Heme-mediated regulation of the N-end rule pathway. (A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. A notation, upward of “hemin” in the middle of diagram, is a “down-regulation” sign that denotes, specifically, a down-regulation of a target macromolecule that is mediated, at least in part, by target's degradation. MetAPs, methionine aminopeptidases. C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate, produced in reactions mediated by nitric oxide (NO), oxygen (O₂) and their derivatives. Oxidized N-terminal Cys is arginylated by ATE1-encoded isoforms of R-transferase (2–4). Type-1 and type-2 primary destabilizing N-terminal residues are recognized by the pathway's E3 Ub ligases, called N-recognins (9). Through their other substrate-binding sites, these E3s also recognize internal (non-N-terminal) degrons in other substrates of the N-end rule pathway, denoted by a larger oval. (B and C) Heme regulatory motifs (HRMs) in R-transferases. Numbers refer to position of the last residue of a shown sequence in the corresponding protein. A disulfide between Cys-71 and Cys-72 refers to hemin-mediated inhibition of ATE1. (D) The mouse ATE1^1B7A isoform (2), with locations of significant Cys-containing motifs.

Heme binds to arginyl-transferase and inhibits its activity. (A) Hemin-mediated inhibition of arginylation by mouse ATE1^1B7A (black bars) and S. cerevisiae ATE1 (red bars). (B) Comparison of mouse ATE1^1B7A inhibition by Fe²⁺-heme (blue bars) and Fe³⁺-heme (hemin) (yellow bars). Red bar, a partial rescue of hemin-inhibited ATE1 by 10 mM 2-mercaptoethanol. (C) Hemin was added either to buffer alone (curve 1), or to lysozyme, which does not bind to hemin (curve 2), or to purified ATE1^1B7A (curve 3), followed by gel filtration (to remove unbound hemin) and spectroscopic measurements. (D) Determination of hemin's affinity for the purified wild-type ATE1^1B7A (open circles) and its ATE1_C411A mutant (filled circles) (see SI Fig. 7 A–C for unprocessed data).

Detection and mapping of hemin-induced disulfide in arginyl-transferase. (A and B) mass spectrometric (MS) analysis of specific tryptic peptides from wild-type ATE1^1B7A that had been either treated or untreated with hemin, and thereafter processed as described in C and Hemin-Induced Disulfide in R-Transferase. Black circles denote NEM/D-NEM moieties conjugated to Cys residues. (C) The logic and design of experiments described in A and B and in SI Fig. 10 C–F. d-NEM, deuterated NEM. SDS-mediated denaturation of a test protein is denoted by the transition from “folded” polypeptide chains to “wavy” ones.

Hemin induces degradation of arginyl-transferase in vivo. (A) A mouse cell line expressing His₆-tagged Cys-RGS4 (Cys-RGS4h) (3), was exposed for 10 h to hemin (denoted as “h”) in the medium, followed by SDS/PAGE and immunoblotting of cell extracts with anti-RGS4 antibody. (B) Same but a cell line expressing Asp-RGS4h (derived from Ub-Asp-RGS4h). (C) Same but a cell line expressing Arg-Asp-RGS4h (derived from Ub-Arg-Asp-RGS4h). (D) Same but a cell line expressing Ub^G76V-Val-RGS4h. Lane 5, same as lane 1 but in the presence of 5 μM MG132 (proteasome inhibitor). (E) Comparison of ¹⁴C-arginylation of the α-lactalbumin reporter (denoted as “¹⁴C-rep”) in extracts from NIH 3T3 cells (lanes 2 and 3) or 3T3^offATE1^1B7Atap cells (lanes 4 and 5), that had been either untreated or treated for 10 h with 7 μM hemin in the growth medium. Lane 1, no extract added. (F) Relative amounts (determined by immunoblotting with antibody to mouse ATE1) of either untagged ATE1 (lanes 1 and 2) or both ATE1 and ATE1^1B7Atap (lanes 3 and 4), in either NIH 3T3 or 3T3^offATE1^1B7Atap cells, respectively, after incubations with hemin in the medium. (G) Quantitation, by autoradiography of ¹⁴C-lactalbumin, of the data in E. (H) Lanes 1 and 2, ATE1 in NIH 3T3 cells, detected by immunoblotting in the absence of hemin treatment and either the absence or presence of 5 μM MG132. Lanes 3 and 4, same but with hemin at 10 μM for 10 h in the growth medium. (I) Quantitation of ATE1^1B7Atap pulse–chase data that are shown in J. Open circles, no added hemin. Filled circles, 10 μM hemin was added at the end of a 30-min pulse with [³⁵S]methionine (time 0). (J) Pulse–chase assay, with cells stably expressing ATE1^1B7Atap in the absence or presence of added hemin. ATE1^1B7Atap was precipitated by immobilized IgG, which bound to the TAP tag of ATE1^1B7Atap. Lane 1, same as lanes 2–11 but with NIH 3T3 cells, which lack ATE1^1B7Atap.

Hemin inhibits activation of CUP9 binding by the UBR1 ubiquitin ligase. (A) Equal amounts of extract from S. cerevisiae expressing ^fUBR1 were incubated with glutathione-Sepharose beads preloaded with GST-CUP9, in ether the presence or absence of indicated dipeptides (at 1 mM each) (8), and either in the absence or presence of hemin, at indicated levels. The bound proteins were eluted, fractionated by SDS/PAGE and immunoblotted with anti-flag antibody. Lane 1, input sample (3% of extract's amount used in GST-pulldowns). Lane 2, ^fUBR1 and GST-CUP9 in the presence of Ala-Leu and Ala-Arg (note the absence of interaction between UBR1 and CUP9). Lane 3, same but in the presence of Leu-Ala and Arg-Ala. Lanes 4–7, same as lane 3 but in the presence of 1, 10, 100, or 200 μM hemin. Lanes 8 and 9, same as lanes 2 and 3, respectively, but in the absence of 1 mM NaOH (a control, because 1 mM NaOH was present in other samples (lanes 2–7), owing to the NaOH-containing hemin's stock solution). (B and C) A diagram (based in part on the present findings) of interactions amongst UBR1, dipeptides with type-1 or -2 destabilizing N-terminal residues, and the CUP9 transcriptional repressor. See the main text.