N-terminal methionine excision of proteins creates tertiary destabilizing N-degrons of the Arg/N-end rule pathway

Abstract

All organisms begin protein synthesis with methionine (Met). The resulting initiator Met of nascent proteins is irreversibly processed by Met aminopeptidases (MetAPs). N-terminal (Nt) Met excision (NME) is an evolutionarily conserved and essential process operating on up to two-thirds of proteins. However, the universal function of NME remains largely unknown. MetAPs have a well-known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but using CHX-chase assays to assess protein degradation in yeast cells, as well as protein-binding and RT-qPCR assays, we demonstrate here that NME also occurs on nascent proteins bearing Met-Asn or Met-Gln at their N termini. We found that the NME at these termini exposes the tertiary destabilizing Nt residues (Asn or Gln) of the Arg/N-end rule pathway, which degrades proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N terminus, as well as a human tropomyosin-receptor kinase-fused gene (TFG) protein, MN-TFG, bearing a Met-Asn N terminus as physiological, MetAP-processed Arg/N-end rule substrates. Furthermore, we show that the loss of the components of the Arg/N-end rule pathway substantially suppresses the growth defects of naa20Δ yeast cells lacking the catalytic subunit of NatB Nt acetylase at 37 °C. Collectively, the results of our study reveal that NME is a key upstream step for the creation of the Arg/N-end rule substrates bearing tertiary destabilizing residues in vivo.

Keywords: N-end rule; N-terminal acetylation; N-terminal amidase; N-terminal arginylation; N-terminal methionine excision; acetylation; proteasome; protein degradation; ubiquitin; ubiquitin ligase.

Figure 1.

Ubr1 mediates degradation of MN-α2-e^K-ha-Ura3. A, scheme of the degradation of MN-α2-e^K-ha-Ura3 by the Ac/N-end rule pathway. Co-translational deubiquitylation of Ub^R48-MN-α2-e^K-ha-Ura3 yields MN-α2-e^K-ha-Ura3 in vivo. Degradation of the resulting MN-α2-e^K-ha-Ura3 involves NatB Nt-acetylase and Doa10 E3 Ub ligase. B, growth assays of WT, doa10Δ, or ubr1Δ cells expressing MN-α2-e^K-ha-Ura3. The indicated cells were cultured to A₆₀₀ ≈1, serially diluted 5-fold, and then spotted on uracil-containing (SC−Trp) or uracil-lacking (SC−Trp−Ura) plates. The plates were incubated at 30 °C for 3 days. C, CHX chases of MN-α2-e^K-ha-Ura3 in WT, doa10Δ, or ubr1Δ S. cerevisiae for 0, 30, and 60 min. Cell extracts were separated by SDS/10% Tris-glycine PAGE, followed by immunoblotting with anti-ha and anti-tubulin antibodies. D, quantitation of data in C. Data show mean ± S.D. of three independent experiments.

Figure 2.

Ubr1 mediates degradation of Nt-unacetylated MN-α2-GST. A, co-translational deubiquitylation of Ub^R48-MN-α2-GST produces MN-α2-GST in vivo. The resulting MN-α2-GST is Nt-acetylated by NatB complex. B, CHX chases of MN-α2-GST in WT, doa10Δ, ubr1Δ, and ubr1Δ doa10Δ S. cerevisiae for 0, 10, and 30 min. C, quantitation of data in B. D, same as in B, but with WT, naa20Δ, ubr1Δ, and naa20Δ ubr1Δ S. cerevisiae. E, quantitation of data in D. The graphs in C and E show quantitation of data as mean ± S.D. from three independent experiments. F, same as in B but for 0 and 30 min in ubr1Δ doa10Δ cells expressing ^fUbr1 from the P_GAL1 promoter. The yeast cells were grown at 30 °C to A₆₀₀ = ∼0.8 in SRaf medium, followed by the addition of either galactose (for UBR1 induction) or glucose (for UBR1 repression). G, same as in F but in naa20Δ ubr1Δ doa10Δ cells expressing vector only, ^fUbr1, or catalytically inactive mutant ^fUbr1^C1220S. H, extracts of naa20Δ ubr1Δ doa10Δ cells expressing MN-α2-GST and ^fUbr1 or ^fUbr1^C1220S were subject to immunoprecipitation with anti-FLAG–agarose. The bound proteins were analyzed via SDS-PAGE and immunoblotting with anti-GST or anti-FLAG antibodies. The bottom panel shows the 1% input that was used for immunoprecipitation. I, same as in H but extracts of NAA20 or naa20Δ S. cerevisiae (ubr1Δ doa10Δ) cells expressing MN-α2-GST and ^fUbr1^C1220S. J, co-targeting scheme of MN-α2-GST via the Ubr1- and NatB/Doa10-mediated degradation by the Ub-proteasome system (UPS).

