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

Enhanced protein recovery after electrotransfer using square wave alternating voltage

Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene difluoride (PVDF) membrane able to be scanned by MALDI. Acquired data were used to recreate a virtual multidimensional image. Voltage used during this protein electroblotting technique was an unusual square wave alternative voltage (SWAV). The goal of the current study is to evaluate quantitatively the efficiency of the SWAV compared with a classical electroblot process on intact proteins. The effect of the pulsed electric field and the buffer composition were compared to a standard continuous transblotting process defined as the gold standard. Combination of the pulsed asymmetric electric field with 3-(cyclohexylamino)-1-propane-sulfonique acid (CAPS) buffers showed an average 65% increase of protein recovery. Moreover, a strongest effect is observed for high M(r) proteins. In conclusion, the present study highlighted a positive influence of the "shaking" effect of the asymmetric alternative voltage on gel protein extraction

C. elegans RPM-1 Regulates Axon Termination and Synaptogenesis through the Rab GEF GLO-4 and the Rab GTPase GLO-1

SummaryC. elegans RPM-1 (for Regulator of Presynaptic Morphology) is a member of a conserved protein family that includes Drosophila Highwire and mammalian Pam and Phr1. These are large proteins recently shown to regulate synaptogenesis through E3 ubiquitin ligase activities. Here, we report the identification of an RCC1-like guanine nucleotide exchange factor, GLO-4, from mass spectrometry analysis of RPM-1-associated proteins. GLO-4 colocalizes with RPM-1 at presynaptic terminals. Loss of function in glo-4 or in its target Rab GTPase, glo-1, causes neuronal defects resembling those in rpm-1 mutants. We show that the glo pathway functions downstream of rpm-1 and acts in parallel to fsn-1, a partner of RPM-1 E3 ligase function. We find that late endosomes are specifically disorganized at the presynaptic terminals of glo-4 mutants. Our data suggest that RPM-1 positively regulates a Rab GTPase pathway to promote vesicular trafficking via late endosomes

The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation

Abstract Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

International audienceProtein N(α) -terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six N(α) -acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein N(α) -termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays N(ε) -acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947)

Downregulation of N-terminal acetylation triggers ABA-mediated drought responses in Arabidopsis

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0.N-terminal acetylation (NTA) catalysed by N-terminal acetyltransferases (Nats) is among the most common protein modifications in eukaryotes, but its significance is still enigmatic. Here we characterize the plant NatA complex and reveal evolutionary conservation of NatA biochemical properties in higher eukaryotes and uncover specific and essential functions of NatA for development, biosynthetic pathways and stress responses in plants. We show that NTA decreases significantly after drought stress, and NatA abundance is rapidly downregulated by the phytohormone abscisic acid. Accordingly, transgenic downregulation of NatA induces the drought stress response and results in strikingly drought resistant plants. Thus, we propose that NTA by the NatA complex acts as a cellular surveillance mechanism during stress and that imprinting of the proteome by NatA is an important switch for the control of metabolism, development and cellular stress responses downstream of abscisic acid

NatB-mediated N-terminal acetylation affects growth and biotic stress responses

International audienceN-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, and naa25-1) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement naa20-1. Quantitative N-terminomics of approximately 1,000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N-termini starting with the initiator methionine followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity towards osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors