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
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ABSTRACT |
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The yeast Saccharomyces cerevisiae
contains three types of N 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).
-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. We
show that the NatC N
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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-, nat1-
, nat3-
, or
mak3-
deletions: mutationally altered iso-1-cytochromes c (iso-1) (2, 5, 6), mutationally
altered
-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-
or nat1-
mutants (NatA substrates);
proteins with Met-Glu or Met-Asp termini and subclasses of proteins
with Met-Asn termini are not acetylated in nat3-
mutants
(NatB substrates); and subclasses of proteins with Met-Ile,
Met-Leu, Met-Trp, or Met-Phe termini are not acetylated in
mak3-
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-
and mak3-
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- strains on glycerol medium
is because of lack of N-terminal acetylation of the mitochondrial
proteins Kdg1p (
-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.
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MATERIALS AND METHODS |
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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. 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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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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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
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 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.
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RESULTS AND DISCUSSION |
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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 PCR-based 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
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;
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+ 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.
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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 ~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-
, mak10-
, mak31-
, and
nat3-
deletions in which the iso-1 contained ~5-10%
of the unacetylated form.
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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- mutant) but
not in B-13739 (mak3-
mutant). The Met-Glu-Phe
(CYC1-853) iso-1 was not acetylated in B-11863
(nat3-
) mutant but remained acetylated in the B-7723 (nat1-
), B-9072 (mak3-
), and B-13274
(mak31-
) mutants. The Met-Ile-Arg (CYC1-1162)
iso-1 was not acetylated in the B-9022 (mak3-
) mutant but
was acetylated in the B-9024 (nat1-
) and B-11865
(nat3-
) mutants. The Ser-Glu-Phe (CYC1-987)
iso-1 was acetylated in B-12332 (mak10-
) and B-13276
(mak31-
) mutants but not in B-10689
(nat1-
). Also, CYC1-987 iso-1 from B-10672 (mak3-
) and B-12045 (nat3-
) 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-
) 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 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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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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are:
N-terminal, N-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.
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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] |