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

Bogdan Polevoda and Fred ShermanDagger

From the Department of Biochemistry and Biophysics, University of Rochester Medical School, Rochester, New York 14642

Received for publication, December 19, 2000, and in revised form, March 26, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The yeast Saccharomyces cerevisiae contains three types of Nalpha -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 Nalpha -terminal regions. We show that the NatC Nalpha -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The two cotranslational processes, cleavage of N-terminal1 methionine residues and N-terminal acetylation, are the most common modifications, occurring on the vast majority of eukaryotic proteins. Methionine residues at the N termini are cleaved from nascent chains of most proteins, and subsequently N-terminal acetylation occurs on certain of the proteins, either those containing or lacking the methionine residue (reviewed in Ref. 1). As summarized in Table I, Saccharomyces cerevisiae contains three types of N-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-terminal regions (1, 2). In addition, the NatA N-terminal acetyltransferase contains another subunit, Nat1p, which associates with the catalytic subunit, Ard1p, and which is required for activity (3, 4).

                              
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Table I
The three types of N-terminal acetyltransferases

The substrate specificities for each of NatA, NatB, and NatC were deduced by considering the lack of acetylation of the following groups of protein in mutants containing one or another of the ard1-Delta , nat1-Delta , nat3-Delta , or mak3-Delta deletions: mutationally altered iso-1-cytochromes c (iso-1) (2, 5, 6), mutationally altered beta -galactosidases (7), abundant proteins (2, 8, 9), ribosomal proteins (10), and 20 S proteasome subunits (11). Subclasses of proteins with Ser, Ala, Gly or Thr termini are not acetylated in ard1-Delta or nat1-Delta mutants (NatA substrates); proteins with Met-Glu or Met-Asp termini and subclasses of proteins with Met-Asn termini are not acetylated in nat3-Delta mutants (NatB substrates); and subclasses of proteins with Met-Ile, Met-Leu, Met-Trp, or Met-Phe termini are not acetylated in mak3-Delta mutants (NatC substrates). We wish to emphasize that acetylation occurs only on subsets of these proteins, and the presence of the specified termini does not guarantee N-terminal acetylation. In addition, a special subclass of NatA substrates with Ser-Glu, Ser-Asp, Ala-Glu, or Gly-Glu termini, previously designated NatD substrates and now designated NatA' substrates, may not be completely acetylated in nat3-Delta and mak3-Delta mutants (1, 2, 11).

MAK3, encoding the catalytic subunit of NatC, is required for the N-terminal acetylation of the killer viral major coat protein, Gag, with a mature Ac-Met-Leu-Arg-Phe terminus. MAK3 was first identified from mak3-deficient mutants that did not assemble or maintain the L-A dsRNA viral particle (12). Mak3-deficient mutants also have reduced growth on media containing nonfermentable carbon sources as the sole source of energy, such as glycerol or ethanol. Tercero et al. (7) suggest that the diminished growth of mak3-Delta strains on glycerol medium is because of lack of N-terminal acetylation of the mitochondrial proteins Kdg1p (alpha -ketoglutarate dehydrogenase), Fum1p (fumarate dehydratase), and Mrp1p (a mitochondrial ribosomal protein), that all contain Met-Leu-Arg-Phe termini, similar to the L-A Gag protein. NatC substrates are rare, and none were encountered among 55 abundant proteins (2) or among 68 ribosomal proteins (10), but two were uncovered, Pup2p and Pre5p, among 14 20 S proteasome subunits, both containing Met-Phe N termini (11).

There are 29 MAK genes defined on the basis of their requirement for stable propagation of M1 dsRNA (13). Their modes of action are diverse, with the bulk of the MAK genes involved in the 60 S ribosomal subunit biogenesis. Mak3p, Mak10p, and Mak31p co-purify, suggesting that they constitute three subunits of a complex (14). Moreover, protein-protein interactions between Mak3p and Mak10p, as well as between Mak31p and Mak10, were detected in a two-hybrid screen (15). Significantly, of all of the MAK genes, only MAK3, MAK10, and MAK31 were found necessary for L-A virus propagation (16-18). Furthermore, similar to mak3 mutants, mak10 mutants grew slowly on media containing nonfermentable carbon sources (19). Although the biological function of the MAK31 gene was not previously known, Séraphin (20) suggested that Mak31p is an Sm-like protein based on protein sequence similarity. Sm-like proteins are small nuclear ribonucleoproteins (snRNP) found associated with U1, U2, and U5 snRNAs, as well as with U4/U6 double snRNP and the U4/U6/U5 triple snRNP. snRNP are involved in various functions including pre-mRNA splicing, histone mRNA 3'formation, 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 ~12.5 kDa and because mass spectrometry can be used conveniently to determine molecules less than 30 kDa with an accuracy of ~1 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
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Genetic Nomenclature-- Using standard genetic nomenclature, MAK3, for example, designates the normal wild-type allele; mak3-Delta 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. The analysis of N-terminal acetylation was carried out with a series of isogenic strains originally derived from B-7528 (cyc1-31 MATa cyc7-67 ura3-52 lys5-10). The series of strains used for producing altered iso-1 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.

