Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys 79 in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys 79 methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated. Molecular & Cellular Proteomics

All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13 CD 3 -methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methylPSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-␤-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl- Post-translational methylation is a widespread protein modification, which predominantly occurs on lysine and arginine residues (1). Protein-lysine methyltransferases catalyze the methylation of lysine residues; these enzymes facilitate the incorporation of methyl groups into the N atoms of lysine residues to produce either mono-, di-, or tri-methyllysine (MML, 1 DML, and TML, respectively). Protein-arginine methyltransferases catalyze the methylation of arginine residues; these enzymes primarily act upon N G atoms to produce mono, asymmetric di-, or symmetric di-methylarginine, although the enzyme-mediated modification of N ␦ atoms to produce ␦-MMA has also been reported in Saccharomyces cerevisiae (2).Traditionally, lysine and arginine methylation have been closely associated with histone proteins, and their crucial roles in modifying chromatin structure have been extensively studied (3). In recent years, however, a growing number of large scale methylation site discovery experiments have indiFrom the ‡New South

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor β-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five.