EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples

Mascot Distiller processing method for protein N-terminal acetylation quantitation. (PDF 24 kb

Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes

Visualization and analysis of molecular scanner peptide mass spectra

AbstractThe molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel

Naa50/San-dependent N-terminal acetylation of Scc1 is potentially important for sister chromatid cohesion

The gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-alpha-acetylation (NTA) and epsilon-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs andKATs, respectively), although the possibility that the sameGCN5-relatedN-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localizedGNATs, which possess a dual specificity. All characterizedGNATfamily members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinctKAand relaxedNTAspecificities. Furthermore, inactivation ofGNAT2 leads to significantNTAorKAdecreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation processin vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryoticGNATs may also possess these previously underappreciated broader enzymatic activities