The Yeast N{alpha}-Acetyltransferase NatA Is Quantitatively Anchored to the Ribosome and Interacts with Nascent Polypeptides

The majority of cytosolic proteins in eukaryotes contain a covalentlylinked acetyl moiety at their very N terminus. The mechanismby which the acetyl moiety is efficiently transferred to a largevariety of nascent polypeptides is currently only poorly understood.Yeast N-acetyltransferase NatA, consisting of the known subunitsNat1p and the catalytically active Ard1p, recognizes a widerange of sequences and is thought to act cotranslationally.We found that NatA was quantitatively bound to ribosomes viaNat1p and contained a previously unrecognized third subunit,the N-acetyltransferase homologue Nat5p. Nat1p not only anchoredArd1p and Nat5p to the ribosome but also was in close proximityto nascent polypeptides, independent of whether they were substratesfor N-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associatedcomplex) and the Hsp70 homologue Ssb1/2p interact with a varietyof nascent polypeptides on the yeast ribosome. A direct comparisonrevealed that Nat1p required longer nascent polypeptides forinteraction than NAC and Ssb1/2p. ${Delta}$ nat1 or ${Delta}$ ard1 deletion strainswere temperature sensitive and showed derepression of silentmating type loci while ${Delta}$ nat5 did not display any obvious phenotype.Temperature sensitivity and derepression of silent mating typeloci caused by ${Delta}$ nat1 or ${Delta}$ ard1 were partially suppressed by overexpressionof SSB1. The combination of data suggests that Nat1p presentsthe N termini of nascent polypeptides for acetylation and mightserve additional roles during protein synthesis.

INTRODUCTION

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Introduction

N-Terminal acetylation of proteins is a common phenomenon ineukaryotes. About 90% of mammalian proteins and 50% of the solubleproteins in lower eukaryotes are N-terminally acetylated (7).It is generally accepted that N-terminal acetylation of mammalianproteins occurs cotranslationally when newly synthesized polypeptideshave just emerged from the ribosomal exit tunnel (16, 32, 34,57). Proteins with serine and alanine N termini are the mostfrequently acetylated substrates, and these residues along withmethionine, glycine, and threonine account for more than 95%of the N-terminally acetylated residues (40). Very little isknown about the composition, function, and importance of mammalianN-acetyltransferases. NatH, a human homologue, was recentlyfound to be overexpressed in papillary thyroid carcinomas (9).

Three N-terminal acetyltransferase complexes termed NatA, NatB,and NatC have been identified in Saccharomyces cerevisiae (28,33, 39, 50). They differ in substrate specificity and subunitcomposition, but all contain a catalytic subunit homologousto the GNAT family of acetyltransferases (30, 41). NatA is themajor N-terminal acetyltransferase in the yeast cytosol, responsiblefor the acetylation of serine, alanine, threonine, and glycine(4, 39, 48). NatA has been shown to contain two subunits, Nat1p,a protein of 98 kDa, and the 27-kDa GNAT homologue Ard1p (28,33). Both subunits are required for activity, and deletion strainsof either ${Delta}$ nat1 or ${Delta}$ ard1 fail to N-terminally acetylate the sameset of substrate proteins (28). In agreement with these observations, ${Delta}$ nat1 and ${Delta}$ ard1 strains display the same set of phenotypes, suchas reduced sporulation efficiency, failure to enter G₀ underspecific conditions, defect in silencing of the silent mating-typeloci, and decreased survival after heat shock (28, 39, 54, 55).The pleiotropic phenotypes are thought to reflect functionaldefects of various target proteins lacking proper acetylationat their N termini. However, there are surprisingly few examplesdemonstrating the biological importance of N-terminal proteinacetylation (40). For NatA, no clear-cut example has been described.It was discussed that the mating defect of a ${Delta}$ nat1 or ${Delta}$ ard1 strainmight be due to the lack of N-acetylation of the silent informationregulator protein Sir3p (47). However, experimental evidenceis missing so far. Recent evidence suggests that the multiplephenotypes observed in the absence of NatB might be caused bythe lack of acetylation of actin and tropomyosin (38). A well-documentedexample has been reported for Mak3p, the catalytic subunit ofNatC. N-Acetylation by Mak3p is essential for the coat proteinassembly of the L-A double-stranded RNA virus in Saccharomycescerevisiae (49, 50).

