Figure 1.

Conserved S92 is phosphorylated by protein kinase C isoforms. (A) Schematic diagram of the MCU. The MCU consists of a mitochondrial targeting sequence (MTS), N-terminal domain (NTD), linker helix domain (LHD), two transmembrane domains (TM1 and TM2), a TM linker (L), and two coiled-coils (CC). (B) Surface and ribbon diagrams of the MCU NTD coloured by scoring the residue conservation from 230 MCU NTD homologues using the ConSurf server. Highly conserved and variable residues are shown in red and green, respectively. The β-strands (β1 − β6), α-helices (α1, α2), and loops (L1 − L8) are shown in arrows, cylinders, and lines, respectively. (C) Detailed view of the highly conserved L2-L4 loop regions in the MCU NTD (PDB ID, 4XTB). The residues and hydrogen bonds are denoted in stick and dashed lines (red). (D–F) In vitro kinase assays of MCU NTD_WT (residues 75–165) (D,E) and MCU NTD^AA_S92 (F). Autoradiography analysis of MCU NTD_WT (residues 75–165) and MCU NTD^AA_S92 proteins that were incubated with protein kinase A (PKA), protein kinase C (PKC) isoforms (PKC mixture of α, β, and γ isoforms with lesser δ and ζ; PKC_βII; PKC_δ; PKC_ε), and [γ-³²P]ATP (P-32). We designed all Ser/Thr (T76, S87, S92, T100, S105, S107, S129, S138, T139, and T157) mutants of the MCU NTD except the S92 (MCU NTD^AA_S92). Full autoradiography results in Supplementary Fig S4. The reaction samples were resolved by SDS-PAGE, and visualized by autoradiography. Data are representative of three independent experiments.