Fig. 3.

TFPβ_HH is involved in the membrane association of TFPβ. (A) The cryo-EM density map before the detergent-free mask is applied, with the structure model docked into the map. The TFPβ_HH regions in TFPβ are highlighted with a blue rectangle. The noncontinuous density shown in the red oval below the TFP tetramer presumably corresponds to the detergent molecules. (B) TFPβ_HH is important for the liposome binding of TFPβ. Cardiolipin-containing liposomes were mixed with the indicated proteins and incubated for 1 h, before the addition of Optiprep reagent to a final concentration of 35%. After centrifugation, 200-μL aliquots were taken out from different layers, from top to bottom, and analyzed by Western blot using an anti-TFPα antibody and an anti-His antibody that recognizes the His tag on TFPβ. Liposomes are enriched in the top layers after centrifugation. (The TFPα in the WT and mutant TFP complexes are untagged and therefore appear smaller on the gel compared with the His-tagged TFPα.) (C) TFPβ_HH is important for the membrane association of TFPβ in cells. C-terminally V5-tagged TFPα and C-terminally Flag-tagged TFPβ were expressed in HEK293A cells as indicated. The mitochondria of these cells were isolated, and the soluble proteins were separated from the mitochondria membranes by sonication and ultracentrifugation. COX2 is an inner mitochondrial membrane protein, and LRP130 is a mitochondrial matrix protein. Mito, mitochondria; mem, membrane.