Newly synthesized polypeptides exit the large ribosomal subunitthrough a tunnel. In yeast, several proteins are located closeto the tunnel exit. Two of them, NAC (nascent polypeptide-associatedcomplex) and the Hsp70 homologue Ssb1/2p (Ssb1p and Ssb2p, two99% identical proteins with identical function and similar expressionlevels), have been shown to be in close proximity to a varietyof nascent polypeptides (14, 36, 43). Recently, it was foundthat the ribosome-associated complex (consisting of the Hsp70homologue Ssz1p and the Hsp40 homologue zuotin) is requiredfor efficient cross-linking of the nascent polypeptide to Ssb1/2p(14). During the course of these experiments an additional high-molecular-masscross-link product was observed. Here, we identify this high-molecular-masscross-link to the nascent polypeptide as Nat1p, the noncatalyticsubunit of NatA (41).

Purification and biochemical analysis of the cross-link-inducingfactor revealed that the NatA complex contains a third subunit.This previously uncharacterized subunit, termed Nat5, like Ard1p,belongs to the GNAT family of acetyltransferases. We find thatNat1p interacts not only with the nascent polypeptide chainbut also with the ribosome, thereby anchoring the NatA complexquantitatively to ribosomes.

MATERIALS AND METHODS

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Materials and Methods

Yeast strains and plasmids. Standard yeast genetic techniques were used (46). MH272-3f a/ ${alpha}$ (ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 ade2/ade2 HMLa/HMLa)is the parental wild-type strain of the derivatives used inthis study (13, 20). The generation of the deletion strainslacking ARD1 was done either by replacing the entire gene withthe kanMX module (52) or by replacing the 306-bp AflII/XbaIfragment in the middle of ARD1 with URA3. NAT1 was deleted byreplacing the 2,384-bp XhoI/NruI fragment with LEU2. NAT5 wasdeleted by replacing the 225-bp Bpu10I/BbsI fragment with ADE2.The resulting strains are ${Delta}$ ard1 (IDA7, MH272-3f ${alpha}$ ard1::kanR; IDA7B,MH272-3f ${alpha}$ ard1::URA3), ${Delta}$ nat5 (IDA8, MH272-3f ${alpha}$ nat5::ADE2), ${Delta}$ nat1(IDA9, MH272-3f ${alpha}$ nat1::LEU2), and ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 (IDA789, MH272-3fanat1::LEU2 ard1::kanR nat5::ADE2). ${Delta}$ nat1 ${Delta}$ ssb1 ${Delta}$ ssb2 (IDA56C9, MH272-3fnat1::LEU2 ssb1::ADE2 ssb2::HIS3) was generated by mating IDA56C ${alpha}$ (ssb1::ADE2 ssb2::HIS3) with IDA9a (MH272-3fa nat1::LEU2) followedby sporulation and tetrad dissection. PCR with appropriate primercombinations and Western blotting with anti-ard1, anti-nat5,and anti-nat1 sera were used to confirm deletion strains. Totest ARD1, NAT5, and NAT1 as multicopy suppressors, two methodswere applied. Either the genes were cloned into multicopy vectorsYEplac195 (2µm, URA3), YEplac112 (2µm, TRP1) (15),or pRS423 (2µm, HIS3) (6) or the genes were cloned downstreamof the GAL1-10 promoter of vectors of the pESC series (Stratagene).As the anti-nat5 serum available did not produce strong signalsin immunoblots, NAT5 under control of the GAL promoter was fusedto an N-terminal FLAG tag for better detection. Resulting multicopyplasmids are 2µm-ARD1, 2µm-NAT5, and 2µm-NAT1.Galactose-inducible constructs are designated pESC-ARD1, pESC-NAT5,and pESC-NAT1. The plasmid overexpressing SSB1 is based on YEplac195(2µm, URA3) and was previously described (14). Yeast straints-187 harbors the S518F point mutant of prt1 encoding translationinitiation factor eIF3. ts-187 is defective in initiation oftranslation at 37°C (19, 53). To generate monosomes in vivo,ts-187 was incubated at 37°C for 20 min as described previously(19). The mating efficiency was tested and quantified as describedpreviously (8). Strains derived from W303 (MATa ade2-101 his3-11,15trp1-1 leu2-3,112 ura3-1), JRM5 (W303 ${Delta}$ ard1), and AMR1 (W303 ${Delta}$ nat1) were obtained from Rolf Sternglanz and are described elsewhere(3, 28). NAT5 was deleted by replacing the 225-bp Bpu10I/BbsIfragment with ADE2, resulting in W303 nat5::ADE2.

