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1.
Saccharomyces cerevisiae contains three N-terminal acetyltransferases (NATs), NatA, NatB, and NatC, composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p (NatA); Nat3p and Mdm20p (NatB); and Mak3p, Mak10, and Mak31p (NatC). The overall patterns of N-terminally acetylated proteins and NAT orthologous genes suggest that yeast and higher eukaryotes have similar systems for N-terminal acetylation. The differential expression of certain NAT subunits during development or in carcinomas of higher eukaryotes suggests that the NATs are more highly expressed in cells undergoing rapid protein synthesis. Although Mak3p is functionally the same in yeast and plants, findings with TE2 (a human Ard1p ortholog) and Tbdn100 (a mouse Nat1p ortholog) suggest that certain of the NAT subunits may have functions other than their role in NATs or that these orthologs are not functionally equivalent. Thus, the vertebrate NATs remain to be definitively identified, and, furthermore, it remains to be seen if any of the yeast NATs contribute to other functions.  相似文献   

2.
N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.  相似文献   

3.
N-terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al. (Gautschi et al. [2003] Mol Cell Biol 23: 7403) previously demonstrated with peptide crosslinking experiments that NatA is bound to ribosomes. In our studies, biochemical fractionation in linear sucrose density gradients revealed that all of the NATs are associated with mono- and polyribosome fractions. However only a minor portion of Nat3p colocalized with the polyribosomes. Disruption of the polyribosomes did not cause dissociation of the NATs from ribosomal subparticles. The NAT auxiliary subunits, Nat1p and Mdm20p, apparently are required for efficient binding of the corresponding catalytic subunits to the ribosomes. Deletions of the genes corresponding to auxiliary subunits significantly diminish the protein levels of the catalytic subunits, especially Nat3p, while deletions of the catalytic subunits produced less effect on the stability of Nat1p and Mdm20p. Also two ribosomal proteins, Rpl25p and Rpl35p, were identified in a TAP-affinity purified NatA sample. Moreover, Ard1p copurifies with Rpl35p-TAP. We suggest that these two ribosomal proteins, which are in close proximity to the ribosomal exit tunnel, may play a role in NatA attachment to the ribosome.  相似文献   

4.
The yeast Saccharomyces cerevisiae contains three types of N(alpha)-terminal acetyltransferases, NatA, NatB, and NatC, with each having a different catalytic subunit, Ard1p, Nat3p, and Mak3p, respectively, and each acetylating different sets of proteins with different N(alpha)-terminal regions. We show that the NatC N(alpha)-terminal acetyltransferases contains Mak10p and Mak31p subunits, in addition to Mak3p, and that all three subunits are associated with each other to form the active complex. Genetic deletion of any one of the three subunits results in identical abnormal phenotypes, including the lack of acetylation of a NatC substrate in vivo, diminished growth at 37 degrees C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.  相似文献   

5.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the state of N-terminal acetylation of 68 ribosomal proteins from a normal strain of Saccharomyces cerevisiae and from the ard1-Delta, nat3-Delta, and mak3-Delta mutants (), each lacking a catalytic subunit of three different N-terminal acetyltransferases. A total 30 of the of 68 ribosomal proteins were N-terminal-acetylated, and 24 of these (80%) were NatA substrates, unacetylated in solely the ard1-Delta mutant and having mainly Ac-Ser- termini and a few with Ac-Ala- or Ac-Thr- termini. Only 4 (13%) were NatB substrates, unacetylated in solely the nat3-Delta mutant, and having Ac-Met-Asp- or Ac-Met-Glu- termini. No NatC substrates were uncovered, e.g. unacetylated in solely mak3-Delta mutants, consistent with finding that none of the ribosomal proteins had Ac-Met-Ile-, Ac-Met-Leu-, or Ac-Met-Phe- termini. Interestingly, two new types of the unusual NatD substrates were uncovered, having either Ac-Ser-Asp-Phe- or Ac-Ser-Asp-Ala- termini that were unacetylated in the ard1-Delta mutant, and only partially acetylated in the mak3-Delta mutant and, for one case, also only partially in the nat3-Delta mutant. We suggest that the acetylation of NatD substrates requires not only Ard1p and Nat1p, but also auxiliary factors that are acetylated by the Mak3p and Nat3p N-terminal acetyltransferases.  相似文献   