                              
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Table II
Yeast strains

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 µg of the oligonucleotides (Table III) that have minimal mismatches.

                              
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Table III
Oligonucleotides used in the construction and testing of disrupted genes
The position of the first nucleotide is presented, where A of the ATG initiation codon is assigned position 1. The underlined sequences correspond to segments in the plasmid pFA6-kanMX4. ORF, open reading frame.

Iso-1 Content-- The amounts of iso-1 in yeast strains were estimated by low temperature (-196 °C) spectroscopic examinations of intact cells (26) and comparing the intensities of the Calpha 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-Delta ::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-Delta ::URA3 disruption was prepared from the plasmid pAA1132 (also designated JM111) (3). The correct nat1-Delta disruptions were identified with Oligo 5 and 6. The fragment required for producing the mak3-Delta ::URA3 disruption was prepared from the plasmid pJC11C (also designated pAA1131) (31). The correct mak3-Delta disruptions were identified with Oligo 7 and 8. mak10-Delta and mak31-Delta 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-Delta and Oligo 15 and 16 for mak31-Delta , 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 BioRex70 column chromatography, 100-200 mesh and 200-400 mesh (Bio-Rad), respectively, in potassium phosphate buffer, pH 7.0, with a 0-1.0 M potassium chloride linear gradient. After chromatography the protein samples were dialyzed against 0.1 M potassium phosphate and, if necessary, concentrated by Centricon-3 (Amicon-Millipore, Bedford, MA). For MALDI-TOF experiments the protein samples were either dialyzed against H2O or the phosphate buffer was replaced by H2O using Centricon-3.

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 µl 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
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Phenotypes of mak Deletion Mutants-- It has been previously reported that mak3-Delta (31) and mak10-Delta (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 PCR-based disrupters (see Table III for corresponding oligonucleotides): normal (B-8462), mak3-Delta (B-9072), mak10-Delta (B-12333), and mak31-Delta (B-13275). As shown in Fig. 1, mak3-Delta , mak10-Delta , and mak31-Delta mutants grew poorly on YPG medium at 37 °C, although growth on YPD medium at 37 °C was normal, thus displaying the Nfs- 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- and Raf+/-, the diminished growth on YPGal and YPRaf media is a secondary consequence of the Nfs- phenotype; rho - 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-Delta (B-9022), mak10-Delta (B-12333), and mak31-Delta (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-Delta , mak10-Delta , and mak31-Delta deletion mutants did not differ significantly from the corresponding normal MAK+ strains, as estimated by low temperature spectroscopic examination of intact cells (Table IV). It is noteworthy that the mak3-Delta (B-9022), mak10-Delta (B-12333), and mak31-Delta (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.


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Fig. 1.   Serial 1/10 dilutions of the following isogenic series of strains grown at 37 °C on YPD and YPG media for 3 days: Normal, B-8462; mak3-Delta , B-9072; mak10-Delta , B-12333, and mak31-Delta , B-13275.

                              
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Table IV
N-terminal acetylation of altered iso-1 from normal and mutant strains
 Percent acetylation values shown in parentheses are from Polevoda et al. (2). The nucleotide mismatches with the cyc1-31 sequence are indicated by underlines.

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-Delta , mak10-Delta , mak31-Delta , nat1-Delta , and nat3-Delta 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 ~42 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 NatA' substrate from CYC1-987 strains having one or another of the mak3-Delta , mak10-Delta , mak31-Delta , and nat3-Delta deletions in which the iso-1 contained ~5-10% of the unacetylated form.

                              
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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).


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Fig. 2.   Examples of MALDI-TOF mass spectra of iso-1 from the NatC, NatA', and NatA series. The NatC series, CYC1-1162 (Ac-Met-Ile-Arg), is represented on the left side of the figure by B-8462 (Normal), B-9022 (mak3-Delta ), B-12333 (mak10-Delta ), and B-13275 (mak31-Delta ), while the NatA' series CYC1-987 (Ac-Ser-Glu-Phe) is represented on the right side of the figure by B-10645 (Normal), B-10672 (mak3-Delta ), B-12332 (mak10-Delta ), B-13276 (mak31-Delta ), and B-12045 (nat3-Delta ). Also shown are examples from a NatA series, CYC1-1383 (Ac-Ser-Glu-Ile) with B-12479 (Normal) and B-13393 (nat1-Delta ) (bottom left). The diminished masses of iso-1 from B-9022, B-12333, B-13275, and B13393 indicate the lack of acetylation. Minor peaks at the appropriate positions indicate low levels unacetylated forms in the B-10672 (mak3-Delta ), B-12332 (mak10-Delta ), B-13276 (mak31-Delta ), and B-12045 (nat3-Delta ) strains. The masses were determined with the Voyager-DE STR linear time-of-flight mass spectrometer, except for those of iso-1 from the B-8462, B-13275, and B-10645 strains, which were determined with the Bruker ProFLEX III MALDI-TOF mass spectrometer.