Figure 3.

Degradation of Nt-unacetylated MN/MQ-α2-GSTs require Nta1, Ate1, and Ubr1. A, determination of the Nt sequences of MN-α2-GST purified from naa20Δ ubr1Δ doa10Δ S. cerevisiae. B, CHX chases of MZ-α2-GST for 0, 10, and 30 min in naa20Δ S. cerevisiae. Z = Asn, Gln, Asp, Glu, or Lys. C, Quantitation of Data in B. D, same as in B but with MN-α2-GST for 0, 20, and 40 min in naa20Δ, ate1Δ naa20Δ, or nta1Δ naa20 Δ S. cerevisiae. E, quantitation of data in D. F, same as in D but for 0, 20, and 60 min with MQ-α2-GST. G, Quantitation of Data in F. The graphs show quantitation of data as mean ± S.D. of three independent experiments.

Figure 4.

MetAPs create the tertiary N-degrons of the Arg/N-end rule pathway. A, parsimonious scheme of the NME-mediated degradation of MN-α2-GST and MQ-α2-GST. B, CHX chases of MN-α2-GST in map1Δ naa20Δ S. cerevisiae for 0 and 15 min in either the presence or absence of a Map2 inhibitor fumagillin (final amount, 5 μg/ml). C, same as in B but with MQ-α2-GST.

Figure 5.

Human MN-TFG is degraded by the Arg/N-end rule pathway. A, schematic representation of human UBR1^1–1031 in Y2H bait vector and human TFG in Y2H prey vectors to be screened. B, UBR box, the N-domain and the RING domain of human UBR1. Fragments of UBR1 used to map its TFG-binding region are depicted below the diagram. C, interaction of TFG with UBR1 fragments upon Y2H assays. D, in vivo detection of UBR1^1–1031–TFG interactions in ATE1 NTA1, ate1Δ, and nta1Δ S. cerevisiae using Y2H assay. S. cerevisiae cells co-expressing bait and prey plasmids were serially diluted 5-fold and spotted on SC (−Leu/Trp) or SC(−Leu/Trp/His/Ade) plates (see “Experimental procedures” for details). E, CHX chases of C-terminal triply FLAG-tagged human TFG (TFG_f3) in Ate1^+/+ WT and Ate1^−/− KO MEF cells for 0, 4, 8, and 12 h. F, graph represents quantitation of data in E with mean ± S.D. of three independent experiments. G, relative levels of human TFG_f3 mRNAs in WT and Ate1^−/− KO MEF cells using RT-qPCR. The data presented are mean ± S.D. in triplicate for each sample. H, steady-state levels of TFG_f3 in HeLa cells with or without the proteasome inhibitor MG132.

Figure 6.

MQ-Rad16 is degraded by the Arg/N-end rule pathway. A, in vivo detection of human UBR1^93–191–MQ-Rad16 interaction in ATE1 NTA1, ate1Δ, or nta1Δ S. cerevisiae (ubr1Δ) using the Y2H assay. B, CHX chases of MQ-Rad16_ha in map1Δ naa20Δ S. cerevisiae for 0 and 20 min in either the presence or absence of fumagillin (final amount, 5 μg/ml). C, CHX chases of MQ-Rad16_ha, for 0, 20, and 40 min, in WT, naa20Δ, ubr1Δ, or ubr1Δ naa20Δ S. cerevisiae. D, same as in C but in naa20Δ, ate1Δ naa20Δ, or nta1Δ naa20Δ S. cerevisiae. E, graph shows quantitation of data in D as mean ± S.D. of three independent experiments.

Figure 7.

Interplay between the Arg/N-end rule and the NatB-mediated Ac/N-end rule pathway. A, growth of WT, naa20Δ, ate1Δ naa20Δ, nta1Δ naa20Δ, or ubr1Δ naa20Δ S. cerevisiae. Equal amounts of indicated cells were 5-fold serially diluted and spotted on YPD plates. The plates were incubated at 30 or 37 °C for 3 days. B, diagram representing the functional and mechanical cooperation between the Arg/N-end rule and the NatB-mediated Ac/N-end rule pathways. The Ac/N-end rule pathway directly targets Nt-acetyl tag of Nt-acetylated AcMN/AcMQ-starting proteins. However, Nt-unmodified but otherwise identical MN/MQ-starting proteins are vulnerable to NME and thereby degraded by the cascades of the Arg/N-end rule pathway.