Structural and functional studies of import and intermembrane space transfer of mitochondrial membrane proteins – MitoMemProtImp

Aiming for a comprehensive description of membrane-protein import from cytosolic targeting to the insertases, we will study in this project a variety of different membrane proteins, of both the alpha-helical and beta-barrel families, including mitochondrial mitochondrial membrane proteins with soluble domains.
We use nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, molecular-dynamics simulations and other in-vitro biophysical methods in order to obtain a structural view of the structures of these membrane proteins bound to different chaperones. The result of this in-vitro approach will be a comprehensive description of the binding sites, dynamics and structure of several complexes. Based on these results, we design mutants which shall impact the binding affinities, structures and dynamics. We will verify those mutants in vitro, and, in parallel, test those proteins in vivo. By membrane-protein import assays in yeast, we will establish the importance of the binding sites that we identified in vitro, and thereby establish the biological relevance of the structures.

Chaperones are essential for assisting protein folding and for transferring poorly soluble proteins to their functional locations within cells.
In a first study, we have deciphered how the two mitochondrial inter-membrane space chaperones differ. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding of how additional interactions enable client specificity is sparse. Here, we decipher what determines binding of two chaperones (TIM8·13 and TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1 and Tim23, which has an additional disordered hydrophilic domain. Combining NMR, SAXS, and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity versus specificity.
We then investigated furthermore how the essential TIM9·10·12 chaperone, assembles. Interestingly, we identified that the sub-units of this hexameric chaperone are in continuous exchange between monomeric and hexameric states.
In a further study, the French and German partners have revealed that a class of recently identified mitochondrial membrane proteins, the mitochondrial pyruvate carriers, are imported via an import pathway that had previously been described for other proteins.

Having advanced on the mechanisms of chaperone function in the mitochondrial inter-membrane space, we are extending now to understanding how these chaperones interact with receptor domains for further integration of the membrane proteins into the membrane.

(1) Sucec, I., Wang, Y., Dakhlaoui, O., Weinhäupl, K., Jores, T., Costa, D., Hessel, A., Brennich, M., Rapaport, D., Lindorff-Larsen, K., Bersch, B., Schanda P. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Sci. Adv. 2020, 6, 51: eabd0263
(2) Weinhäupl, K., Wang, Y., Brennich, M., Lindorff-Larsen, K., Schanda P*. Architecture and assembly dynamics of the essential mitochondrial chaperone complex TIM9·10·12. Structure, 2021, in press.
(3) Rampelt H, Sucec I, Bersch B, Horten P, Perschil I, Martinou J-C, van der Laan M, Wiedemann N, Schanda P, Pfanner N.* The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments. BMC Biology, 18:2

Mitochondria are essential for Fe-S cluster biogenesis, crucial for apoptosis, involved in numerous metabolic pathways and well known for their role in ATP synthesis. All these processes depend on beta-barrel channels in the outer membrane and alpha-helical metabolite carriers in the inner membrane. Similar to the vast majority of mitochondrial proteins, all these channels and transporters are encoded in the nucleus and translated on cytosolic ribosomes. Classical mitochondrial precursor proteins contain an N-terminal presequence which is required and sufficient for targeting and import and afterwards it is cleaved off to produce the mature protein. The goal of this project is a comprehensive description of the import of mitochondrial membrane protein precursors with internal targeting signals. After targeting to the translocase of the outer membrane (TOM) and translocation these hydrophobic precursors are sorted through the aqueous intermembrane space. Subsequently, beta-barrel precursors are inserted with the help of the sorting and assembly machinery (SAM) into the outer and alpha-helical metabolite carriers by the carrier translocase (TIM22) into the inner membrane. Major open questions are the specific targeting mechanisms of the three Tom receptors for membrane proteins with internal targeting signals, the structural basis of protein chaperoning in the intermembrane space by the TIM chaperone system and the transfer to the membrane insertase complexes.
By an integrated structural biology and biochemical approach, we will analyze the contribution of the different redundant Tom receptors for the targeting of beta-barrel proteins and metabolite carriers. Moreover, we will determine the mechanism of membrane protein precursor transfer by the TIM chaperone system from the TOM complex through the aqueous intermembrane space to the SAM and TIM22 complexes. We will determine the the structure and dynamics of the complexes formed by the membrane protein precursors and the receptor domains and chaperones by combining NMR, SAXS and other biophysical approaches, determine relative binding affinities, and study the preprotein transfer from chaperones to the Sam50-POTRA domain and to the Tim54 receptor of the TIM22 complex at the structural level. These biophysical and structural approaches will be complemented by generation of site-specific point mutants of the receptor and chaperone proteins and their in vivo and in vitro analysis by growth assays and import experiments into isolated mitochondria. Taken together, we will complement the knowledge for the import of the classical mitochondrial precursor proteins by an in-depth description of the import of the two most abundant membrane protein classes of mitochondria and this will reveal important principles also for chloroplasts and Gram negative bacteria.