Functional analysis of the Letm1 proteins

Mdm38p/LETM1 is a nuclear encoded member of a novel, conserved mitochondrial protein family. A crucial function of the LETM1 proteins is the control of the cation/proton exchange across the mitochondrial inner membrane and thus the maintenance of volume homeostasis. Inactivation of LETM1 abolished essential mitochondrial functions by deregulating ion and volume homeostasis and triggering mitophagy. We provided compelling evidence indicating that LETM1 has an essential function for the mitochondrial K+ /H+ exchange. In spite of the fact that the synthetic K+ /H+ exchanger nigericin reverted all mitochondrial defects caused by the inactivation of LETM1, the nature of the substrate has become controversial, since new data suggested that LETM1 may have a role in mitochondrial Ca2+/H+ exchange as well. This debate also raised the interesting possibility that mitochondrial K+ and Ca2+ homeostasis could be linked, with K+ fluxes regulating Ca2+ trans-mitochondrial exchange by modulating the membrane potential. Moreover, it is still not clear whether LETM1, with one single transmembrane domain, can catalyze the exchange per se (possibly by forming oligomers) or rather is an essential co-factor of a still unknown exchanger. Since haploinsufficiency of human LETM1 correlates with seizures in Wolf-Hirschhorn syndrome patients, the unambiguous identification of the LETM1 substrate(s), and the distinction between its role as regulator or executor of the exchange activity are of prime importance and will be addressed here combining various techniques available in the applicant`s or collaborating laboratories.

Mitochondria are essential organelles providing the cells with energy, metabolites and precursor molecules for DNA and amino acids. When mitochondria become defective, they turn into a danger for the whole cell and lead to numerous diseases. In their predominant function in energy conservation by coupling respiration to oxidative phosphorylation, mitochondria generate an electrochemical gradient that represent a strong driving force for the influx of intracellular cations. This driving force needs to be counter balanced by cation exchangers to prevent the overload of cations. However, the molecular identities of most of the cation exchangers are still under investigation. Most recently, NCLX was identified as mitochondrial Ca2+/Na+ exchanger.Potassium is an osmotic and the most abundant cellular cation. Major work of our group has shown that the crucial function of the mitochondrial membrane protein LETM1 is to maintain the equilibrium between cytosolic and mitochondrial K+ concentration by regulating the mitochondrial potassium exchanger (KHE). This is a fundamental process to maintain the volume and functionality of mitochondria and ensure the cellular vitality in all eukaryotic organisms. In line with this notion, total depletion of LETM1 is lethal, and haploinsuficiency, loss of LETM1 on one allele, in human is associated with the severe phenotype of seizures in the Wolf Hirschhorn Syndrome. Recently, conflicting data challenged this proposed function and claimed that LETM1 proteins would rather constitute the Ca2+/H+ exchanger (HCX).The goal of this project was to solve this conflict and explore in more details the function of LETM1. For the first time, we conducted detailed K+ and Ca2+ transport assays in human cells or isolated mitochondria and in a cell free system. K+ extrusion is mediated by the unselective KHE that also transports Na+ while Ca2+ is released by NCLX, the Ca2+/Na+ and most likely the still unknown HCX. Passive swelling assays confirmed that LETM1 gene silencing in human cells reduced the mitochondrial KHE activity. Moreover, data showed that LETM1 depletion did not affect mitochondrial Ca2+/H+ exchange, while it significantly decreased mitochondrial Ca2+/Na+ exchange. However, NCLX expression levels remained constant. Accordingly, this work has revealed that the mitochondrial balance of K+, Na+ or H+ is required for the functionality of NCLX. Additional evidence was obtained by a complementary chemical approach: using quinine, an inhibitor of the KHE, in wild-type cells resulted likewise in loss of NCLX activity. Altogether, the data collected in this project strengthened the notion that LETM1 is primarily an essential component of the KHE, and defective mitochondrial Ca2+ release a consequence of KHE inactivation.A LETM1 mutant collection was generated and one pathogenic point mutant, previously identified in a patient with sever cardiomyopathy, was characterized as a loss-of-function mutation, since its overexpression in cells with reduced LETM1 expression was not able to rescue the LETM1 depletion phenotype. This finding calls for further functional investigation of the pathogenic mutation.The additional goal of this project was to identify interactors of LETM1 required to form a functional KHE, given that we had found LETM1 to be part of a high molecular weight protein complex. To this purpose we adjusted an organellar approach, miniaturized a proteomic mass spectrometry protocol and obtained an optimized new method to identify mitochondrial interaction networks. Our method allowed to start with very little content of isolated mitochondrial material, which is a critical point in cell culture. Importantly, we validated our method by identifying the known MCU core interactome. Applying the validated miniaturized proteomic approach, we found new high confidence LETM1-interaction partners. Among the potential candidates, we confirmed the interaction of LETM1 with a novel mitochondrial multi- pass protein belonging to a conserved protein family with functions in intracellular calcium. Based on this finding, we plan to follow up on our preliminary data.Altogether, this FWF funded work has substantiated that LETM1 is primarily an essential component of the mitochondrial KHE and clarified how LETM1 indirectly affects mitochondrial Ca2+ fluxes. Additionally, it has identified an important novel LETM1 interactor displaying all required features to accomplish trans-mitochondrial cation transport. Thus, data from this work contribute in better understanding the function of key players in mitochondrial cation homeostasis.