Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake.

ABSTRACT

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca2+-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca2+-uptake function. Thus, it is essential for the Ca2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca2+-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.