Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca2+ Uptake Pathways

Markus Waldeck-Weiermair,Claire Jean-Quartier,Rene Rost,Muhammad Jadoon Khan,Neelanjan Vishnu,Alexander I Bondarenko,Hiromi Imamura,Roland Malli,Wolfgang F Graier

doi:10.1074/jbc.m111.244517

Markus Waldeck-Weiermair, Claire Jean-Quartier + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m111.244517

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Abstract

Cytosolic Ca2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2+/H+ antiporter that achieved mitochondrial Ca2+ sequestration at small Ca2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca2+ but strongly diminished the transfer of entering Ca2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca2+ uptake at low Ca2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca2+ uptake pathways in intact endothelial cells.

Highlights

28444 JOURNAL OF BIOLOGICAL CHEMISTRY intact cells [1,2,3,4,5,6], the strong functional and even physical interaction of mitochondria with their cellular environment became evident [7,8,9]
(16) could be recently identified in one given cell, 2) uncoupling proteins 2 and 3 (UCP2/3)2 were described to be involved in mitochondrial Ca2ϩ uptake in intact cells [17], 3) with the mitochondrial calcium uptake 1 (MICU1) protein a novel modulator of mitochondrial Ca2ϩ uptake was recently described in HeLa cells [18], and 4) the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2ϩ/Hϩ exchanger that achieves a slow but highly sensitive mitochondrial Ca2ϩ loading [19]
leucine zipper EF handcontaining transmembrane protein 1 (Letm1) Is Expressed in Endothelial Cells and mRNA Levels of Letm1 Can Be Efficiently Reduced by a Combination of Two siRNAs—Using respective primers, the expression of Letm1 was verified in the human umbilical vein endothelial cell line EA.hy926 (Fig. 1A)

Summary

Introduction

28444 JOURNAL OF BIOLOGICAL CHEMISTRY intact cells [1,2,3,4,5,6], the strong functional and even physical interaction of mitochondria with their cellular environment became evident [7,8,9]. In cells that were transiently transfected with siRNA against Letm1, no inhibitory effect on mitochondrial Ca2ϩ sequestration in response to intracellular Ca2ϩ release was found (Fig. 2A).

Results

Conclusion