Fig. 3: Heme export from isolated mitochondria is stimulated by GAPDH and involves FLVCR1b-GAPDH association and heme transfer to GAPDH. | Nature Communications

Fig. 3: Heme export from isolated mitochondria is stimulated by GAPDH and involves FLVCR1b-GAPDH association and heme transfer to GAPDH.

From: Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH

Fig. 3

Mitochondria purified from mouse brain and liver underwent incubation in buffer alone or containing δ-ALA, ADP, and mitochondria-free HEK293T cell supernatant to stimulate their in vitro heme biosynthesis. Heme content of the reisolated mitochondria as indicated by A the hemochromogen assay, t = 12.20, DF = 4 and B by the intensity of the visible spectra 420 nm peak. C Representative fluorescence microscope images of mitochondria indicating relative levels of GAPDH-FLVCR1b association (HADHA, green; PLA, pink) before or after the 30 min incubation with δ-ALA, ADP, and cell supernatant followed by reisolation, n = 3. D Quantification of the PLA results from (C) and two additional independent trials, using Volocity 6.5.1 (Quorum Technologies) image analysis software. t = 5.217, DF = 4. E, F kinetics of 14C-heme release from mitochondria into solution and loss of mitochondrial 14C-heme content, respectively, after adding purified GAPDH or GST proteins to reisolated mitochondria that had undergone incubation with 14C δ-ALA, ADP, and cell supernatant. G Heme transfer to FlAsH-HA-TC-GAPDH upon addition of reisolated mitochondria that had been incubated with δ-ALA, ADP, and cell supernatant. FIAsH-HA-TC-GAPDH in buffer was the control, and fluorescence emission was monitored at 528 nm. H Effect of adding GAPDH on heme transfer from reisolated mitochondria to target protein FlAsH-apo-TC-sGCβ. FIAsH-apo-TC-sGCβ in buffer received either nothing (control), reisolated mitochondria that had been incubated with δ-ALA, ADP, and cell supernatant, or these mitochondria plus GAPDH added after five additional min. Fluorescence readings began for all samples after the GAPDH addition (Time = 0). A, D, E, F, Mean ± SD of three independent trials. G, H Representative of three independent trials, mean ± SD of triplicates. Significance: ** p < 0.01, *** p < 0.001 vs. the compared group based on a two-tailed t test. p-values: A Mito vs Mito + δALA p = 0.0003. D. Mito vs Mito + δALA p = 0.0064. Abbreviations: Mito Mitochondria, Mito + δALA Mitochondria + δ aminolevulinic acid, HA-TC Hemagglutinin-Tetra cysteine-.

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