Purification of NatA. Cytosol was prepared as described previously (10) from a 12-literculture of YRG16 (MATa ura3 leu2 his3 trp1 ade2 HMLa egd2::ADE2egd1::URA3) (13) and was centrifuged for 1.5 h at 200,000 xg at 4°C. The ribosomal pellet was resuspended in 20 mMHEPES-KOH (pH 7.4), 700 mM potassium acetate, 5 mM magnesiumacetate, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and wascentrifuged again for 1.5 h at 200,000 x g at 4°C. The ribosome-freesupernatant, termed ribosomal salt wash, was diluted with 6volumes of 40 mM HEPES-KOH (pH 7.4) and loaded onto a ResourceQ(6-ml) anion-exchange column (Amersham-Pharmacia). Bound proteinswere eluted with a 100 to 800 mM 25-ml linear potassium acetategradient in 40 mM HEPES-KOH (pH 7.4). NatA eluted at a potassiumacetate concentration of 450 to 550 mM. NatA-containing fractionswere pooled and diluted with 1.5 volumes of 40 mM HEPES-KOH(pH 7.4) and applied to a MonoQ (5/5) anion-exchange column(Amersham-Pharmacia). Bound proteins were eluted with a 200to 600 mM 22-ml linear potassium acetate gradient in 40 mM HEPES-KOH(pH 7.4). NatA eluted with a 250 to 350 mM concentration ofpotassium acetate. NatA-containing fractions were pooled andloaded onto a Hi-Load (16/60) Superdex 200 prep grade gel filtrationcolumn (Amersham-Pharmacia) preequilibrated with 40 mM HEPES-KOH(pH 7.4) and 150 mM potassium acetate. NatA eluted at a volumeof 66 ml, and these fractions were stored at -80°C and usedfor further experiments.

Identification of the NatA subunits. Purified NatA complex was transferred to a polyvinylidene difluoridemembrane. Ard1p and Nat5p were identified by N-terminal Edmandegradation. Nat1p was N-terminally blocked and was identifiedby mass spectrometry of a tryptic digest. To estimate the relativeamounts of Ard1p, Nat5p, and Nat1p, the purified NatA complexwas separated by reverse-phase high-performance liquid chromatography(HPLC) (Nucleosil 500-5, C₃PPN; Macherey-Nagel) by applyinga 30 to 60% 6-ml gradient of 0.09% trifluoroacetic acid-CH₃CNin 0.08% trifluoroacetic acid-H₂O. The molar ratios of the differentsubunits in the preparation were calculated by using the peakarea of the HPLC profile and the following calculated molarextinction coefficients at 280 nm: ${varepsilon}$ (Nat1p), 105,680 M^-1 cm^-1; ${varepsilon}$ (Ard1p), 21,050 M^-1 cm^-1; and ${varepsilon}$ (Nat5p), 13,370 M^-1 cm^-1.

Generation of antibodies. Polyclonal antibodies specifically recognizing Ard1p (anti-ard1),Nat5p (anti-nat5), or Nat1p (anti-nat1) were generated againstHis-tagged versions of the proteins expressed in Escherichiacoli (EUROGENTEC, Seraing, Belgium).

