Mechanism of MCUB‐Dependent Inhibition of Mitochondrial Calcium Uptake

Mitochondrial Ca²⁺ levels are regulated to balance stimulating respiration against the harm of Ca²⁺ overload. Contributing to this balance, the main channel transporting Ca²⁺ into the matrix, the mitochondrial Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ uptake. Surprisingly, mutations to MCUB that affect interactions with accessory subunits or the conduction pore all failed to rescue Ca²⁺ uptake, suggesting the mechanism of inhibition may involve more global domain rearrangements. Second, using concatemeric tetramers with varying MCU:MCUB ratios, we find that MCUB incorporation does not abolish conduction, but rather inhibits Ca²⁺ influx proportional to the amount of MCUB present in the channel. Reducing rather than abolishing Ca²⁺ 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 Ca²⁺ transport, creating a precise, tunable mechanism for cells to regulate mitochondrial Ca²⁺ uptake.