In Experiment #1, cells were untreated, and in Experiment #2 cells were treated with 1 mM H2O2 for 30 min before harvesting cells. 1: Quantification and statistical analysis for growth tolerance in canavanine (Physique 4B and D), thialysine (Physique 4figure supplements 3 and ?and4),4), and hydrogen peroxide (Determine 4E and F). elife-58155-fig4-data1.xlsx (69K) GUID:?0365C29D-A038-49CF-8D80-A4404C899ED4 Supplementary file 1: Corresponds to Figure 2figure supplement 1. Yeast cells (NHY318 background) expressing either wildtype or S57D ubiquitin were cultured in heavy (H; expressing wildtype ubiquitin) or light (L, expressing S57D ubiquitin) SILAC media to the?mid-log phase and treated with 1 mM H2O2 for 30 min before harvesting cells. Following cell lysis and digestion of lysates with trypsin for 24 hr, ubiquitin-remnant peptides were enriched (see Materials and methods) and analyzed by mass spectrometry. Three biological replicate experiments were analyzed. Since the peptide corresponding to K63-linked poly-ubiquitin also harbors the residue mutated in phosphomimetic (S57D) ubiquitin, K63 linkages are a blind spot for SILAC measurements in these experiments. n.d. indicates not detected. elife-58155-supp1.docx (12K) GUID:?AB751064-0905-428C-896C-E2B3162859CA Supplementary file 2: Corresponds to Figure 3A and Physique 3figure supplement 2. For yeast Ser57 ubiquitin kinases, we analyzed consensus phosphorylation motifs as decided from a previous study based on in vitro activity analysis on peptide libraries (Mok et al., 2010). Values in parentheses are the quantified selectivity values, based on site preference of in vitro activity. Only amino acids selected at a position with a value? 2 are shown. elife-58155-supp2.docx (13K) GUID:?EE7FCEEA-E53A-41BF-B981-738395A83917 Transparent reporting form. elife-58155-transrepform.docx (245K) GUID:?A85E5D0B-E339-427C-8E6A-731262004EF6 Data Availability StatementAll data generated or analyzed during this study are included in the manuscript and supporting files. Abstract Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is usually partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is Ergoloid Mesylates usually Ergoloid Mesylates further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in ions are tabulated and MS-observed masses are shown in the spectra. Physique 2figure supplement 2. Open in a separate window SILAC-MS fragmentation spectra of unmodified (top) and Ser57-phosphorylated (bottom) peptides of ubiquitin isolated from yeast cells grown in the presence (light) or absence (heavy) of H2O2.Cells were grown in SILAC media (supplemented with light or heavy lysine and arginine) to the?mid-log phase (OD600 of 0.6C0.7) and treated with 0.6 mM H2O2 for 30 min. Chromosomally expressed 3xFLAG-tagged ubiquitin (from Ergoloid Mesylates the and loci) was isolated by affinity purification and digested with trypsin. Phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC), separated by a capillary reverse-phase analytical column, and analyzed on a Q Exactive mass spectrometer. Theoretical masses of ions are tabulated and MS-observed masses are shown in the spectra. To identify candidate ubiquitin kinases, we screened for Ser57 phosphorylation activity by co-expressing ubiquitin and yeast kinases in and immunoblotting lysates using an antibody specific for Ser57 phosphorylated ubiquitin. Initially, we focused on candidate kinases for which mutants exhibit phenotypes corresponding to those observed for cells expressing S57A or S57D ubiquitin. We found that co-expression of ubiquitin with the kinase Vhs1 resulted in immunodetection of Ser57 phosphorylated ubiquitin (Physique 3A and Physique 3figure supplement 1). Vhs1 is usually part of the yeast family of Snf1-related kinases (Tumolo et al., 2020), and additional screening revealed three other kinases in this family that phosphorylated ubiquitin at the Ser57 position: Sks1 (which is usually 43% identical to Vhs1) (Physique 3B), Gin4 and Kcc4 (Physique 3figure supplement 2). A previous study reported consensus phosphorylation motifs for Vhs1, Gin4, and Kcc4, and all bear similarity to the amino acid sequence surrounding Ser57 in ubiquitin (Supplementary file 2; Mok et al., 2010). Open in a separate window Physique 3. A subset of the Snf1-related family of kinases phosphorylates ubiquitin at the Ser57 position.(A) Anti-phospho-Ser57 western blot of Rosetta 2 (DE3) lysates co-expressing ubiquitin and yeast kinases. (B) Anti-phospho-Ser57 western blot of Rosetta 2 (DE3) lysates co-expressing ubiquitin (wildtype, S57A, or S65A variants) and Sks1, a paralog of Vhs1. (C and D) In vitro reconstitution of ubiquitin Ser57 phosphorylation Rabbit Polyclonal to AZI2 using purified recombinant Vhs1 (C) and Sks1 (D). Ubiquitin monomers as well as Ergoloid Mesylates linear (M1-linked) dimers and trimers were included in equal amounts. Numerical values above each lane represent reaction time for the sample. (E and F) SILAC-MS of IP-enriched 3XFLAG ubiquitin from yeast cells (JMY1312 background) with either empty vector or with vector for overexpression of Sks1 (E) or Vhs1 (F). Ergoloid Mesylates Black and red dots.