NatC Nα-terminal Acetyltransferase of Yeast Contains Three Subunits, Mak3p, Mak10p, and Mak31p*

The yeast Saccharomyces cerevisiaecontains three types of Nα-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 Nα-terminal regions. We show that the NatC Nα-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 °C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.

tRNA processing, rRNA maturation, and telomeric DNA synthesis (21). However, Mak31p is more divergent compared with other yeast Sm-like proteins, and it is the only member lacking a glycine or cysteine at position 107 as numbered according to the alignment of Sm domains. Furthermore, Mak31p did not precipitate any of the tested RNAs (20).
We have investigated the requirements of the Mak3p, Mak10p, and Mak31p subunits for acetylation with the yeast iso-1 system. Because the N-terminal region of iso-1 is dispensable for biosynthesis, function, and mitochondrial import (22,23), N-terminal processing can be investigated freely with essentially any alteration. In fact, altered forms of iso-1 proved to be ideally suited for investigating the specificity of N-terminal methionine cleavage and N-terminal acetylation (2,5,6). In addition, because the mass of iso-1 is ϳ12.5 kDa and because mass spectrometry can be used conveniently to determine molecules less than 30 kDa with an accuracy of ϳ1 Da, we have used this method to determine acetylation of mutant forms of iso-1.
In this study, we have demonstrated that each of the three subunits, Mak3p, Mak10p, and Mak31p, is absolutely required for N-terminal acetylation of a NatC type of substrate in vivo. Deletion of any of the corresponding genes prevented acetylation of a NatC type of altered iso-1 but not of a NatA or NatB types of iso-1. We have also shown that all three deletion strains showed similar phenotypes, including slower growth on nonfermentable carbon sources at elevated temperatures. Thus, all three subunits (Mak3p, Mak10p, and Mak31p) of the complex are required for NatC activity.

MATERIALS AND METHODS
Genetic Nomenclature-Using standard genetic nomenclature, MAK3, for example, designates the normal wild-type allele; mak3-⌬ designates the deletion or disruption of the gene; and Mak3p designates the protein encoded by MAK3. CYC1-853, CYC1-987, etc., designate   genes encoding different mutant forms of functional iso-1, whereas cyc1-3 and cyc1-115 designate alleles lacking or encoding nonfunctional forms of iso-1. The cyc7-67 allele denotes a partial deletion of the CYC7 gene that results in complete deficiency of iso-2-cytochrome c.
Yeast Strains and Media-Unless stated otherwise, yeast was grown at 30°C in YPD or YPG medium or SD medium containing appropriate supplements (24). The strains used in this study are listed in Table II (2). The nucleotide mismatches with the cyc1-31 sequence are indicated by underlines.
a The substrate type designates the substrates that are exemplified by the N-terminal sequence of the altered iso-1 shown in column 3 (see Table I). b The results were obtained with the strains listed in Table II. c Designates the level (%) of iso-1-cytochrome c in comparison with the isogenic CYC1 ϩ strain.

