Mechanism of MCUB-dependent inhibition of mitochondrial calcium uptake.

Mitochondrial Ca levels are regulated to balance stimulating respiration against the harm of Ca overload. Contributing to this balance, the main channel transporting Ca into the matrix, the mitochondrial Ca uniporter, can incorporate a dominant-negative subunit (MCUB). MCUB is homologous to the pore-forming subunit MCU, but when present in the pore-lining tetramer, inhibits Ca transport. Here, using cell lines deleted of both MCU and MCUB, we identify three factors that contribute to MCUB-dependent inhibition. First, MCUB protein requires MCU to express. The effect is mediated via the N-terminal domain (NTD) of MCUB. Replacement of the MCUB NTD with the MCU NTD recovers autonomous expression but fails to rescue Ca uptake. Surprisingly, mutations to MCUB that affect interactions with accessory subunits or the conduction pore all failed to rescue Ca uptake, suggesting the mechanism of inhibition may involve global rearrangements. Second, using concatemeric tetramers with varying MCU:MCUB ratios, we find that MCUB incorporation does not abolish conduction, but rather inhibits Ca influx proportional to the amount of MCUB present in the channel. Reducing rather than abolishing Ca transport is consistent with MCUB retaining the highly-conserved selectivity filter DIME sequence. Finally, we apply live-cell Förster resonance energy transfer to establish that the endogenous stoichiometry is 2:2 MCU:MCUB. Taken together, our results suggest MCUB preferentially incorporates into nascent uniporters, and the amount of MCUB protein present linearly correlates with the degree of inhibition of Ca transport, creating a precise, tunable mechanism for cells to regulate mitochondrial Ca uptake.