PMC

mTORC1 associates with ISCU protein. A, immunoprecipitation (IP) with anti-FLAG M2 beads was performed with 293T cells transfected with control vectors or plasmids expressing WT or S14A mutant. Immunopurified protein complexes were probed with anti-mTOR, anti-Raptor, or as an immunoprecipitation quality control, anti-FLAG antibodies. B, as described in A, the same cells were used for immunoprecipitation with anti-Raptor antibody followed by Western blot (WB) analysis. C, as stated in A, immunopurified ISCU-FLAG WT or S14A mutant were analyzed by Western blot with anti-phosphorylated serine (pSer) or anti-FLAG antibodies. The relative amount of WT, S14A, mTOR, or Raptor protein in the immunoprecipitation reactions was examined by Western blot with the input.

mTORC1 inhibition destabilizes ISCU protein. A, 3T3-L1 cells control (Con) or serum-starved overnight (SS) were analyzed by Western blot and Taqman RT-PCR assays. B, 3T3-L1 cells were incubated with 200 nm rapamycin (Rapa) or dimethyl sulfoxide (DMSO; Con) in complete medium for 4 h and further analyzed by Western blot and Taqman RT-PCR assays. C, MLg cells, incubated with 200 nm rapamycin in complete medium for 7 h, were analyzed by Western blot and Taqman RT-PCR assays. D, MLg cells were treated with 20 μm rapamycin and/or 25 μm cycloheximide (CHX) for 2 h. The resultant cell pellets were subjected to Western blot analysis. E, MLg cells, pretreated with or without 10 μm MG132 for 0.5 h, were further co-incubated with 200 nm rapamycin or dimethyl sulfoxide for 4 h. The resultant cell pellets were analyzed by Western blot.

mTORC1 inhibition-mediated degradation of ISCU decreases ISC assembly. A and B, MLg cells were incubated with rapamycin for 7 h in complete medium followed by Western blot (A) and aconitase activity (B) assays. *, p < 0.05 versus control (Con). C and D, 293T or HeLa cells were incubated with 200 nm rapamycin (Rapa) for 2 h following serum starvation overnight. The resultant cell pellets were analyzed by Western blot (C) and aconitase activity (D) assay. *, p < 0.05 versus HeLa control; #, p < 0.05 versus 293T control. E, Mlg cells were infected with lentiviruses control or Raptor shRNA and further selected with puromycin. Puromycin-resistant cells were analyzed by Western blot. F and G, cell line 4 stated in E, were further analyzed with Western blot (F) and aconitase activity (G) analyses. *, p < 0.05 versus control. S6K, S6 kinase; Rapa, rapamycin.

mTORC1 phosphorylation of Ser-14 enhances the protein stability of ISCU. A, after 30 h post-transfection, 293T cells expressing WT or mutant S14A were subjected to the cellular fractions isolation followed by Western blot analysis. SOD2 and tubulin served as quality controls for mitochondrial and cytosolic fractions, respectively. B, the whole cell lysates isolated from 293T cells expressing WT or mutant S14A were analyzed by Western blot. C, signals of immunoreactive bands for precursor (slow-migrating) and mature (fast-migrating) form in B were quantified with NIH ImageJ software, *, p < 0.05 versus WT. The total levels of ISCU represent the combined signals of precursor and mature form. D, in 20 h post-transfection, 293T cells expressing WT or S14A mutant were further treated with 5 μm MG132 in complete medium for 24 h and followed by Western blot (WB) analysis. E, in 16 h post-transfection, 293T cells expressing ISCU-FLAG WT, S20A, S14A, or S29A were subjected to immunoprecipitations (IP) with anti-FLAG M2 beads. Immunopurified proteins were analyzed by in vitro kinase (upper panel) and Western blot (lower panel) assays. F, after 30 h post-transfection, 293T cells expressing ISCU-FLAG WT, S20A, S14A, or S29A were analyzed by Western blot. G, in 20 h post-transfection, HeLa cells expressing WT or cMTS-ISCU-FLAG were serum-starved for 3 h followed by 200 nm rapamycin incubation for 2 or 3 h. Cell pellets were analyzed by Western blot. SE, short exposure; LE, long exposure; Rapa, rapamycin.