TABLE V Molecular masses and percent acetylation of altered iso-1 prepared from normal and mutant strains
The "observed" mass refers to the mass obtained experimentally, whereas the "expected" mass refers to the values deduced from the structure either having or lacking the acetyl group as indicated in the table. Percent acetylation values shown in parentheses are from Polevoda et al. (2). The percent acetylation of samples from B-10672 and B-12045, determined in this study by mass spectrometry, correct the values previously determined by high pressure liquid chromatography (2). were constructed by first using synthetic oligonucleotides to generate CYC1 mutations encoding the desired iso-1 and second by separately disrupting each of the MAK3, MAK10, MAK31, NAT1, or NAT3 genes (see below). Strains are designated as "normal" if they contain the full complement of normal NAT genes.
Mating Efficiencies-Quantitative matings were determined by plating serial dilutions of logarithmically growing yeast cells onto SD plates containing a lawn of the tester mating strain, B-6925, and determining the frequencies of prototrophic diploid colonies arising after incubation for 3 days. Dilutions of the haploid strains were also plated on YPD plates to determine total number of cell. Mating efficiencies were expressed as the ratio of the number of diploid colonies to the number of haploid cells plated on the lawn of the tester strain.
Construction of Iso-1 Mutants-Strains with altered forms of iso-1 were conveniently produced by transforming yeast directly with synthetic oligonucleotides as described previously by Yamamoto et al. (25), using the cyc1-31 mutant B-7528 (Table II) and 50 g of the oligonu-cleotides (Table III) that have minimal mismatches.
Iso-1 Content-The amounts of iso-1 in yeast strains were estimated by low temperature (Ϫ196°C) spectroscopic examinations of intact cells (26) and comparing the intensities of the C ␣ band at 547 nm with the corresponding band of strains having known amounts of cytochrome c.
Construction of Deletion Mutants-Standard molecular biological procedures were performed as described previously (27). The NAT1, NAT3, MAK3, MAK10, and MAK31 genes were disrupted by replacing a portion of the genes with either the kanMX4 or URA3 genes and using the appropriate fragment for gene replacements. The PCR-generated fragment required for producing the nat3-⌬::kanMX4 disruption was prepared by the method of Baudin et al. (28) using the pFA6-kanMX4 plasmid (29) as template and the primers Oligo 1 and 2 (Table III). The correct disruptions in the transformants were identified by PCR using the set of primers Oligo 3 and 4. The fragment required for producing the nat1-⌬::URA3 disruption was prepared from the plasmid pAA1132 (also designated JM111) (3). The correct nat1-⌬ disruptions were identified with Oligo 5 and 6. The fragment required for producing the mak3-⌬::URA3 disruption was prepared from the plasmid pJC11C (also designated pAA1131) (31). The correct mak3-⌬ disruptions were identified with Oligo 7 and 8. mak10-⌬ and mak31-⌬ disruptions were made by PCR-based technique using disrupter Oligo 9 and 10, and Oligo 13 and 14, respectively. The correct disruptions were identified with Oligo 11 and 12 for mak10-⌬ and Oligo 15 and 16 for mak31-⌬, respectively.
Purification of Iso-1-The strains were grown and the cells treated with ethyl acetate as described previously (30). Iso-1-cytochromes c were purified using two subsequent rounds of weak cation exchange Mass Spectrometric Analysis-MALDI samples were prepared by mixing one part of the protein sample with one part of the sinapinic acid matrix at a concentration of 10 mg/ml in 30% acetonitrile and applying 1 l of this mixture to the sample probe. For some samples, 1% trifluoroacetic acid was added to the sample before mixing with matrix in order to enhance the MALDI signal. Samples were allowed to air dry before inserting them in either a Voyager-DE STR linear time-of-flight mass spectrometer (PE Biosystems, Framingham, MA) at the Microchemical Protein/Peptide Core Facility, University of Rochester or a Bruker ProFLEX III MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA) at the Mass Spectrometry Facility, Department of Chemistry, Louisiana State University (Baton Rouge, LA). Positive ion mass spectra were recorded using 25 keV of total acceleration energy and with a grid voltage of 93.5%. External mass calibration was performed using a sample containing insulin, thioredoxin, and apomyoglobin in sinapinic acid matrix or using horse cytochrome c.

