[HTML][HTML] Analysis of the structure and function of EMRE in a yeast expression system

T Yamamoto, R Yamagoshi, K Harada… - … et Biophysica Acta (BBA …, 2016 - Elsevier
T Yamamoto, R Yamagoshi, K Harada, M Kawano, N Minami, Y Ido, K Kuwahara, A Fujita…
Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2016Elsevier
The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel,
and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory
subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression
system for the mammalian calcium uniporter subunits is useful for investigating their
functions. We here established a yeast expression system for the native-form mouse MCU
and 4 other subunits. This expression system enabled us to precisely reconstitute the …
Abstract
The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca2 + uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca2 + uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca2 + uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca2 + uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.
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