Overall, the analysis of iso-1 acetylation from the complete sets of isogenic strains gave rise to expected results (Tables IV and V). The decrease of the molecular mass of Ser-Glu-Ile (CYC1-1383) iso-1 by ~42 Da, indicative of the lack of acetylation, was observed in B-13393 (nat1-Delta mutant) but not in B-13739 (mak3-Delta mutant). The Met-Glu-Phe (CYC1-853) iso-1 was not acetylated in B-11863 (nat3-Delta ) mutant but remained acetylated in the B-7723 (nat1-Delta ), B-9072 (mak3-Delta ), and B-13274 (mak31-Delta ) mutants. The Met-Ile-Arg (CYC1-1162) iso-1 was not acetylated in the B-9022 (mak3-Delta ) mutant but was acetylated in the B-9024 (nat1-Delta ) and B-11865 (nat3-Delta ) mutants. The Ser-Glu-Phe (CYC1-987) iso-1 was acetylated in B-12332 (mak10-Delta ) and B-13276 (mak31-Delta ) mutants but not in B-10689 (nat1-Delta ). Also, CYC1-987 iso-1 from B-10672 (mak3-Delta ) and B-12045 (nat3-Delta ) were partially acetylated (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-Delta ) and B-13275 (mak31-Delta ) 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 N-terminal 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) comparisons revealed that species containing orthologs of the yeast Mak3p include Schizosaccharomyces pombe, C. elegans, D. melanogaster, Mus musculus, Arabidopsis thaliana, and H. sapiens. Also proteins with high similarity to Mak3p are present in Archaeoglobus fulgidus, Aeropyrum pernix, Campylobacter coli, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Pyrococcus horikoshii, Sulfolobus solfataricus, Ureaplasma urealyticum, and Escherichia coli. 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).


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Fig. 3.   Amino acid sequence alignments of Mak3p (A) and Mak31p (B) orthologs. Protein sequences were aligned using Multalin, version 5.4.1 (35). Highly conserved residues are highlighted in black, and moderately conserved residues are highlighted in gray. Consensus symbols are: !, any one of IV; $, any one of LM; %, any one of FY; #, any one of NDQEBZ. The protein accession numbers (GenBankTM and PIR) are as follows: A. thaliana, AtT01245, AtAAG51452 (plant); C. elegans, CeAAB65989, CeAAB28212 (invertebrate); D. melanogaster, DmCAA17683, DmAAF47567 (invertebrate); H. sapiens, HsBAB14397, HsAAD56232 (mammal); M. musculus, MmBAB27439 (mammal); M. sativa, MmCAA44975 (plant); S. cerevisiae, Mak3p, Mak31p (fungus); S. pombe, SpT39482, SpT41178 (fungus); T. acidophilum, TaCAC11206 (archaea).

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 alpha -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 Mak3p·Mak10p·Mak31p 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.

    ACKNOWLEDGEMENTS

We are thankful to Dr. Gurrinder Bedi, Microchemical Protein/Peptide Facility University of Rochester, and Dr. Tracy D. McCarley, Department of Chemistry Louisiana State University (Baton Rouge, LA), for help in mass spectrometric analysis.

    FOOTNOTES

* This work was supported by National Institutes of Health Research Grant GM12702 and in part by Grant RR14682 from the National Center for Research Resources of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, P. O. Box 712, University of Rochester Medical School, Rochester, NY 14642. Tel.: 716-275-6647; Fax: 716-275-6007; E-mail: Fred  Sherman@urmc.rochester.edu.

Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M011440200

    ABBREVIATIONS

The abbreviations used are: N-terminal, Nalpha -terminal; NAT, N-terminal acetyltransferase; iso-1, iso-1-cytochrome c or iso-1-cytochrome c proteins; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; snRNP, small nuclear ribonucleoproteins; PCR, polymerase chain reaction; Oligo, oligonucleotide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1. Polevoda, B., and Sherman, F. (2000) J. Biol. Chem. 275, 36479-36482[Free Full Text]
2. Polevoda, B., Norbeck, J., Takakura, H., Blomberg, A., and Sherman, F. (1999) EMBO J. 18, 6155-6168[Abstract/Free Full Text]
3. Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S., Gribskov, M., Colavito-Shepanski, M., Grunstein, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075[Abstract]
4. Park, E.-C., and Szostak, J. W. (1992) EMBO J. 11, 2087-2093[Abstract]
5. Tsunasawa, S., Stewart, J. W., and Sherman, F. (1985) J. Biol. Chem. 260, 5382-5391[Abstract]
6. Moerschell, R. P., Hosokawa, Y., Tsunasawa, S., and Sherman, F. (1990) J. Biol. Chem. 265, 19638-19643[Abstract/Free Full Text]
7. Tercero, J. C., Dinman, J. D., and Wickner, R. B. (1993) J. Bacteriol. 175, 3192-3194[Abstract]
8. Boucherie, H., Sagliocco, F., Joubert, R., Maillet, I., Labarre, J., and Perrot, M. (1996) 17, 1683-1699
9. Garrels, J. I., McLaughlin, C. S., Warner, J. R., Futcher, B., Latter, G. I., Kobayashi, R., Schwender, B., Volpe, T., Anderson, D. S., Mesquita-Fuentes, R., and Payne, W. E. (1997) Electrophoresis 18, 1347-1360[Medline] [Order article via Infotrieve]
10. Arnold, R., Polevoda, B., Reilly, J. P., and Sherman, F. (1999) J. Biol. Chem. 274, 37035-37040[Abstract/Free Full Text]
11. Kimura, Y., Takaoka, M., Tanaka, S., Sassa, H., Tanaka, K., Polevoda, B., Sherman, F., and Hirano, H. (2000) J. Biol. Chem. 275, 4635-4639[Abstract/Free Full Text]
12. Tercero, J. C., and Wickner, R. B. (1992) J. Biol. Chem. 267, 20277-20281[Abstract/Free Full Text]
13. Wickner, R. B. (1996) Microbiol. Rev. 60, 250-265[Free Full Text]
14. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Séraphin, B. (1999) Nat. Biotechnol. 17, 1030-1032[CrossRef][Medline] [Order article via Infotrieve]
15. Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. M. (2000) Nature 403, 623-627[CrossRef][Medline] [Order article via Infotrieve]
16. Sommer, S. S., and Wickner, R. B. (1982) Cell 31, 429-441[Medline] [Order article via Infotrieve]
17. Wickner, R. B., and Toh-e, A. (1982) Genetics 100, 159-174[Abstract/Free Full Text]
18. Fujimura, T., and Wickner, R. B. (1987) Mol. Cell. Biol. 7, 420-426[Medline] [Order article via Infotrieve]
19. Lee, Y-J., and Wickner, R. B. (1992) Genetics 132, 87-96[Abstract/Free Full Text]
20. Séraphin, B. (1995) EMBO J. 14, 2089-2098[Abstract]
21. Mattaj, I. W., Tollervey, D., and Séraphin, B. (1993) FASEB. J. 7, 47-53[Abstract/Free Full Text]
22. Baim, S. B., Pietras, D. F., Eustice, D. C., and Sherman, F. (1985) Mol. Cell. Biol. 5, 1839-1846[Medline] [Order article via Infotrieve]
23. Wang, X., Dumont, M. E., and Sherman, F. (1996) J. Biol. Chem. 271, 6594-6604[Abstract/Free Full Text]
24. Sherman, F. (1991) Methods Enzymol. 194, 3-21[Medline] [Order article via Infotrieve]
25. Yamamoto, T., Moerschell, R. P., Wakem, L. P., Ferguson, D., and Sherman, F. (1992) Yeast 8, 935-948[Medline] [Order article via Infotrieve]
26. Sherman, F., and Slonimski, P. P. (1964) Biochim. Biophys. Acta 90, 1-15
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
28. Baudin, A., Ozier, K. O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve]
29. Wach, A., Brachat, A., Pohlmann, R., and Philippsen, P. (1994) Yeast 10, 1793-1808[Medline] [Order article via Infotrieve]
30. Sherman, F, Stewart, J. W., Parker, J. H., Inhaber, E., Shipman, N. A., Putterman, G. J., Gardisky, R. L., and Margoliash, E. (1968) J. Biol. Chem. 243, 5446-5456[Abstract/Free Full Text]
31. Tercero, J. C., Riles, L. E., and Wickner, R. B. (1992) J. Biol. Chem. 267, 20270-20276[Abstract/Free Full Text]
32. Deleted in proof
33. Altshul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
34. Isono, K., and Isono, S. (1980) Mol. Gen. Genet. 177, 645-651[Medline] [Order article via Infotrieve]
35. Corpet, F. (1988) Nucleic Acids Res. 16, 10881-10890[Abstract]


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