007). It is tempting to speculate that the same conformational change takes place during translocation of proteins in the mitochondria. Such a conformational change would not only reorient the two helices in respect to the core of the C-domain but also change the relative orientation of N- and C-terminal domains. Since the two domains have different interaction partners within the TIM23 complex, such a change could rearrange the entire complex. The importance of this proposed conformational change in Tim44 is supported by the data presented here. The function of the full-length Tim44 could be reconstituted from its individual domains only very poorly. Also, there is obviously a very strong evolutionary pressure to keep the two domains of Tim44 within one polypeptide chain. N+C strain had to be kept at all times on the selective medium - even after only an overnight incubation on a nonselective medium the full-length protein reappeared (our unpublished observation), likely due to a recombination event between two plasmids.

Tim44 can be crosslinked to translocating proteins. Our data revealed that it is the C-terminal domain of Tim44 that interacts with proteins entering the matrix from the translocation channel in the inner membrane. A direct interaction of the same domain with Tim17 would optimally position the C-terminal domain to the outlet of the translocation channel. This raises an interesting possibility that translocating precursor proteins may play an important role in the above postulated conformational changes of Tim44.

A missense mutation Pro308Gln in human Tim44 is associated with familial oncocytic thyroid carcinoma. The corresponding mutation in yeast, Pro282Gln, destabilized the protein but produced no obvious growth phenotype or an in vivo import defect (our unpublished observations), suggesting that the yeast system is more robust. This observation is in agreement with the notion that mutations that would severely affect the function of the TIM23 complex would likely be embryonically lethal in humans. Still, the disease caused by a mutation in the C-terminal domain of human Tim44 speaks for an important role of this domain in the function of the entire TIM23 complex. Furthermore, the mutation maps to the short loop between A3 and A4 helices in the C-terminal domain of Tim44. Based on the crystal structure of Tim44, it was previously suggested that the mutation could affect the conformational flexibility of the A1 and A2 helices (Handa et al., 2007), intriguingly providing further support for the above postulated conformational changes of Tim44.

Based on the previously available data and the results presented here, we put forward the following model to describe how translocation of precursor proteins through the channel in the inner membrane is coupled to their capture by the ATP-dependent import motor at the matrix face of the channel (Figure 7). Tim44 plays a central role in this model. We envisage that two domains of Tim44 are connected by the central segment that contains membrane-recruitment helices, like two cherries on the stalks (Figure 7 insert). This central segment of Tim44 recruits the protein to the cardiolipin-containing membranes. There, through direct protein–protein interactions, the C-terminal domain of Tim44 binds to Tim17 and the N-terminal domain to mtHsp70 and to Tim14-Tim16 subcomplex (1). In this way, Tim44 functions as a central platform that connects the translocation channel in the inner membrane with the import motor at the matrix face. Additional interactions likely stabilize the complex, in particular that between the N-terminal domain of Tim44 and Tim23 (Ting et al., 2014) as well as the one between Tim17 and the IMS-exposed segment of Tim14 (Chacinska et al., 2005). In the resting state, the translocation channel is closed to maintain the permeability barrier of the inner membrane. During translocation of proteins (2), the translocation channel in the inner membrane has to open to allow passage of proteins. Opening of the channel will likely change the conformation of Tim17 that could be further conveyed to the C-terminal domain Tim44. It is tempting to speculate that this conformational change is transduced to the N-terminal domain of Tim44 through the central, membrane-bound segment of Tim44, leading to relative rearrangements of the two domains of Tim44. This change would now allow Tim14-Tim16 complex to stimulate the ATPase activity of mtHsp70 leading to stable binding of the translocating protein to mtHsp70. mtHsp70, with bound polypeptide, will then move into the matrix, opening a binding site on Tim44 for another molecule of mtHsp70 (3). We speculate that the release of mtHsp70 with bound polypeptide from the N-terminal domain of Tim44 will send a signal back to the C-terminal domain of Tim44 and further to the translocation channel. Multiple cycles of mtHsp70 are required to translocate the entire polypeptide chain into the matrix. Once the entire polypeptide has been translocated, the translocation channel will revert to its resting, closed state, bringing also Tim44 back to its resting conformation (1). Thus, the translocation channel in the inner membrane and the mtHsp70 system at the matrix face communicate with each other through rearrangements of the two domains of Tim44 that are stimulated by translocating polypeptide chain.

Figure 7

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Author details

1. Rupa Banerjee

   Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität, Munich, Germany

   Contribution

   RB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Christina Gladkova

   Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität, Munich, Germany

   Contribution

   CG, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Koyeli Mapa

   Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität, Munich, Germany

   Contribution

   KM, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Gregor Witte

   Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany

   Contribution

   GW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Dejana Mokranjac

   Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität, Munich, Germany

   Contribution

   DM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   dejana.mokranjac@med.uni-muenchen.de

   Competing interests

   The authors declare that no competing interests exist.

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