Fig. 1: Phylogeny, localization and topology of Put6 and Put7.

a Phylogenetic tree focused on 15 selected species across different eukaryotic phyla showing the presence of MCUR1 and mitochondrial Ca²⁺ uniporter components MCU, MICU1, and EMRE. b Schematic showing predicted domain organization of Put6, Put7, MCUR1, and CCDC90B proteins. MTS, mitochondria targeting sequence; TM, transmembrane helix; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens. c, d Put6 and Put7 are localized to the mitochondrial fraction. SDS-PAGE immunoblot analysis of different subcellular fractions prepared from Put6-V5 and Put7-V5 expressing cells. Por1 and Pgk1 were used as mitochondrial and cytosolic markers, respectively. Whole-cell protein lysate is depicted as “Total”, P and S refer to pellet and supernatant, respectively, and the number following P and S refers to the centrifugation speed × 1000 g. Data are representative of two independent experiments. e, f Put6 and Put7 are integral membrane proteins. Mitochondria containing Put6-V5 and Put7-V5 were exposed to alkaline treatment at pH 11.5. Membrane integrated (P, pellet) and soluble (S) material were separated by centrifugation and analyzed by immunoblotting. Cox2 and Coa6 were used as markers for integral membrane and soluble proteins, respectively. Data are representative of two independent experiments. g, h N-terminal soluble domains of Put6 and Put7 are matrix facing. Mitochondria expressing Put6-V5 and Put7-V5 were resuspended in isotonic (SEM), hypotonic-swelling (SW), and Triton X-100 (TX-100)-containing buffer and were treated with (+) or without (−) proteinase K (PK). After 30 min incubation, samples were analyzed by immunoblotting using antibodies against V5 and the marker proteins, Tom70 (OMM), Tim50 (IMS), and Tim44 (matrix). Data are representative of three independent experiments. i Model showing the localization and topology of Put6 and Put7 proteins. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane. Source data are provided as a Source data file.