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. 2011 Feb 1;108(5):1763-70.
doi: 10.1073/pnas.1014402108. Epub 2011 Jan 5.

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

Annemarthe G Van der Veen  1 , Kenji Schorpp, Christian Schlieker, Ludovico Buti, Jadyn R Damon, Eric Spooner, Hidde L Ploegh, Stefan Jentsch
Affiliations

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

Annemarthe G Van der Veen et al. Proc Natl Acad Sci U S A. .

Abstract

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Urm1 is conjugated on oxidative stress in a thiocarboxylate-dependent manner. (A) Schematic illustration of Urm1. The C-terminal glycine of WT Urm1 (WT) is modified by MOCS3 to a thiocarboxylate. Deletion of the C-terminal glycine of Urm1 (ΔG) prevents thiocarboxylate formation. (B) HA-tagged Urm1 WT or ΔG was immunoprecipitated from stably transduced HeLa cells and separated on a polyacrylamide gel (Left) or a polyacrylamide gel supplemented with APM (Right). Transient sulfur-mercury interactions between thiol-containing proteins and APM result in reduced electrophoretic mobility. (C) HA-Urm1 WT-expressing HeLa cells were treated with different stressors (described in SI Materials and Methods) and lysed in 1% SDS. Total cell lysates were resolved by SDS/PAGE and subjected to anti-HA immunoblotting. (D) HA-Urm1 WT and HA-Urm1 ΔG HeLa cells were treated with 400 μM diamide (Dia) or 5 mM H2O2 for 10 min and lysed in 1% SDS. HA-Urm1 was immunoprecipitated to enrich for adducts, resolved by SDS/PAGE, and subjected to anti-HA immunoblotting. (E) Immunoblot of HA-tagged Urm1 protein conjugates after treatment (1 h) of yeast cells with NEM (10 mM) or Dia (20 mM). HA-Urm1 is expressed from its chromosomal locus in WT or Δuba4 cells. (Lower) Dpm1 was used as a loading control. (F) Urmylation of myc-tagged Ahp1. Cells expressing either Ahp1-myc or HA-Urm1 or both in combination were grown to an OD600 of 1.0, and 10 mM NEM was added for 1 h. Blots were probed with myc (Upper) or Dpm1-specific (Lower) antibodies. (G) Ahp1 urmylation in vivo depends on both catalytically active cysteine residues in Uba4. The Δuba4 strains were transformed with integrative plasmids expressing myc-tagged Uba4 variants. The blots were probed with antibodies specific for HA (Top, HA-Urm1), myc (Middle, myc-Uba4), and Dpm1 (Bottom, loading control).
Fig. 2.
Fig. 2.
Urmylation requires a thioester intermediate and gives rise to a covalently linked product that is not acyl disulfide-linked. (A) HA-Urm1 WT cells were treated with diamide (Dia) or H2O2, and HA-Urm1 was immunoprecipitated as described in Fig. 1D. Immunoprecipitates were boiled in sample buffer in the absence or presence of DTT and analyzed by anti-HA immunoblotting. (B) NH2OH pretreatment prevents urmylation. HA-Urm1 WT cells were pretreated for 10 min in PBS with or without 50 mM NH2OH before administration of Dia or H2O2. (C) Postlysis addition of NH2OH does not affect Urm1 adducts. HA-Urm1 WT cells were treated with Dia or H2O2, and HA-Urm1 was immunoprecipitated. Immunoprecipitates were incubated with or without 50 mM NH2OH before boiling in DTT-containing sample buffer.
Fig. 3.
Fig. 3.
Diamide induces conjugation of recombinant thiocarboxylated Urm1 (but not thiocarboxylated EGFP) to target proteins in vitro. (A) Schematic illustration of recombinant HA-tagged Urm1 with either a carboxylate (COOH) or thiocarboxylate (COSH) at its C terminus. (B) Analysis of recombinant HA-Urm1-COOH and HA-Urm1-COSH on a polyacrylamide gel in the absence (Left) or presence (Right) of APM. (C) HA-Urm1 was immobilized on anti-HA agarose and treated with either SDS lysis buffer or a HeLa cell extract in the presence or absence of diamide for 1 h at 4 °C. Immunoprecipitates were boiled in DTT-containing sample buffer, resolved by SDS/PAGE, and detected by anti-HA immunoblotting. (D) Recombinant HA-EGFP-COOH and HA-EGFP-COSH were immunoprecipitated and treated as described in C and compared with HA-Urm1-COSH treated with both HeLa extract and diamide. Note that half of the HA-Urm1-COSH reaction was analyzed, whereas the entire HA-EGFP reaction was loaded onto the gel. *Contaminant in the purified EGFP fraction.
