An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A

A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.     

ARD1 and NAT1 proteins form a complex that has N-terminal acetyltransferase activity.

Two yeast genes, ARD1 and NAT1, are required for the expression of an N-terminal protein acetyltransferase. This activity is required for full repression of the silent mating type locus HML, for sporulation, and for entry into G0. While the NAT1 gene product is thought to be the catalytic subunit of the enzyme, the role of the ARD1 protein has remained unclear. We have used epitope tagged derivatives of ARD1 and NAT1 to provide biochemical evidence for the formation of an ARD1-NAT1 complex, and to show that both proteins are required for the N-terminal acetyltransferase activity. We also present evidence for the formation of ARD1-ARD1 homodimers. Deletion analysis suggests that the C-terminal region of ARD1 may be involved in the formation of both ARD1-ARD1 and ARD1-NAT1 complexes.     

一个新的人类N末端乙酰转移酶基因的克隆及其表达

乙酰化修饰普遍发生于真核生物的各种蛋白质 ,对蛋白质的稳定性和活性产生重大影响。酵母N末端乙酰基转移酶NatA由NAT1和ARD1两个亚基组成。任何一个亚基缺陷都导致NatA活性丧失 ,酵母表现出在接合型转换、细胞周期调控及染色体稳定性等方面的异常。通过同源克隆方法 ,克隆了与酵母NAT1高度同源的一个人类基因。经GenBank查询 ,为新基因 ,命名为HNAT1。原位杂交结果显示 ,在成年小鼠曲精管内 ,该基因的表达强烈。其表达强度与细胞类型和细胞所处时相有关。该基因在精子发生过程中可能具有重要作用     

N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome

N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1 mutant than in the normal strain.     

The action of N-terminal acetyltransferases on yeast ribosomal proteins

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.     

Composition and function of the eukaryotic N-terminal acetyltransferase subunits

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.     

N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins

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.     

An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development

We previously identified mNAT1 (murine N-terminal acetyltransferase 1) as an embryonic gene that is expressed in the developing brain and subsequently down-regulated, in part, by the onset of N-methyl-d-aspartate (NMDA) receptor function. By searching the data base we discovered a second closely related gene, mNAT2. mNAT1 and mNAT2 are highly homologous to yeast NAT1, a gene known to regulate entry into the G0 phase of the cell cycle. However, in the absence of further characterization, including evidence that mammalian homologues of NAT1 encode functional acetyltransferases, the significance of this relationship has been unclear. Here we focus on mNAT1. Biochemical analysis demonstrated that mNAT1 and its evolutionarily conserved co-subunit, mARD1, assemble to form a functional acetyltransferase. Transfection of mammalian cells with mNAT1 and mARD1 followed by immunofluorescent staining revealed that these proteins localize to the cytoplasm in both overlapping and separate compartments. In situ hybridization demonstrated that throughout brain development mNAT1 and mARD1 are highly expressed in areas of cell division and migration and are down-regulated as neurons differentiate. Finally, mNAT1 and mARD1 are expressed in proliferating mouse P19 embryonic carcinoma cells; treatment of these cells with retinoic acid initiates exit from the cell cycle, neuronal differentiation, and down-regulation of mNAT1 and mARD1 as the NMOA receptor 1 gene is induced. The results provide the first direct evidence that vertebrate homologues of NAT1 and ARD1 form an evolutionarily conserved N-terminal acetyltransferase and suggest that expression and down-regulation of this enzyme complex plays an important role in the generation and differentiation of neurons.     

N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome

The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain.     

NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation

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.     

Nat3p and Mdm20p are required for function of yeast NatB Nalpha-terminal acetyltransferase and of actin and tropomyosin

NatB Nalpha-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with Met-Glu- or Met-Asp- termini and subclasses of proteins with Met-Asn- and Met-Met- termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-Delta and mdm20-Delta mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNA-damaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.     

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.     

N-Terminal Acetylation by NatC Is Not a General Determinant for Substrate Subcellular Localization in Saccharomyces cerevisiae

N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.     

The stress-induced Tfs1p requires NatB-mediated acetylation to inhibit carboxypeptidase Y and to regulate the protein kinase A pathway

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.     

Lysine 3 acetylation regulates the phosphorylation of yeast 6-phosphofructo-2-kinase under hypo-osmotic stress

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.     

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.     

Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB

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.