In vitro transcription, translation, preparation of RNCs, and cross-link assay. Yeast translation extract was prepared as described previously(11) from strain JK93d ${alpha}$ (20) or YRG16a (13). Translation reactionswere performed in the presence of [³⁵S]methionine (Amersham-Pharmacia).Ribosome-bound nascent chains for cross-link experiments wereproduced as described previously (10). Different lengths ofDNA fragments for transcription reactions were generated byPCR with plasmids pSP65-mdh1 (carrying yeast MDH1), pSP65-rpl4(carrying yeast RPL4A), or pDJ100 (carrying the sequence forprepro ${alpha}$ -factor as a template) (14). In the cases of RPL4A andprepro ${alpha}$ -factor, the reverse primer changed the penultimate aminoacid to methionine for better detection. Point mutants of Rpl4A[Rpl4A-S(2)K] and of prepro ${alpha}$ -factor [prepro ${alpha}$ -R(2)K], in whichthe respective amino acids were changed to lysine, were usedwhen indicated. Translation reactions were primed with truncatedmRNA encoding proteins of different lengths, leading to thegeneration of the following ribosome-nascent chain complexes(RNCs): the N-terminal 50, 70, or 87 amino acids of prepro ${alpha}$ -factoror prepro ${alpha}$ -factor R(2)K; the N-terminal 50, 70, or 86 aminoacids of yeast ribosomal protein Rpl4A or Rpl4A-S(2)K; and theN-terminal 40, 45, 50, 55, 60, 80, 85, 104, 150, or 200 aminoacids of mitochondrial malate dehydrogenase (Mdh1p). For thedetailed determination of the minimal length required for cross-linkformation to the different ribosome-bound factors, the precursorof Mdh1p was chosen as a nascent polypeptide, since this proteincontains 13 lysines evenly distributed within the first 200amino acids (see Fig. 4D). In some experiments, prepro ${alpha}$ -R(2)Kwas used to increase cross-link efficiency.

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FIG. 4. NatA contacts a variety of ribosome-bound nascent chains. (A) Cross-link reactions were performed with low-salt-treated RNCs carrying the prepro ${alpha}$ -factor, Rpl4A, or Rpl4A-S(2)K as a nascent chain. Aliquots of the cross-link reactions were subjected to immunoprecipitation under denaturing conditions. Anti-ssb1 ( ${alpha}$ -ssb1), anti-nat1 ( ${alpha}$ -nat1), or preimmune serum (pre) was used for the immunoprecipitations. The material bound to the respective antibody was run on 10% Tris-Tricine gels and subsequently analyzed by autoradiography. (B) RNCs carrying Rpl4A-S(2)K were generated and subsequently separated from soluble proteins by centrifugation through a sucrose cushion under low-salt conditions (LS-Sup and LS-ribos), under high-salt conditions (HS-Sup and HS-ribos), and after treatment with puromycin (HP-Sup and HP-ribos) as described in Materials and Methods. Each sample was subjected to a cross-link reaction and subsequently to immunoprecipitation under denaturating conditions with anti-nat1. The material bound to anti-nat1 (upper panel) and a total of the cross-linking reaction (lower panel) was run on a 10% Tris-Tricine gel and subsequently analyzed by autoradiography. Note, as shown in lane 1, a fraction of Rpl4A-S(2)K was not associated with the ribosome under low-salt conditions. This fraction does not contain NatA, which is retained in the LS-ribos fraction (compare Fig. 2B). (C) Low-salt RNCs carrying different length yeast malate dehydrogenase (mdh1), prepro ${alpha}$ -R(2)K, or Rpl4A-S(2)K as indicated were subjected to cross-linking reactions. The numbers indicate the length of the nascent chains in amino acids (aa). Each sample was split into three aliquots and subjected to immunoprecipitation reactions under denaturating conditions with anti-nat1, anti-ssb1, or anti-beta-nac antibodies. The material bound to the respective antibody was run on 10% Tris-Tricine gels and subsequently analyzed by autoradiography. Nat1p-CL, cross-link of the nascent chain to Nat1p; Ssb1/2p-CL, cross-link of the nascent chain to Ssb1/2p; beta-NAC-CL, cross-link of the nascent chain to beta-NAC. (D) Protein sequences of the nascent chains used for cross-linking experiments.

