Fig. 4: TANGO2 enables FLVCR1b heme export to GAPDH and its delivery to target proteins.

HEK293T cells treated with nothing, scrambled siRNA (control), or TANGO2 siRNA for 48 h were then transfected to express HA-TC-GAPDH or IDO1 as indicated. A kinetics of mitochondrial heme transfer to FlAsH-HA-TC-GAPDH in HEK293T cells that had undergone the indicated treatments, after stimulating their heme biosynthesis with δ-ALA/Fe added at time = 0. Effect of TANGO2 knockdown on delivery of mitochondrial heme to two client proteins in cells, as judged by B it blocking the fluorescence decrease of FlAsH-TC-sGCβ versus time or C blocking the gain in heme-dependent IDO1 activity (L-Kynurenine accumulation, F = 447.8, DF = 6) and the gain in IDO1 ¹⁴C-heme content (F = 110.5, DF = 6). D Fluorescence microscope images of HEK293T cells treated with control or TANGO2 siRNA for 48 h and then incubated with δ-ALA/Fe for the indicated times. Cells stained with DAPI for nuclei (blue), HADHA antibody for mitochondria (green), and antibodies against GAPDH and FLVCR1b to indicate their association by PLA (pink). E Quantification of the PLA results from (D) and three additional trials, using Volocity 6.5.1 (Quorum Technologies) software. Normalization of PLA signals utilized antibodies against FLVCR1 and laminin. Control cells, F = 19.84, DF = 6 and TANGO2-silenced cells, F = 5.352, DF = 6. A, B Representative of three independent trials, values  = mean ± SD of triplicates. C, E Three independent trials, mean ± SD. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the compared group based on a one-way ANOVA test. ns, not significant. p-values: C In Kynurenin assay, Control siRNA+no Heme vs. Control siRNA+Heme p = <0.0001, Control siRNA+Heme vs. siFLVCR1b+Heme p = <0.000. In heme count, Control siRNA+no Heme vs. Control siRNA+Heme p = <0.0001, Control siRNA+Heme vs. siFLVCR1b+Heme p = 0.0001. Abbreviations: HA-TC hemagglutinin-tetra cysteine, δ-ALA/Fe δ-aminolevulinic acid and ferric citrate, μM micromolar.