N ␣ -terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys-and Val-N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-⌬ nat1-⌬ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-⌬ nat1-⌬ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys-termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.Ard1 ͉ COFRADIC ͉ N-terminal acetylation ͉ Nat1 ͉ NatA P rotein N ␣ -terminal acetylation (here referred to as Nacetylation) is one of the most common covalent modifications of eukaryotic proteins, in which an acetyl group is transferred from acetyl-CoA to the ␣-amino group of protein N-terminal residues. N-acetylation occurs cotranslationally on nascent polypeptide chains and almost all N-acetylations in Saccharomyces cerevisiae are catalyzed by 1 of 3 major Nterminal acetyltransferase (NAT) complexes, NatA, NatB or NatC, consisting of catalytic subunits Ard1p, Nat3p, and Mak3p, respectively, and 1 or more auxiliary subunits (1). Yeast NatA, the major and best studied NAT, is composed of the catalytic subunit Ard1p in complex with Nat1p (2). Nat1p is responsible for anchoring Ard1p to the ribosome, thus facilitating cotranslational N-acetylation (3). Both subunits are required for optimal acetyltransferase activity and yeast strains lacking either one of the subunits display the same phenotypes, indicating that both genes are also functionally linked (4). The yeast NatA, NatB and NatC complexes differ in their substrate specificities. NatA substrates represent by far the largest group and contain proteins with Ser-, Ala-, Gly-, Val-, Cys-or Thr-N termini, whereas NatB and NatC act on diff...

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Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

Focal amplifications of chromosome 3p13-3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon. However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses.

We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

The advent of high-throughput proteomics has enabled the identification of ever increasing numbers of proteins. Correspondingly, the number of publications centered on these protein identifications has increased dramatically. With the first results of the HUPO Plasma Proteome Project being analyzed and many other large-scale proteomics projects about to disseminate their data, this trend is not likely to flatten out any time soon. However, the publication mechanism of these identified proteins has lagged behind in technical terms. Often very long lists of identifications are either published directly with the article, resulting in both a voluminous and rather tedious read, or are included on the publisher's website as supplementary information. In either case, these lists are typically only provided as portable document format documents with a custom-made layout, making it practically impossible for computer programs to interpret them, let alone efficiently query them. Here we propose the proteomics identifications (PRIDE) database (http://www.ebi.ac.uk/pride) as a means to finally turn publicly available data into publicly accessible data. PRIDE offers a web-based query interface, a user-friendly data upload facility, and a documented application programming interface for direct computational access. The complete PRIDE database, source code, data, and support tools are freely available for web access or download and local installation.