Molecular mechanisms of transport of proteins across the mitochondrial inner membrane
Final Report Abstract
The majority of mitochondrial proteins are synthesized in the cytosol as precursor proteins and are translocated to the final place of their function within the organelle with the help of the TOM and TIM23 complexes in the outer and inner mitochondrial membranes, respectively. DFG-funded projects MO1944/1-1 and 1-2 focused on elucidating the molecular mechanisms of protein translocation across mitochondrial inner membrane mediated by the TIM23 complex. The TIM23 complex uses the energy of membrane potential across the inner membrane and ATP in the matrix to translocate precursor proteins across and insert them into the inner membrane. Functionally, the TIM23 complex consists of receptors exposed to the intermembrane space that recognize precursor proteins as soon as they appear at the outlet of the TOM complex, translocation channel through which the precursor proteins cross the inner membrane and import motor at the matrix face of the channel that uses the energy of ATP hydrolysis to complete translocation across the inner membrane. Aspects of the TIM23 complex that were addressed in this project and the data obtained are summarized below. In one line of research, we revealed an intricate network of protein-protein interactions in the IMS that enables coordinated translocation of proteins across TOM and TIM23 complexes and identified residues in the IMS-exposed segments of Tim50 and Tim23 that mediate, directly or indirectly, the interaction between the two proteins and are essential for early steps of protein translocation across the mitochondrial inner membrane. Towards our goal to understand the structure of the translocation channel of the TIM23 complex, we identified highly conserved GxxxG motifs within the first two transmembrane segments of Tim23 that are essential for the interaction with Tim17 and translocation of proteins across the inner membrane. To understand how the energy of ATP hydrolysis is converted into unidirectional translocation of proteins across the inner membrane, we focused on answering the question how the translocation channel and the import motor of the TIM23 complex communicate with each other. Our data show that the two domain structure of Tim44 is essential for this process. We proposed a model in which the communication is based on intricate rearrangements within Tim44 which are stimulated by the translocating polypeptides and conveyed between the two domains of Tim44 through its two central helices that recruit the protein to the inner membrane. We went on to demonstrate a critical role of Tim17 in the same process and revealed distinct functions of different transmembrane segments of Tim17. The first two are involved in interaction with Tim23 and may contribute to the channel formation, whereas the second two, and the matrix-exposed loop between them in particular, are critical for recruitment of Tim44. Thus, we uncovered how precursor proteins are handed over from the translocation channel to the import motor. Finally, our analysis of the conformation of mtHsp70, the ATP consuming subunit of the import motor, revealed that it is predominantly found in the substrate-bound state in intact mitochondria. Combined with biochemical experiments, our data suggest a model in which the availability of free mtHsp70 limits import of proteins into mitochondria, providing a mechanistic explanation to how cells use protein import to sense the function of their mitochondria and thus couple biogenesis and quality control of this fascinating organelle.
Publications
- (2013) Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET. EMBO J. 32(11): 1639-1649
Sikor, M., Mapa, K., von Voithenberg, L.V., Mokranjac, D., and Lamb, D.C
(See online at https://doi.org/10.1038/emboj.2013.89) - (2015) Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria. J. Mol. Biol. 427(5): 1075-1084
Waegemann, K., Popov-Čeleketić, D., Neupert, W., Azem, A., and Mokranjac, D.
(See online at https://doi.org/10.1016/j.jmb.2014.07.015) - (2015) GxxxG motifs hold the TIM23 complex together. FEBS J. 2824(11): 2178-2186
Demishtein-Zohary, K., Marom, M., Neupert, W., Mokranjac, D., and Azem, A.
(See online at https://doi.org/10.1111/febs.13266) - (2015) Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein. eLife e11897
Banerjee, R., Gladkova, C., Mapa, K., Witte, G., and Mokranjac, D.
(See online at https://doi.org/10.7554/eLife.11897) - (2017) Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50. Sci. Adv. 3: e1700532
Malhotra, K., Modak, A., Nangia, S., Daman, T.H., Günsel, U., Robinson, V., Mokranjac, D., May, E.R. and Alder, N.
(See online at https://doi.org/10.1126/sciadv.1700532) - (2017) Role of Tim17 in coupling the import motor to the translocation channel of the mitochondrial presequence translocase. eLife e22696
Demishtein-Zohary, K., Günsel, U., Marom, M., Banerjee, R., Neupert, W., Azem, A. and Mokranjac, D.
(See online at https://doi.org/10.7554/eLife.22696) - (2019) A mutagenesis analysis of Tim50, the major receptor of the TIM23 complex, identifies regions that affect its interaction with Tim23. Sci. Rep. 9(1): 2012
Dayan, D., Bandel, M., Günsel, U., Nussbaum, I., Prag, G., Mokranjac, D., Neupert, W., and Azem, A.
(See online at https://doi.org/10.1038/s41598-018-38353-1) - (2020) In Vivo Dissection of the Intrinsically Disordered Receptor Domain of Tim23. J. Mol. Biol. 432(10): 3326-3337
Günsel, U., Paz, E., Gupta, R., Mathes, I., Azem, A., and Mokranjac D.
(See online at https://doi.org/10.1016/j.jmb.2020.03.031)
