Fig. 4. TFAM is hydroxylated by EGLN3 at proline 53/66 causing pVHL recognition.

a, Autoradiograms showing recovery of ³⁵S-labelled VHL protein bound to HA-immunoprecipitated (IP) full-length TFAM that was first subjected before to hydroxylation by EGLN3 WT or EGLN3-H196A catalytic mutant. b, Immunoprecipitation using anti-hydroxyproline antibody (HydroxyP) from 293FT cells that were transiently transfected with plasmids encoding Flag-TFAM and Flag-EGLN3 WT or catalytically dead mutant (H196A) with or without DMOG treatment. Immunoblots show co-immunoprecipitation of Flag-TFAM and Flag-EGLN3. In a and b, n = 3 biological independent experiments. c, Schematic of the hydroxylation assay using the biotinylated synthetic TFAM peptide 31–70. d, Autoradiograms showing recovery of ³⁵S-labelled VHL protein bound to biotinylated TFAM peptide 31–70. Before pulldown, peptides were incubated with EGLN1, EGLN2, EGLN3 or EGLN3 catalytic mutant (Mut) generated by IVT or unprogrammed reticulocyte lysate (−). Expression of IVT-produced EglN proteins in each reaction was verified by immunoblot. n = 3 biological independent experiments. e,f, Mass spectrometry of biotinylated TFAM peptide 31–70 was subjected to EGLN3 hydroxylation assay. Representative fragmentation spectra of hydroxylated Biotin-KP(ox)VSSYLR (e) and hydroxylated Biotin-EQLP(ox)IFKA (f). g, Autoradiograms of EGLN3 hydroxylation and ³⁵S-VHL capture as shown in using biotinylated TFAM peptides containing proline-to-alanine substitutions, or no substitution (WT). h, Autoradiograms showing recovery of ³⁵S-labelled VHL protein (WT) or corresponding disease mutants (as indicated) bound to biotinylated TFAM peptides synthesized with double hydroxyl-prolines on prolines 53 and 66 (TFAM-P-OH-53/66). Synthetic biotinylated HIF1α peptide (residues 556 to 575) with hydroxylated proline 564 (HIF1α-P-OH) was included as a control. Biotinylated TFAM naïve peptide was used as negative controls. i, Autoradiograms showing recovery of ³⁵S-labelled VHL protein (WT) or corresponding disease mutants (as indicated) bound to biotinylated TFAM peptides synthesized with double hydroxyl-prolines on prolines 53 and 66 (TFAM-P-OH-53/66). Synthetic biotinylated HIF2α peptide (residues 521 to 543) with hydroxylated proline 531 (HIF2α-P-OH) was included as a control. Biotinylated TFAM and HIF2α naïve peptides were used as negative controls. j, Peptide pulldown using biotinylated TFAM-P-OH-53/66 peptide incubated with whole-cell lysates from 786-O cells expressing either HA-VHL-WT or HA-VHL disease mutant. Biotinylated TFAM and HIF2α naïve peptides were used as negative controls. In g–j, n = 3 biological independent experiments.

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