Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Spatio-temporal expression pattern of the NatB complex, Nat5/Mdm20 in the developing mouse brain: implications for co-operative versus non-co-operative actions of Mdm20 and Nat5

The NatB complex, Nat5/Mdm20 acetyltransferase mediates N-acetylation to control cell cycle progression and actin dynamics in yeast. As yet, little is known about the expression pattern of Mdm20 and Nat5 in multi-cellular organisms. Here we show that Mdm20 is highly expressed in mouse embryonic brain. At E11.5, Mdm20 was widely expressed in both neural progenitors and early differentiating neurons, whereas Nat5 was expressed in Sox1/3+/Mdm20+ neural progenitors. By E14.5, both Mdm20 and Nat5 were downregulated in most ventricular zone neural progenitors, whereas both proteins were found in differentiating neurons and co-expression was maintained at E18.5 in derivatives of these cells, such as midbrain dopaminergic (DA) neurons and septal neurons. These data suggest that Nat5/Mdm20 complex-mediated acetylation may play a role in the proliferation and differentiation of neural progenitors. Intriguingly, our data also showed that Mdm20 is not always co-expressed with Nat5 in all differentiated neurons, for example deep cerebellar neurons. Moreover, detailed examination of the subcellular localization of Mdm20 and Nat5 in cultured Nat5+/Mdm20+ midbrain DA neurons revealed that Mdm20 is also not necessarily co-localized with Nat5 within neurons. Given that Nat5 is only a known member of Nat family protein that interacts with Mdm20, our data imply that Mdm20 may function either with an unidentified Nat protein partner(s) or possibly in a Nat-independent manner.     

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.     

Identification of the human N(alpha)-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression

Protein N(alpha)-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein N(alpha)-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific N(alpha)-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G(0)/G(1)-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G(0)/G(1)-phase and increased the fraction of cells in the sub-G(0)/G(1)-phase. In summary, we show for the first time a vertebrate NatB protein N(alpha)-acetyltransferase complex essential for normal cell proliferation.     

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.     

Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes

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.     

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.     

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.     

The Chaperone-Like Protein HYPK Acts Together with NatA in Cotranslational N-Terminal Acetylation and Prevention of Huntingtin Aggregation

The human NatA protein N^α-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G₀/G₁ phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.N^α-terminal acetylation is among the most common protein modifications in eukaryotes, occurring on ∼50% of Saccharomyces cerevisiae proteins and ∼80% of human proteins (12). In yeast, four types of N^α-terminal acetyltransferases (NATs) have been defined (NatA-NatD), while a fifth type, NatE, has been hypothesized (21, 32-34, 38). For humans, NatA, NatB, NatC, and NatE were recently presented (2, 4, 18, 39, 40). A revised NAT-subunit nomenclature was recently introduced in order to have identical names for orthologous subunits from different species, and each gene was denoted NAA (N^α-acetyltransferase) followed by a number depending on Nat type and the type of subunit (catalytic/auxiliary) (32). The major human NAT complex, hNatA, is composed of the catalytic subunit hNaa10p (previously named hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH) (4). Human NatA is evolutionarily conserved from the yeast complex in terms of subunit composition and substrate specificity (12, 26, 28). However, in contrast to yeast cells, human cells potentially contain several distinct NatA complexes due to the presence of two genes for each of the two NatA subunits, NAA10 and NAA15 (6, 8). Protein N-terminal acetylation occurs on the ribosome when the nascent polypeptide emerges (21, 29, 30, 41, 42). Proteins with Ser, Thr, Gly, Ala, Val, or Cys N termini are potential substrates of NatA (12), while NatB and NatC potentially acetylate specific classes of substrates that still carry the initiator Met (34). The biological importance of the human NatA complex was evident from knockdown experiments where induction of apoptosis and growth arrest of cells in the G₁/G₀ phase were the resulting phenotypes (9, 11, 20, 25). The phenotypes induced by hNatA depletion most likely reflect the fact that one or more specific substrate proteins lack proper N^α acetylation, in view of the fact that a large quantitative proteomic analysis of the acetylation status of protein N termini in hNaa15p-hNaa10p knockdown cells revealed a decrease in the level of N^α acetylation of some partially acetylated substrates compared to that in control cells (12).To further characterize the human NatA complex, we looked for the presence of stable interaction partners of hNaa15p and hNaa10p. Here we present data identifying the Huntingtin (Htt) yeast two-hybrid protein K (HYPK) as a novel factor involved in cotranslational NatA acetylation. HYPK, originally identified in a yeast two-hybrid screen during a search for potential interaction partners for the Huntingtin protein (19), was recently found to reduce Htt polyglutamine (polyQ) aggregation upon overexpression (36). However, the role of the endogenous HYPK protein has yet to be revealed. We demonstrate that endogenous HYPK (i) stably interacts with the hNaa10p-hNaa15p NatA N-terminal-acetyltransferase complex and with ribosomes, (ii) is required for normal N-terminal acetylation of a NatA substrate, (iii) is important for cell survival independent of Htt polyQ, and (iv) is important for the prevention of Htt polyQ aggregation. Furthermore, NatA is essential for the proper expression of HYPK protein and modulates Htt polyQ aggregation.     

Mutation of an Arabidopsis NatB N-Alpha-Terminal Acetylation Complex Component Causes Pleiotropic Developmental Defects

N-α-terminal acetylation is one of the most common, but least understood modifications of eukaryotic proteins. Although a high degree of conservation exists between the N-α-terminal acetylomes of plants and animals, very little information is available on this modification in plants. In yeast and humans, N-α-acetyltransferase complexes include a single catalytic subunit and one or two auxiliary subunits. Here, we report the positional cloning of TRANSCURVATA2 (TCU2), which encodes the auxiliary subunit of the NatB N-α-acetyltransferase complex in Arabidopsis. The phenotypes of loss-of-function tcu2 alleles indicate that NatB complex activity is required for flowering time regulation and for leaf, inflorescence, flower, fruit and embryonic development. In double mutants, tcu2 alleles synergistically interact with alleles of ARGONAUTE10, which encodes a component of the microRNA machinery. In summary, NatB-mediated N-α-terminal acetylation of proteins is pleiotropically required for Arabidopsis development and seems to be functionally related to the microRNA pathway.     

Synthetic lethal screen of NAA20, a catalytic subunit gene of NatB N-terminal acetylase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae NatB N-terminal acetylase contains a catalytic subunit Naa20 and an auxiliary subunit Naa25. To elucidate the cellular functions of the NatB, we utilized the Synthetic Genetic Array to screen for genes that are essential for cell growth in the absence of NAA20. The genome-wide synthetic lethal screen of NAA20 identified genes encoding for serine/threonine protein kinase Vps15, 1,3-beta-glucanosyltransferase Gas5, and a catabolic repression regulator Mig3. The present study suggests that the catalytic activity of the NatB N-terminal aceytase is involved in vacuolar protein sorting and cell wall maintenance.     

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.     

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.     

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 DNAJB6 and DNAJB8 Protein Chaperones Prevent Intracellular Aggregation of Polyglutamine Peptides

Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington''s disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.     

Autophagy Genes Protect Against Disease Caused by Polyglutamine Expansion Proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington’s disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

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.