Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.).

Markus Waldeck-Weiermair,Corina T Madreiter-Sokolowski,Benjamin Gottschalk,Warisara Parichatikanond,Rene Rost,Christiane Klec,Wolfgang F Graier,Roland Malli

doi:10.1038/srep15602

Markus Waldeck-Weiermair, Corina T Madreiter-Sokolowski + Show 6 more

Open Access

https://doi.org/10.1038/srep15602

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Journal: Scientific Reports	Publication Date: Oct 22, 2015
Citations: 49	License type: CC BY 4.0

Affiliation: Medical University of Graz

Abstract

Mitochondrial Ca2+ uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca2+ uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca2+ channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca2+ signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca2+ rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca2+ uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca2+ rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca2+ concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

Highlights

Mitochondrial Ca2+ uptake is a vital process that controls distinct cell and organelle functions
In order to dynamically monitor whether and, if so, how intracellular Ca2+-mobilization by an inositol 1,4,5-trisphosphate(IP3-)generating agonist affects the arrangement of mitochondrial calcium uptake 1 (MICU1) in intact cells, Förster energy transfer (FRET) imaging was applied in cells co-expressing MICU1 fused to either cyan fluorescent protein (MICU1CFP) or yellow fluorescent protein (MICU1-YFP)
Because MICU1 has been shown to assemble in hexamers in the absence of Ca2+ and rearranges to dimers upon Ca2+ binding[23], we assumed that under resting conditions the expressed MICU1-CFP and MICU1-YFP chimeras exist as hexamers, facilitating FRET from CFPs to YFPs (Fig. 1A)

Summary

Introduction

Mitochondrial Ca2+ uptake is a vital process that controls distinct cell and organelle functions. While mitochondrial calcium uniporter regulator 1 (MCUR1)[8], leucine zipper/EF hand-containing transmembrane-1 (Letm1)[9,10,11] and the novel uncoupling proteins 2 and 3 (UCP2/3)[12,13,14], which have been shown to contribute to certain modes of mitochondrial Ca2+ uptake, have not been found in MCU containing protein complexes[5], other types of proteins have been identified and characterized as regulatory components of the MCU machinery. The kinetics of the rearrangement of the MICU1 multimers upon cytosolic Ca2+ elevation and whether or not the mitochondrial matrix Ca2+ concentration or membrane potential (ψ mito) have an impact on the structural reorganization of MICU1 remain elusive

Methods

Results

Conclusion