TMBIM5, an important piece of the mitochondrial exchange puzzle discovered

TMBIM5 – Important piece of the mitochondrial exchange puzzle discovered
A. Scheme illustrating workflow for miniaturized AP-MS experiments, left to right: whole cells or isolated mitochondria were lysed or solubilized, respectively. The cell/mitochondrial lysates were used for affinity purification (AP) using the STREP tag and tandem affinity purification (TAP) using STREP and HA tag found on the bait protein. Eluates of the AP and control experiments were reduced, alkylated, and digested by trypsin. Peptides are purified on a C18 stage tip and then run on an LTQ Orbitrap Velos. Protein identifications were made by internal tools using MASCOT and Phenyx and the removal of nonspecific interactors done using the CRAPome. Created with B. MCU was selected as a model protein, the functional complex consists of the five proteins above (MCU, MCUb, MICU1, MICU2, EMRE). Note that an additional tissue-specific tertiary interaction partner (MICU3) is only expressed at very low levels in HEK293 cells (Diego De Stefani, personal communication). Illustration adapted from Sancak et al (2013). C. All high-confidence interaction partners of LETM1 are shown as nodes. Node color indicates SAINT score, a probability-based measure of interaction confidence. See also Appendix Fig S1A–C. D. Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane protein complexes. The middle and right panel of the co-IPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN–PAGE conducted in TMBIM5WT and KO. Credit: EMBO reports (2022). DOI: 10.15252/embr.202254978

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Essential in this context are so-called Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, with the exception of the mitochondrial Ca2+/H+ exchanger (CHE).

A research team from the University of Veterinary Medicine Vienna has now put an end to the search with the identification of the protein TMBIM5 as the long-sought mitochondrial CHE. The discovery promises a better understanding of diseases and may enable the development of new treatments.

Ion homeostasis is crucial for mitochondrial function. The dynamic balance of cations is achieved by a set of integrated transport systems for K+, Na+ and Ca2+. Loss of this balance between cation uptake and release has serious consequences and can ultimately lead to . Intracellularly, mitochondria are important sinks of Ca2+.

The role of mitochondrial Ca2+ buffering has been extensively studied, yet some of the players in maintaining Ca2+ balance have not been identified. One of the missing pieces in this molecular puzzle is the Na+-independent Ca2+ efflux pathway, a putative Ca2+/H+ exchanger (CHE). This exchanger, which has been sought since the 1970s, is critical for maintaining mitochondrial Ca2+ levels and pH homeostasis.

One of the CHE candidate proteins was LETM1, a protein that was initially characterized as the mitochondrial K+/H+ exchanger (KHE). Important questions about its function, however, have until recently remained unanswered. A research team at Vetmeduni therefore searched for partners of LETM1 and found the interactor Transmembrane BAX Inhibitor Motif containing protein 5 (TMBIM5).

After identifying TMBIM5, the researchers validated its physical interaction with LETM1. According to the first authors of the study, Shane Austin, Ronald Mekis and Sami Mohammed from the Department of Physiology and Biophysics at Vetmeduni, "Biochemical assays in demonstrate that TMBIM5 is essential for the H+-dependent mitochondrial Ca2+ release and that a mutation in the pH-sensing domain of TMBIM5 completely or severely reduces this function."

"Assays in proteoliposomes confirm pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE."

"This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease and to the development of drugs modulating Ca2+ exchange," says the study's last author, Karin Nowikovsky of the Department of Physiology and Biophysics at Vetmeduni.

According to the experts, further studies are now needed to understand how LETM1 and TMBIM5 link mitochondrial K+ and Ca2+ cycles and to shed more light on the regulatory mechanism of LETM1 and its interaction partners maintaining mitochondrial ion .

The research was published in EMBO reports.

More information: Shane Austin et al, TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria, EMBO reports (2022). DOI: 10.15252/embr.202254978

Journal information: EMBO Reports

Citation: TMBIM5, an important piece of the mitochondrial exchange puzzle discovered (2022, November 25) retrieved 27 January 2023 from
This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.

Explore further

New insights into mitochondrial nucleoid dynamics


Feedback to editors