Characterization of the New LETM1 Interaction Partner TMBIM5

Mitochondria are indispensable organelles for the life of eukaryotic cells. Their functions are essential in maintaining cell survival by delivering energy from sugars, fats and proteins, producing metabolites required for the building of proteins and nucleotides, assembling iron-sulfur clusters necessary as essential cofactors of numerous enzymes and playing crucial roles in cell death decisions. To ensure functional and dynamic mitochondria, their volume and cation homeostasis must be tightly regulated. Consistent with the vital role of mitochondria, LETM1 is an essential gene for survival and fitness. LETM1 is highly conserved throughout the eukaryotic kingdom and encodes a mitochondrial protein of the inner membrane. Though LETM1 regulates the mitochondrial matrix volume and K+ homeostasis, its function is still debated. LETM1 has been proposed to control the mitochondrial K+/H+ or the mitochondrial Ca2+/H2+ exchange. A common property of cation/proton exchangers is the presence of several transmembrane (TM) regions to form a channel. Because LETM1 displays only one or two TM regions, it would need to form a multiprotein complex with itself or with another unknown protein. We determined the core interaction network of LETM1 and identified and validated TMBIM5 as interaction protein partner of LETM1. TMBIM5 belongs to a conserved protein family with a proposed role in regulating intracellular Ca2+ homeostasis, and is the only member to localize at the mitochondria. It is a membrane protein with 6-8 proposed transmembrane domains. Besides the notion that it maintains the mitochondrial structure and protects from apoptosis, the function of TMBIM5 is not well understood. The objectives of the herein proposed study are to o unravel the functional interplay between TMBIM5 and LETM1 o explore the role of TMBIM5 in mitochondrial Ca2+ fluxes o clarify the protein architecture of TMBIM5. Here, we propose to investigate in detail whether both proteins function in dependence of their interaction: For example, whether one acts as a regulatory subunit and the other as a transporter. Or whether both proteins have different transport functions, depending on whether they form a protein complex or not. To determine which functions depend on the interaction, a set of LETM1 and TMBIM5 protein mutants should be generated and their interaction and transport activity analysed. Clarifying these points will shed light in the functions and interaction of LETM1 and TMBIM5 and advance our understanding of the regulation of mitochondrial Ca2+ homoeostasis. The expected results will also elucidate how TMBIM5 may link mitochondrial Ca2+ homeostasis, Cytochrome c stability and maintenance of cristae structure under control and apoptosis stress conditions.

Mitochondria play a central role in cellular functions beyond their traditional image as energy powerhouses. These organelles serve as vital metabolic hubs involved in gene expression, immune responses, and many other essential processes for organismal well-being. To maintain functional and dynamic mitochondria, tight regulation of volume and cation homeostasis is crucial. The interplay of transport systems and the bioenergetic state governs these processes. Among them, the mitochondrial homeostasis of K+ is essential for osmotic integrity and is heavily dependent on the K+/H+ exchanger (KHE) activity mediated by LETM1. Similarly, Ca2+ homeostasis in mitochondria regulates signal transduction and metabolism, and excessive Ca2+ overload is prevented by Na+-dependent and independent transport systems. Although many mitochondrial players in K+ and Ca2+ transport are known, the identity of the Ca2+/H+ exchanger (CHE) has remained a missing piece in this molecular puzzle. Our research project identified the physical interaction between LETM1 and TMBIM5, a member of the protein family involved in intracellular Ca2+ handling. Building upon this discovery, our primary focus was to elucidate the function of TMBIM5 in mitochondrial cation regulation and its interplay with LETM1. To achieve our research objectives, we employed a range of complementary approaches, including genetic knockouts, Ca2+ and K+ transport assays in intact and permeabilized cells, isolated mitochondria, and cell-free assays using proteoliposomes. These efforts, supported by expert collaborations, allowed us to thoroughly investigate the functions of TMBIM5. A major milestone in our project was the identification of TMBIM5 as the elusive mammalian mitochondrial CHE. This critical discovery shed new light on the controversial debate surrounding LETM1's role as a putative CHE and provided insights into the feedback loop between mitochondrial Ca2+ homeostasis, ATP metabolism, and cell death, where TMBIM5 plays a crucial role. Our findings highlighted the significance of TMBIM5 in maintaining mitochondrial bioenergetics, cristae architecture, and controlling permeability pore transitions. Moreover, making use of mutational analyses, we explored the interconnections between the mitochondrial KHE and CHE, as well as the protein complex formation between LETM1 and TMBIM5. By unravelling the molecular identity of the mitochondrial CHE, our research project advances the field of mitochondrial research, offering exciting prospects for future investigations. The discoveries made during this study not only deepen our understanding of mitochondrial physiology but also pave the way for exploring the clinical implications of mitochondrial cation dysregulation caused by LETM1 and TMBIM5's dysfunctions. This research contributes to the broader efforts of elucidating the intricate mechanisms of cation homeostasis governing mitochondrial functions and their impact on cellular and organismal health.