Nutrient starvation enhances NME3-GFP enrichment at the mitochondrial contact interface. (A) The enrichment of NME3-GFP at mitochondrial contact interface upon nutrient starvation. The dynamic distribution of NME3-GFP was observed in Cos-7 cells cultured in control or nutrient-starved media. Arrows indicate the contact interface between two closely positioned mitochondria. Scale bar, 10 μm. Bars for inset images, 2 μm. (B) Effect of nutrient starvation on the enrichment of NME3-GFP at the mitochondrial contact interface. The GFP intensity of these constructs was converted to a heat map. (C) The intensity of GFP-tagged proteins at the mitochondrial contact interface was quantified, normalized with the non-contact area, compared to the intensity enrichment of cells cultured in control medium. Scale bar, 2 μm. (D) Data are expressed as mean ± SD of three independent experiments analyzed with Student’s t test. ****P < 0.0001. The following event of two contact mitochondria was analyzed. (E) A hypothetical mechanism for PLD6 and NME3-mediated tethering of CL-externalized mitochondria. When two mitochondria with CL externalized onto its outer membrane encounter, PLD6 could catalyze CL into PA in trans at the mitochondrial contact interface. The PA then enriches NME3 via intercalating the amphipathic helix of N-terminal 17 amino acids of NME3 into the lipid packing defects. Through its oligomeric structure and membrane binding ability, NME3 functions as a selective tethering factor to stabilize mitochondrial apposition thus facilitate fusion of damaged mitochondria.