Extended Data Fig. 5: Determination of NCLX orientation and the comparison of NCLX assembly and the Ca²⁺-binding site in different conformational states.

a, Protease digestion of NCLX. After isolating mitoplasts from HEK cells expressing C-terminally 1D4-tagged human NCLX, adding proteinase K (Pro-K) causes a downward shift of the NCLX band, as detected by an anti-1D4 antibody (image on the left). This suggests that (1) the C-terminal 1D4 tag dwells within the matrix and is therefore protected from Pro-K digestion, and (2) Pro-K cuts an NCLX area in the intermembrane space to cause the observed band shift. As the loop between TM5 and TM6 is large and unstructured, we hypothesized that this loop is digested by Pro-K. Accordingly, we introduced a TEV protease (TEVP) site after V278 in the TM5-6 loop and digested this construct in mitoplasts using TEVP. This manoeuvre produces a band at a similar location as the band generated by Pro-K digestion of WT NCLX, suggesting that Pro-K and TEVP both can cut the TM5-6 loop and that this loop is in the intermembrane space. Two subunits in the mitochondrial Ca²⁺ uniporter complex, MCU and EMRE, were used as controls. The green bands represent native MCU proteins. As most of MCU’s protein mass is in the matrix, it is not affected by Pro-K or TEVP. The EMRE protein has its C-terminal end exposed to the intermembrane space. Therefore, Pro-K was able to digest a 1D4 tag attached to EMRE’s C-terminus, as reflected by the disappearance of the Western blot signal created by the anti-1D4 antibody (image on the right). The experiment was performed with four independent biological replicates, all yielding similar results. Molecular weight marker unit: kDa. For gel source data, see Supplementary Fig. 1. b, A schematic of NCLX orientation and transmembrane topology. Key residues in panels a and c are highlighted in blue. The orange dotted lines surrounding M196 indicate linkers that were engineered to make M196 more exposed. c, TEVP digestion of NCLX. TEVP sites were introduced into NCLX in positions after the indicated residues. Those sites, whose digestion by TEVP is unaffected by DDM (e.g., D350 and M196), are located in the intermembrane space. By contrast, those sites, which require DDM to be fully digested by TEVP (e.g., M584 and V51), are inside the matrix. In the presence of DDM, cleavage of the TEVP site after M584 causes the disappearance of the band (lane 9 from the left). This is because the fragment that contains the 1D4 tag is too small (~10 amino acids) and would migrate out of the gel. Digestion of the TEVP site after V51 causes a band shift (lane 12 from the left). This is because the digested fragment that contains NCLX residue 52–584 and the C-terminal 1D4 tag is smaller than the undigested NCLX (lanes 10 and 11). For gel source data, see Supplementary Fig. 1. d, A summary of TEVP digestion results. The Western signal ratios of digested NCLX with or without DDM in panel c are presented. A ratio close to 1 indicates that DDM does not have effects on proteolysis, while a ratio close to 0 indicates that the TEV site is in the matrix, protected by the inner mitochondrial membrane. Data are shown as means ± s.e.m. Numbers in parentheses indicate the number of independent biological replicates. e, The superposition of three classes of Ca²⁺-free NCLX. The transport domains that mediate the oligomerization superimpose well. f, The superposition of three classes of NCLX with Ca²⁺. The transport domains that mediate the oligomerization superimpose well. g, The coordination environment around the Ca²⁺ binding site (cytosol-facing conformation). Ca²⁺ is depicted as a green sphere, and the water molecules are shown as red spheres. h, Superposition of Ca²⁺-coordinating residues in cytosol- and matrix- facing conformations. i, Superposition of cytosol-facing NCLX with Ca²⁺ bound or at low pH without Ca²⁺ (comparison of the protomer on the left and the transport domain on the right).

Source Data