Rescue of mitochondrial morphology and ultrastructure by FAM92A1 and mutants. (A) Schematic diagram of mutated sites in different FAM92A1 mutants. (B) A vesicle coflotation assay was used to map the membrane-binding sites of FAM92A1. (C) EM showed the membrane tubulation of unilamellar vesicles by WT FAM92A1 and the mutants. (D) Quantification of membrane tubulation in C. At least 600 vesicles were used for quantification. (E) Quantification of membrane tubulation in giant unilamellar vesicles (GUVs) by WT FAM92A1 and mutants. (F) Rescue of the cellular ATP levels by WT FAM92A1 and mutants. (G) Rescue of OCR changes caused by FAM92A1 knockdown by expressing WT FAM92A1 or mutants. (H) Representative transmission EM image of mitochondria after expressing WT FAM92A1 and mutants in FAM92A1-depleted cells. Bars, 500 nm. (I–L) Quantification in mitochondrial diameter (I), percentage of mitochondria with lamellar cristae (J), length of cristae (K), and number of lamellar cristae per mitochondrial length (L). At least 200 mitochondria (F), 300 mitochondria (G), 200 cristae (H), and 100 mitochondria (I) were used for analysis. Error bars indicate mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, Student's t test. In F–L, WT FAM92A1 and mutants were transiently expressed for 24 h in FAM92A1-depleted cells after treatment with FAM92A1 siRNA for 20 h. The total time for control or FAM92A1 siRNA treatment was 44 h.