The initial methionine of Rpl4A is cleaved cotranslationally,and the processed protein contains a single methionine at position84. Prepro ${alpha}$ -factor contains a single methionine at position1, which is not cleaved. Approximately 40 amino acids of theribosome-bound nascent chains are covered by the ribosome. Thehomobifunctional cross-linker bis-(sulfosuccinimidyl)-suberate(BS³) (spacer length, 1.14 nm; Pierce) was used for cross-linkingreactions. BS³ covalently cross-links primary amines, like the ${varepsilon}$ -amino group of lysines or the N-amino group of a nascent polypeptide.For cross-linking reactions, RNCs were isolated as describedunder high (presence of 700 mM potassium acetate)- or low (presenceof 120 mM potassium acetate)-salt conditions as described previously(14). Cross-linking reactions (typically 40-µl final volume)were started by the addition of BS³ to a final concentrationof 400 µM and were incubated for 30 min on ice. The reactionwas quenched for 10 min on ice by the addition of glycyl-glycineto a final concentration of 5 mM. Reaction mixtures were analyzedon 10% Tris-Tricine gels (45) followed by autoradiography. Quantificationof ¹²⁵I and ³⁵S on immunoblots or autoradiographies, respectively,was by Aida ImageAnalyzer (Raytest; IsotopenmessgeräteGmbH).

Preparation of ribosomes. In a typical experiment, 60 µl of yeast cytosol was diluted1:2 with either low-salt dilution buffer (20 mM HEPES-KOH [pH7.4], 120 mM potassium acetate, 2 mM dithiothreitol [DTT], 5mM magnesium acetate, 1 mM PMSF) or high-salt dilution buffer(20 mM HEPES-KOH [pH 7.4], 1.4 M potassium acetate, 2 mM DTT,5 mM magnesium acetate, 1 mM PMSF). After removal of an aliquot(cytosol), 40 µl of the samples was layered on top ofa 100 µl of low-salt sucrose cushion (20 mM HEPES-KOH[pH 7.4], 25% sucrose, 120 mM potassium acetate, 2 mM DTT, 5mM magnesium acetate, 1 mM PMSF) or a 100 µl of high-saltsucrose cushion (20 mM HEPES-KOH [pH 7.4], 25% sucrose, 700mM potassium acetate, 2 mM DTT, 5 mM magnesium acetate, 1 mMPMSF). Accordingly, potassium acetate concentrations of between100 and 700 mM were applied to determine the salt sensitivityof NatA interaction with the ribosome. After centrifugationat 200,000 x g in an RC M120 GX ultracentrifuge (Sorvall) for3 h at 4°C, samples were separated into supernatant andpellet and corresponding amounts were analyzed on Tris-Tricinegels followed by immunoblotting. To generate nascent chain-freeribosomes, a mock translation reaction was performed in thepresence of 1 mM puromycin, without the addition of mRNA. Afteran incubation of 30 min at room temperature, potassium acetatewas added to a final concentration of 800 mM and ribosomes (ribosomalsubunits) were separated from cytosolic proteins and releasedpolypeptides by centrifugation on a high-salt sucrose cushionas described above. The nascent chain-free ribosomal pelletwas resuspended in resuspension buffer (20 mM HEPES-KOH [pH7.4], 100 mM potassium acetate, 2 mM DTT, 5 mM magnesium acetate,0.5 mM PMSF, 2 U of RNase inhibitor/ml) and used for furtherexperiments. In a control experiment, the addition of puromycinafter the translation of Rpl4A-S(2)K released all nascent chainsquantitatively from the ribosomal pellet (see Fig. 4B; datanot shown).

