Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant

 

Background:

Most energy used by our cells is produced in mitochondria in the form of ATP. Therefore, mitochondria are full of proteins devoted to this task. Alongside components of the respiratory chain complexes, another protein supporting energy production in mitochondria is the ADP/ATP carrier (Aac in yeast; ANT in humans), a member of the SLC25 solute carrier family. It sits in the inner mitochondrial membrane and imports ADP into the mitochondrial matrix for use by the ATP synthetase, while at the same time exporting the generated ATP for use by the rest of the cell. Available structures of the ADP/ATP carrier show up to three cardiolipin molecules bound to distinct pockets of the protein [1-3]. Cardiolipin is a lipid found predominantly in the inner mitochondrial membrane, where it is known to stabilise several prominent proteins and protein complexes [4, 5]. Even though there were hints that the cardiolipins may support the stability and function of the ADP/ATP carrier[6-9], this preprint is the first to provide experimental verification for this and to link this lipid-protein interaction to a clinically relevant mutation.

 

Model generation:

To investigate how cardiolipin (CL) binding influences the stability and function of the ADP/ATP carrier, mutations were introduced in the previously identified CL binding pockets 1 to 3 of the ADP/ATP carrier in yeast: Aac2. Residues in the binding pocket’s vicinity were mutated to negatively charged amino acids in order to disrupt CL binding by electrostatic repulsion of the CL headgroups. These mutant yeast Aac2 variants did not display defects in expression level or topology. Native mass spectrometry could confirm that three CL-specific lipid binding pockets exist in Aac2, and that the designed mutations indeed weaken binding of CL to the Aac2 carrier.

 

Key findings:

1) CL binding stabilises Aac2’s monomeric structure
As already shown in the authors’ previous study [9], removing CL from the cell leads to the destabilisation of the 140kDa Aac2 monomer into a smear on a blue native-PAGE. The mutations in CL binding pockets generated in the present study destabilise the monomeric structure in the same way, even though CL is present in the cell. This clearly points to a direct role of CL in stabilizing Aac2’s structure via its binding. It should be mentioned, however, that the observed destabilisation only occurs after detergent-mediated extraction from the cell, as conformation-stabilising inhibitors of Aac2 can rescue the destabilising effect when added to the cells.

2) Bound CL is important for Aac2 transport activity, and exerts its strongest effect through binding pocket 2
Native mass spectrometry data revealed that one of the binding pockets has lower affinity towards CL than the other two. The elevated importance of one of the pockets also became apparent in functional studies. Pocket 2 mutants displayed the biggest decrease in ADP/ATP transport activity measured on isolated mitochondria, followed by pocket 3 and 1 mutants, respectively. These findings were further supported by oxygen consumption measurements, which detected lower respiration activity due to reduced ADP import in isolated mitochondria from pocket 2 and 3 mutants compared to WT mitochondria.

3) CL-Aac2 interaction supports respiratory complexes
Cox1-3 are subunits of the respiratory chain complex encoded in mitochondrial DNA. It has previously been shown that ADP/ATP transport modulates the expression of these subunits. Mutants with the most reduced Aac2 activity also showed the lowest expression of Cox1-3. Furthermore, several mutants could not maintain a stable interaction between Aac2 and the respiratory supercomplex seen in WT. Again, the pocket 2 mutants were most affected.

4) An existing pathogenic mutation leads to the loss of CL association with human Ant1 and has an effect on the transporter’s structure and function
Having established that CL binding is important for the structure and function of yeast Aac2, the authors examined an as of yet uncharacterised mutation in the human orthologue ANT1: Leucine 141, localised to pocket 2, replaced by phenylalanine (L141F). The patient harbouring this recessive mutation suffers from mitochondrial myopathy [10].
The pathogenic mutation was introduced in ANT1 in human cells as well as in Aac2 in yeast cells and a very similar phenotype to the previously characterised CL-binding mutants could be observed. This included reduced CL binding in yeast; and reduced monomeric stability, ADP/ATP exchange capacity and oxygen consumption rate in human cells.
Molecular dynamics simulations were performed to investigate CL interactions with ANT1. CL was less likely to associate with pocket 2 of the mutant ANT1 than the WT ANT1 when the pocket was left unoccupied at the start of the simulation. In addition to potentially providing a steric hinderance, the L141F mutation also led to higher fluctuations in other residues of pocket 2 in the simulation, destabilising the CL binding environment. Another computational method was employed to calculate that the binding free energy of CL is around 12 kcal/mol lower for the WT than for the mutant ANT1.

 

What I like about the preprint:

I am interested in inner mitochondrial membrane proteins and lipids, which is why I selected this preprint. There is an increasing number of studies emerging, including structural data, which show that cardiolipin is important for the stability and function of mitochondrial membrane proteins. I liked that the authors tried to answer the question of how exactly cardiolipin may be important. In this preprint, Senoo and co-authors could directly attribute the defects seen in their mutants to the loss of a very specific lipid-protein interaction. Moreover, they could show that the same phenotype can be caused by a pathological mutation in humans, providing strong evidence for the physiological significance of these specific lipid-protein interactions.

 

Questions for the authors:

1) Do you have any ideas on how CL is involved in mechanisms of ADT/ATP transport? For example, how CL binding and unbinding may contribute to conformational changes between the c-state and the m-state? What kind of methods could one employ to dig even deeper into these kind of questions?

2) As the binding between the ADP/ATP carrier and cardiolipin is based on electrostatic interactions, do you think that other negatively charged lipids may be able to compensate for the lack of CL, if present in sufficient concentrations?

3) What did you enjoy the most while working on this study? 🙂

 

References:

1. Pebay-Peyroula, E., et al., Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature, 2003. 426(6962): p. 39-44.
2. Nury, H., et al., Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers. FEBS Lett, 2005. 579(27): p. 6031-6.
3. Ruprecht, J.J., et al., Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism. Proc Natl Acad Sci U S A, 2014. 111(4): p. E426-34.
4. Acoba, M.G., N. Senoo, and S.M. Claypool, Phospholipid ebb and flow makes mitochondria go. J Cell Biol, 2020. 219(8).
5. Paradies, G., et al., Role of Cardiolipin in Mitochondrial Function and Dynamics in Health and Disease: Molecular and Pharmacological Aspects. Cells, 2019. 8(7).
6. Crichton, P.G., et al., Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J Biol Chem, 2015. 290(13): p. 8206-17.
7. Yi, Q., et al., The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state. J Lipid Res, 2022. 63(6): p. 100227.
8. Montalvo-Acosta, J.J., et al., Structure, substrate binding, and symmetry of the mitochondrial ADP/ATP carrier in its matrix-open state. Biophys J, 2021. 120(23): p. 5187-5195.
9. Senoo, N., et al., Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast. Sci Adv, 2020. 6(35): p. eabb0780.
10. Tosserams, A., et al., Two new cases of mitochondrial myopathy with exercise intolerance, hyperlactatemia and cardiomyopathy, caused by recessive SLC25A4 mutations. Mitochondrion, 2018. 39: p. 26-29.

 

doi: https://doi.org/10.1242/prelights.35101

Read preprint (1 votes)