6.
N-terminal acetylation in the yeast Saccharomyces cerevisiae is catalysed by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain the catalytic subunits Ard1p, Nat3p and Mak3p, respectively. Yeast 6-phosphofructo-2-kinase (PFK2) was found to be acetylated at the amino acid lysine 3. The Lys3-Arg mutant was not acetylated and the mutation causes a slight decrease in enzyme activity. PFK2 from yeast cells exposed to hypo-osmotic stress is known to be phosphorylated at Ser8 and Ser652 (Dihazi et al., 2001a). We have taken a mass spectrometric approach to investigate the influence of PFK2 acetylation on its phosphorylation. Wild-type PFK2 and the Lys3-Arg mutant were purified from hypo-osmotically stressed cells and analysed with MALDI-TOF MS for phosphorylation. Wild-type PFK2 without any tag sequence was found to be acetylated and two times phosphorylated at the N-terminal peptide T(1-40) carrying the acetylation. The same results were observed with C-terminally His-tagged PFK2. When the His-tag was added to the N-terminus of the protein PFK2, acetylation was found to be incomplete and only one phosphate was incorporated in the peptide T(1-41). The Lys3-Arg mutant of PFK2 was not at all post-translationally modified at the N-terminal peptide. Our data indicate that Lys3 acetylation affects the N-terminal phosphorylation of PFK2 under hypo-osmotic stress.  相似文献   

7.
N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was N(alpha) acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.  相似文献   

8.
Arylamine N-acetyltransferases (NATs) catalyse acetylation reactions which can result in either detoxification or activation of arylamine carcinogens. The human NAT loci (NAT1, NAT2, and a pseudogene, NATP) have been mapped to human chromosome 8p22, a region frequently deleted in tumours. There are three functional genes in mice (Nat1, Nat2, and Nat3) encoding for three NAT isoenzymes. Different alleles at the Nat2 locus are responsible for the acetylation polymorphism identified in different mouse strains. We show that Nat3 is close to Nat1 and Nat2, by screening of a P1 artificial chromosome (PAC) library and provide cytogenetic evidence for co-localisation of the three genes in chromosome region 8 B3.1-B3.3. The Nat region of mouse and human is homologous. We also provide sequence information and a restriction map in the vicinity of Nat1 and Nat2 and describe a noncoding exon located 6 kb upstream of the Nat2 coding region.  相似文献   

9.
Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine Nϵ-acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. Nα-terminal acetylation occurs on the ribosome where the α amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an Nα-acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.  相似文献   

10.
Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here, we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.  相似文献   

11.
The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.  相似文献   

12.
The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets. We report the x-ray crystal structure of Naa50p bound to a native substrate peptide fragment and CoA. We found that the peptide backbone of the substrate is anchored to the protein through a series of backbone hydrogen bonds with the first methionine residue specified through multiple van der Waals contacts, together creating an α-amino methionine-specific pocket. We also employed structure-based mutagenesis; the results support the importance of the α-amino methionine-specific pocket of Naa50p and are consistent with the proposal that conserved histidine and tyrosine residues play important catalytic roles. Superposition of the ternary Naa50p complex with the peptide-bound Gcn5 histone acetyltransferase revealed that the two enzymes share a Gcn5-related N-acetyltransferase fold but differ in their respective substrate-binding grooves such that Naa50p can accommodate only an α-amino substrate and not a side chain lysine substrate that is acetylated by lysine acetyltransferase enzymes such as Gcn5. The structure of the ternary Naa50p complex also provides the first molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications.  相似文献   

13.
The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Delta exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Delta was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Delta showed an approximately 100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Delta strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Delta. Second, this suppression by tfs1Delta was abolished in the nat3Delta background, indicating that Tfs1p was not functional in the nat3Delta strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.  相似文献   