Constitutively active mTORC1-mediated stabilization of ISCU sensitizes cells to iron deprivation. A, TSC2^+/+ and TSC2^−/− MEFs were examined with Western blot and aconitase activity analyses, *, p < 0.05 versus TSC2^+/+ MEFs. B and C, TSC2^−/−MEFs were incubated with different concentrations of rapamycin in complete medium for 24 h followed by Western blot (B) and aconitase activity (C) assays. D, TSC2^+/+ and TSC2^−/− MEFs were incubated with 15 μm DFO for 24 h and further examined by Western blot. E, after overnight culture, 1000 TSC2^+/+ or TSC2^−/− MEFs per well in 12-well plate were incubated with 15 μm DFO for 24 h. Thereafter, cells were recovered for 5 days followed by crystal violet staining (left panel) or cell counting (right panel). F, 5000 MEFs per well were pretreated with 200 nm rapamycin for 6 h and further combined with DFO for 21 h followed by assays as described in E. G, cells were infected with lentivirus control (Con) or ISCU shRNA and further selected with puromycin. Puromycin-resistant cells were analyzed by Western blot. H and I, as in G, puromycin-resistant cells were treated with 15 μm DFO for 24 h and further stained with crystal violet (H) and cell counting (I). Empty bar, TSC2^+/+; solid bar, TSC2^−/−. In C, the single asterisk indicates p < 0.05 versus 0 nm rapamycin (Rapa) treatment. In E, the single asterisk indicates p < 0.05 versus TSC2^+/+ MEFs with the respective treatment as to TSC2^−/− MEFs. J, graphic abstract. ISCU associates with and is phosphorylated by mTOR in the cytosol. This phosphorylation stabilizes ISCU protein and thus increase ISCU levels in mitochondria, whereas the dephosphorylation destabilizes ISCU protein in cytosol and decreases its mitochondrial levels.

mTORC1 phosphorylates ISCU protein at serine 14. A, sequence alignment of the putative mTOR phosphorylation site in ISCU was derived by the Cluster W method. B, 293T cells were transfected with plasmids expressing ISCU-FLAG WT. In 30 h post-transfection, the cell pellets were analyzed by Western blot with respective antibodies as indicated. C, as stated in B, 293T cells expressing ISCU-FLAG WT were subjected to the isolation of mitochondrial and cytosolic fractions and further analyzed by Western blot. SOD2 and tubulin served as the markers for mitochondrial and cytosolic fractions, respectively. D, 293T cells were transfected with plasmids expressing ISCU-FLAG WT, S14A mutant, or the positive control 4E-BP1-FLAG. After 20 h post-transfection, immunoprecipitation (IP) with anti-FLAG M2 beads was performed and followed by an in vitro kinase assay with or without recombinant mTOR protein (upper panel). To monitor the amount of proteins used, the immunopurified proteins were also probed with anti-FLAG antibody by Western blot (WB) (lower panel). E, as in D, 293T cells expressing WT were pretreated with 200 nm rapamycin (Rapa) for 1 h in complete medium followed by immunoprecipitation and kinase assays with additional 200 nm rapamycin (upper panel). Immunopurified proteins were also analyzed by Western blot (lower panel). F and G, as described in D and E, 293T cells, expressing ISCU-FLAG WT was analyzed in an in vitro kinase assay (F), whereas as a side-by-side comparison, a proportion of the immunoprecipitated protein complex used for lane 1 in F was analyzed by Western blot with anti-FLAG antibodies (G). 293T cells transfected with vectors served as a control (G). DMSO, dimethyl sulfoxide.