RESULTS AND DISCUSSION
Phenotypes of mak Deletion Mutants-It has been previously reported that mak3-⌬ (31) and mak10-⌬ (19) mutants have diminished growth on medium with glycerol or ethanol as a carbon source. These phenotypes were confirmed and others were investigated in this study using the following isogenic series of strains that were prepared from strain B-7528 by making null deletions of MAK3, MAK10, and MAK31 genes by disrupter plasmids (see "Materials and Methods") or PCRbased disrupters (see Table III for corresponding oligonucleotides): normal (B-8462), mak3-⌬ (B-9072), mak10-⌬ (B-12333), and mak31-⌬ (B-13275). As shown in Fig. 1, mak3-⌬, mak10-⌬, and mak31-⌬ mutants grew poorly on YPG medium at 37°C, although growth on YPD medium at 37°C was normal, thus displaying the Nfs Ϫ phenotype (diminished growth on media containing a nonfermentable substrates as the sole carbon and energy source). Similar reduced growth for all of the deletion mutants was detected on YPE (ethanol), YPGal (galactose), and YPRaf (raffinose) media (not shown). (Because the normal strain, B-8462 is Gal Ϫ and Raf ϩ/Ϫ , the diminished growth on YPGal and YPRaf media is a secondary consequence of the Nfs Ϫ phenotype; Ϫ derivatives of B-8462 also did not grow on YPGal and grew poorly on YPRaf media, especially at 37°C.) Also, the mating frequencies of mak3-⌬ (B-9022), mak10-⌬ (B-12333), and mak31-⌬ (B-13275) mutants were the same or similar to the mating efficiency of normal strain B-8462 (not shown), a result that is in contrast to the results with the ard1, nat1, and nat3 mutants, which have reduced mating efficiencies (2,3). In addition, iso-1 levels in the mak3-⌬, mak10-⌬, and mak31-⌬ deletion mutants did not differ significantly from the corresponding normal MAK ϩ strains, as estimated by low temperature spectroscopic examination of intact cells (Table IV). It is noteworthy that the mak3-⌬ (B-9022), mak10-⌬ (B-12333), and mak31-⌬ (B-13275) mutant strains contained the normal complement of all of the cytochromes when grown on YPD medium at 37°C. Thus, the lack of growth at 37°C on media with nonfermentable carbon sources cannot be attributed to a cytochrome deficiency.
Lack of Acetylation of Altered Iso-1 in Deletion Mutants-For this study, we chose four different CYC1 alleles with corresponding altered iso-1 N termini, each representing one class of NAT substrates (Table IV). Iso-1 from the normal strain and from each of the mak3-⌬, mak10-⌬, mak31-⌬, nat1-⌬, and nat3-⌬ mutants were subjected to mass spectrometric analysis. Peaks corresponding to iso-1 were observed at masses between 12.5 and 13 kDa depending on the altered N-terminal sequence. The iso-1 were identified by the masses determined from the spectra, the masses deduced from the gene sequences, and knowledge of previously established modifications. The results of MALDI-TOF determinations of molecular masses for altered iso-1 are presented in Table V. Examples of some spectra are shown in Fig. 2. The lack of protein acetylation leads to a diminished molecular mass of ϳ42 Da, which corresponds to the mass of the acetyl group. In general, the samples were either completely or almost completely acetylated or were completely unacetylated, except for a NatAЈ substrate from CYC1-987 strains having one or another of the mak3-⌬, mak10-⌬, mak31-⌬, and nat3-⌬ deletions in which the iso-1 contained ϳ5-10% of the unacetylated form.
Overall, the analysis of iso-1 acetylation from the complete sets of isogenic strains gave rise to expected results (Tables IV  and V (Fig. 2). All of these results could be predicted from the previously identified NAT substrates (2). Importantly, acetylation was not detected in altered iso-1 Met-Ile-Arg (CYC1-1162) from B-12333 (mak10-⌬) and B-13275 (mak31-⌬) mutants. Thus, the presence of the functional Mak3p, Mak10p, and Mak31p is absolutely required for acetylation of NatC-type substrates in vivo.
Orthologs of Mak3p, Mak10p, and Mak31p-One important question concerns the N-terminal acetyltransferases and the nature of N-terminal acetylation in other eukaryotes. Moerschell et al. (6) point out the similarity in the pattern of Nterminal acetylation of proteins from higher eukaryotes and S. cerevisiae, suggesting that the same systems may operating in all eukaryotes. In addition, Polevoda, Sherman, and colleagues (2) point out the existence of orthologous genes encoding the catalytic subunits of the three N-terminal acetyltransferases, also indicative of the same or similar N-terminal acetyltransferases operating in higher eukaryotes. Species containing orthologs of the yeast Ard1p and Nat3p include Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, as well as others (2).
As shown in Fig. 3A, BLAST (basic local alignment search tool program) (33)  However, it is doubtful that the similar proteins in prokaryotes are N-terminal acetyltransferases that act co-translationally on a wide range of proteins but are rather more similar, for example, to E. coli RimIp that acetylates the N terminus of ribosomal protein S18 (34).
BLAST comparisons also revealed orthologous genes encoding snRNP Sm-like proteins, similar to Mak31p, in S. pombe, Medicago sativa, Thermoplasma acidophilum, C. elegans, D. melanogaster, A. thaliana, and H. sapiens, with 35-55% amino acid sequence similarity spanning almost the entire protein (Fig. 3B). Although it is unclear whether these proteins have Sm or Mak31p functions, the Gly or Cys residues at position 107 conserved in Sm proteins are not found in the these proteins.
Lee and Wickner (19) have previously pointed out the sequence similarity of Mak10p to the variable regions of the T cell receptor ␣-subunit. Also, the Mak10p sequence is similar to open reading frames of unknown function from S. pombe, Rattus norvegicus, C. elegans, H. sapiens, Guillardia theta, D. melanogaster, and A. thaliana, and to a lesser extent to the folate transporter from C. elegans, Flo3p and Gea2p from S. cerevisiae, and a presumable peroxisomal targeting signal receptor from S. pombe (alignment not presented). Clearly, some of these defined proteins are not N-terminal acetyltransferases, and the similarities may reflect the presence of one or two hypothetical transmembrane domains in Mak10p. Nevertheless, it is unknown whether Mak10p is truly a membrane protein.
Conclusions-We conclude that the NatC N-terminal acetyltransferase is a Mak3p⅐Mak10p⅐Mak31p complex and that each of the subunits is required for acetylating NatC-type substrates in vivo, including the killer viral major coat protein, Gag, and an unidentified component responsible for normal utilization of nonfermentable substrates.