Fig 2: BRAWNIN (BR) is a conserved SEP at the inner mitochondrial membrane.a Ribosome protected fragment (RPF) reads from the BRAWNIN gene (C12orf73) in human cell lines. P1 and P2 refer to the 2 potential ORF isoforms generated by alternative splicing. b BR (P1) peptide is conserved throughout vertebrate evolution and possesses an N-terminal hydrophobic region that is predicted to be a transmembrane domain or signal peptide. Underlined regions are peptides detected by mass spec. Regions used as immunogens for raising the polyclonal antibodies used in this study are indicated. c Endogenous BR is detected in HeLa and HEK293T with an a-BR antibody. OE = overexpression. d Western blot of HEK293T lysates transfected with control or BR-targeting siRNAs. e Immunofluorescence of endogenous BR detected by a-BR in HeLa transfected with control or BR-targeting siRNAs. Scale = 5 µM. f Western blot of indicated proteins from subcellular fractions of HEK293T. W = whole cell, M = mitochondria enriched fraction, C = cytosol, P = nuclear fraction and debris, including un-extracted mitochondria; BR?N25 = synthetic BR peptide lacking the N-terminal 25 a.a. g Endogenous BR detected by a-BR in HeLa costained with mitotracker. Scale = 5 µM. h Endogenous BR detected by a-BR in HEK293T costained with mitotracker. Scale = 5 µM. i a-BR and a-citrate synthase (CS) immunofluorescence in mouse skeletal muscle, longitudinal section. Scale = 20 µM. j a-BR immunofluorescence and GFP epifluorescence of Mito-Dendra2 in mouse skeletal muscle, longitudinal section. Scale = 20 µM. k Extraction of endogenous BR from HEK293T mitochondria with buffer alone, Na2CO3 solution or buffer containing 1% Triton X. S = supernatant; P = pellet. l Extraction of endogenous BR from HEK293T mitochondria using increasing concentrations of digitonin. TOMM20, TOMM70, and VDAC1 are outer mitochondrial membrane (OMM) proteins, are MTCO1, ATP5A, and TIM23 are IMM associated. m Proteinase K protection assay of HEK293T mitochondria. The a-BR epitope is C-terminal to the predicted transmembrane domain. TOMM20 and TOMM70 have cytosolic domains. TIM23 has IMS domains. Both CS and ATP5A are in the matrix.

Fig 3: Intracellular A3G is exported via exosome secretion.(A) Detection of A3G protein in cell lysates and exosomes prepared from Huh-7 cells transfected with A3G expression plasmid or vector control. Exosome markers Alix and Rab7, and mitochondria marker VDAC1 were detected in parallel. (B) Treatment with GW4869 or transfection of plasmid expressing nSMase2-targeting shRNA (shnSMase2) inhibited exosome secretion by Huh-7 cells. Exosomes were prepared from 1E7 cells treated as indicated and total protein contents were quantified using BCA assay. DMSO vehicle and unrelated shRNA construct (shneg) were used as controls. (C) Treatment with GW4869 or shnSMase2 inhibited A3G export via exosome secretion and increased intracellular A3G protein level. Huh-7 cells transfected with A3G expression plasmid or vector control and treated as indicated were used for lysate and exosome preparation. In addition to A3G and exosome/mitochondria markers, nSMase2 was also detected to demonstrate knock-down by shnSMase2.