Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO

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

“…We further investigated the basis for the rise and fall in FLVCR1b-GAPDH interaction by expressing the H53A point mutant variant of HA-hGAPDH, which due to it having a 30-fold poorer heme binding affinity, 12 does not accumulate mitochondrially-generated heme in cells that are given δ-ALA/Fe 12,19 . The PLA data in Fig.…”

“…To explore how heme that is generated in mitochondria transfers to GAPDH in the cell, we tested the importance of FLVCR1b, a transmembrane protein exclusively localized in the outer mitochondrial membrane that has previously been implicated in mitochondrial heme export in erythrocytes during erythropoiesis 20 . We performed experiments in a human embryonic kidney cell line (HEK293T cells) transfected to express an HA-tagged human GAPDH reporter protein called TC-hGAPDH 19 , which after being labeled with FlAsH reagent 19,21 signals its heme binding in living cells or in solution by a fluorescence quenching of its FlAsH signal 19 . We determined how siRNA-targeted knockdown of cell FLVCR1b expression impacts heme loading onto TC-GAPDH in living cells in response to our stimulating their mitochondrial heme biosynthesis by providing the heme precursor molecules δ-aminolevulinic acid (δ-ALA) and ferric citrate (Fe) 22 , which we have previously shown causes the total heme level in the HEK293T cells, and the level of heme bound in the TC-hGAPDH, to increase by about 3-fold within 2 h 19 .…”

“…All chemicals were purchased from Sigma unless otherwise noted. 19 , pCMV5-TC-hsGCβ(1-619) 44 , and pCMV3-hIDO1-FLAG (# HG11650-CF, Sino Biologicals). Protein expression was allowed for 48 h.…”

“…Live cell heme binding kinetics using FIAsH-labeled TC-hGAPDH HEK293T cells in black walled 96-well plates (Greiner Bio-One, # 655090) that had been grown in heme-deficient conditions, transfected to express TC-hGAPDH, and had or had not undergone FLVCR1b, PGRMC2, or TANGO2 silencing, were labelled with FlAsH using the method described previously 19 . Briefly, the cell monolayers were washed once with phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L glucose and then given FlAsH-EDT 2 (5 μM) in Opti-MEM for 30 min at 37 °C.…”

“…However, one key question remains unanswered in the overall pathway, namely, how does mitochondrial heme reach GAPDH? Here, we address this question in experiments that utilize a cultured human cell line, isolated mouse mitochondria, and a newly described GAPDH reporter protein 19 whose heme binding in living cells can be followed in real-time based on the change in its fluorescence intensity. Our results identify a transporter protein located in the outer mitochondrial membrane, Feline Leukemia Virus subgroup C Receptor 1b (FLVCR1b), as the sole conduit for mitochondrial heme export to GAPDH in the cells, operating through a mechanism that involves a direct FLVCR1b-GAPDH interaction for heme exchange.…”