Fig. 4.
Fig. 4.
Urm1 is conjugated to lysine residues in MOCS3, ATPBD3, CTU2, and USP15 in vivo. (A) Nontransduced, HA-Urm1 WT, and HA-Urm1 ΔG HeLa cells were transiently transfected with MOCS3-FLAG and after 24 h treated with either diamide (Dia) or H2O2. MOCS3 was immunoprecipitated through its FLAG tag from SDS lysates, boiled in DTT-containing sample buffer, and resolved by SDS/PAGE. Urmylation was evaluated by anti-HA immunoblotting. The total cell lysate of HeLa cells transfected with MOCS3-HA was included to illustrate the difference in mass between nonurmylated (52 kDa) and urmylated (∼65 kDa) MOCS3-FLAG. (B) As described in A, using FLAG-ATPBD3 as the substrate. The total cell lysate of HeLa cells transfected with ATPBD3-HA is shown in the left lane. (C) As described in A, using CTU2-FLAG as the substrate. The total cell lysate from HeLa cells transfected with CTU2-HA is shown in the left lane. (D) HA-Urm1 WT and HA-Urm1 ΔG HeLa cells were treated with H2O2. Samples were processed as in A. Immunoblotting for endogenous USP15 (110 kDa) revealed that USP15 is modified by one or two molecules of WT Urm1 on H2O2 addition. (E) HA-Urm1 WT and HA-Urm1 ΔG cells were transiently transfected with WT MOCS3 (MOCS3 WT-FLAG) or mutant MOCS3 in which all lysines have been replaced by arginines (MOCS3 KtoR-FLAG). Cells were processed as in A. (F) Urm1 functions as a lysine-directed modifier and modifies Ahp1 on lysine K32 in vivo. The Δahp1 strains were transformed with integrative plasmids expressing different myc-tagged Ahp1 variants under the control of their own AHP1 promoter. Yeast cells were grown in the absence or presence of NEM, and samples were taken at an OD600 of 1.0. Blots were probed with antibodies specific for myc (Upper, Ahp1-myc) or Dpm1 (Lower, loading control).
Fig. 5.
Fig. 5.
Urm1 is appended to the nucleocytoplasmic shuttling factor CAS in response to H2O2 treatment. (A) HA-Urm1 WT and HA-Urm1 ΔG HeLa cells were treated with H2O2. Urm1 was retrieved from SDS lysates through its HA tag. Immunoprecipitates and whole-cell lysates (input) were boiled in the presence of DTT and separated on a polyacrylamide gel. Anti-CAS immunoblotting showed that CAS (110 kDa) is modified by one, or possibly two, molecules of WT Urm1 (12.5 kDa) in response to H2O2. (B) HA-Urm1 WT cells were treated with increasing concentrations of H2O2 for 10 min. Cells were processed as in A. (C) Urmylated CAS resides in the cytosol. HA-Urm1 WT cells were left untreated or treated with 5 mM H2O2. Cells were either lysed directly in an SDS-containing buffer (Total) or lysed in a Nonidet P-40–containing buffer to extract the cytoplasm and membrane fraction (Cytoplasm) followed by disruption of the nuclear pellet in an SDS-based buffer (Nuclei). Urm1 conjugates were immunoprecipitated from these fractions through their HA tag, and urmylation of CAS was demonstrated by anti-CAS immunoblotting. (D) Urmylated CAS persists for several hours. HA-Urm1 WT cells were left untreated or treated with H2O2. After treatment, cells were either immediately harvested or washed and recovered for 1 or 4 h in medium lacking the oxidant. Samples were processed as in A.
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