(A) Western blot analysis of WT-MCU response to PEGM, in the absence or presence of DDM detergent, with molecular weight marker positions indicated on left. (B) PEGM treatment of WT, I49C, or I97C EMRE. EMRE’s molecular weight is ~10 kDa. (C) Cartoon illustrating the proposed membrane orientation of MCU and EMRE. The N-terminus of EMRE is fused to the C-terminus of MCU (dashed line). Blue circles: native cysteines. Green boxes: soluble region. (D) WT-MCU or MCU-EMRE fusion protein (Fus) probed with anti-MCU (left) or anti-EMRE (right) antibodies. (E) MCU-EMRE fusion treated with PEGM. In the presence of DDM, PEGM treatment produces 4 bands, representing fusion proteins with various numbers of cysteines modified. (F) Mitochondrial Ca2+ uptake in ME-KO cells with (red) or without (black) expression of the MCU-EMRE fusion protein. See also Figure 2—figure supplement 1.