14.
15.
Abstract Protein N-terminal acetylation by Nα-acetyltransferases (NATs) is an omnipresent protein modification that affects a large number of proteins. The exact biological role of N-terminal acetylation has, however, remained enigmatic for the overall majority of affected proteins, and only for a rather small number of proteins, N-terminal acetylation was linked to various protein features including stability, localization, and interactions. This minireview tries to summarize the recent progress made in understanding the functionality of N-terminal protein acetylation and also focuses on noncanonical functions of the NATs subunits.  相似文献   

16.
The N-terminal acetyltransferase NatB in Saccharomyces cerevisiae consists of the catalytic subunit Nat3p and the associated subunit Mdm20p. We here extend our present knowledge about the physiological role of NatB by a combined proteomics and phenomics approach. We found that strains deleted for either NAT3 or MDM20 displayed different growth rates and morphologies in specific stress conditions, demonstrating that the two NatB subunits have partly individual functions. Earlier reported phenotypes of the nat3Delta strain have been associated with altered functionality of actin cables. However, we found that point mutants of tropomyosin that suppress the actin cable defect observed in nat3Delta only partially restores wild-type growth and morphology, indicating the existence of functionally important acetylations unrelated to actin cable function. Predicted NatB substrates were dramatically overrepresented in a distinct set of biological processes, mainly related to DNA processing and cell cycle progression. Three of these proteins, Cac2p, Pac10p, and Swc7p, were identified as true NatB substrates. To identify N-terminal acetylations potentially important for protein function, we performed a large-scale comparative phenotypic analysis including nat3Delta and strains deleted for the putative NatB substrates involved in cell cycle regulation and DNA processing. By this procedure we predicted functional importance of the N-terminal acetylation for 31 proteins.  相似文献   

17.
Protein N(α)-terminal acetylation (Nt-acetylation) is considered one of the most common protein modification in eukaryotes, and 80-90% of all soluble human proteins are modified in this way, with functional implications ranging from altered protein function and stability to translocation potency amongst others. Nt-acetylation is catalyzed by N-terminal acetyltransferases (NATs), and in yeast five NAT types are identified and denoted NatA-NatE. Higher eukaryotes additionally express NatF. Except for NatD, human orthologues for all yeast NATs are identified. yNatD is defined as the catalytic unit Naa40p (Nat4) which co-translationally Nt-acetylates histones H2A and H4. In this study we identified and characterized hNaa40p/hNatD, the human orthologue of the yeast Naa40p. An in vitro proteome-derived peptide library Nt-acetylation assay indicated that recombinant hNaa40p acetylates N-termini starting with the consensus sequence Ser-Gly-Gly-Gly-Lys-, strongly resembling the N-termini of the human histones H2A and H4. This was confirmed as recombinant hNaa40p Nt-acetylated the oligopeptides derived from the N-termini of both histones. In contrast, a synthetically Nt-acetylated H4 N-terminal peptide with all lysines being non-acetylated, was not significantly acetylated by hNaa40p, indicating that hNaa40p catalyzed H4 N(α)-acetylation and not H4 lysine N(ε)-acetylation. Also, immunoprecipitated hNaa40p specifically Nt-acetylated H4 in vitro. Heterologous expression of hNaa40p in a yeast naa40-Δ strain restored Nt-acetylation of yeast histone H4, but not H2A in vivo, probably reflecting the fact that the N-terminal sequences of human H2A and H4 are highly similar to each other and to yeast H4 while the N-terminal sequence of yeast H2A differs. Thus, Naa40p seems to have co-evolved with the human H2A sequence. Finally, a partial co-sedimentation with ribosomes indicates that hNaa40p co-translationally acetylates H2A and H4. Combined, our results strongly suggest that human Naa40p/NatD is conserved from yeast. Thus, the NATs of all classes of N-terminally acetylated proteins in humans now appear to be accounted for.  相似文献   

18.
19.
N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.  相似文献   

20.
We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.  相似文献   

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