Immunoprecipitation reactions. Immunoprecipitation reactions were performed with protein A-Sepharosebeads (CL-4B; Amersham-Pharmacia) coated with immunoglobulindirected against proteins as indicated in the figure legends.To identify the cross-link partners of the nascent polypeptides,immunoprecipitations were performed under denaturing conditionsas described previously (14). Immunoprecipitations were performedunder native conditions to characterize the subunit compositionof the NatA complex as described previously (13, 44). Startingmaterial for native immunoprecipitations was the ribosomal saltwash (compare to "Purification of NatA" above).

Miscellaneous. Protein concentrations were determined by the Bradford assay(Bio-Rad), with bovine serum albumin as a standard. [¹²⁵I]-labeledprotein A was used to develop the immunoblots (17). Zymolyasewas from Seikagaku, Falmouth, Mass.; all other reagents werefrom Sigma or Roche Molecular Biochemicals. Sequence alignmentswere performed with T-COFFEE, version 1.37 (31).

RESULTS

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Results

Purification of a protein complex that forms a novel, high-molecular-mass cross-link to nascent polypeptides in yeast. Cross-linking to ribosome-bound nascent chains, as e.g., tothe N-terminal 87 amino acids of prepro ${alpha}$ -factor, generated twohigh-molecular-mass cross-link products with an efficiency of15 to 20% (Fig. 1A) (4, 36). The cross-link product of about80 kDa was to stably ribosome-bound Ssb1/2p (14), whereas theprotein forming the 120-kDa cross-link has not been characterizedpreviously. High-salt treatment of RNCs eliminated both the80- and 120-kDa cross-links (data not shown) (14). The ribosome-associatedmaterial that was removed by high-salt treatment (ribosomalsalt wash) served as the starting material for the purificationof the protein forming the 120-kDa cross-link to the nascentpolypeptide. Fractionated ribosomal salt wash was subjectedto cross-linking experiments in the presence of high-salt RNCsand analyzed for the appearance of the 120-kDa cross-link (seeMaterials and Methods). This assay led to the identificationof three copurifying proteins: Nat1p (98 kDa), Ard1p (27 kDa),and Nat5p (20 kDa). Approximation of the molar concentrationsin the purified complex suggested that the three polypeptideswere present in a 1:1:1 ratio (Fig. 1B) (for quantificationrefer to Materials and Methods). The finding that Nat1p, Ard1p,and Nat5p were present in equimolar amounts, combined with publisheddata on the size of the NatA complex (24, 33), suggests thatthe complex is trimeric.

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FIG. 1. Nat1p, a subunit of yeast NatA interacts with RNCs. (A) RNCs carrying 87 N-terminal amino acids of ³⁵S-labeled prepro ${alpha}$ -factor (prepro ${alpha}$ 87) were incubated in the absence (-) or presence of the homobifunctional cross-linker BS³ as indicated. Aliquots were run on 10% Tris-Tricine gels and were subsequently analyzed by autoradiography. The amounts of prepro ${alpha}$ 87 and the cross-link products (CL) were quantified by densitometry to approximate the cross-linking efficiency. The upper panel (120 kDa CL, 80 kDa CL) shows a four-times-longer exposure than the lower panel (prepro ${alpha}$ 87); the signal was linear in relation to the exposure time. The amount of prepro ${alpha}$ 87 was set to 100%. (B) Subunits of the cross-link-inducing complex were separated by reverse-phase HPLC and analyzed on a Coomassie-stained gel. The amount of protein contained in the different fractions was determined by using the peak area of the HPLC profile and the calculated molar extinction coefficients (see Materials and Methods). (C) Cross-link reactions were performed with low-salt treated RNCs carrying ³⁵S-labeled prepro ${alpha}$ 87 as a nascent chain. Aliquots of the cross-link reactions were subjected to immunoprecipitation under denaturing conditions with preimmune serum (PI), anti-nat1 ( ${alpha}$ -nat1), anti-ard1 ( ${alpha}$ -ard1), and anti-nat5 ( ${alpha}$ -nat5) antibodies. Shown is the material bound to the respective antibody (IP-Pel). Immunoprecipitation of the 120-kDa cross-link with anti-nat1 was quantitative (data not shown).

Immunoprecipitation reactions were performed to confirm theidentity of the 120-kDa cross-link to Nat1p. The 120-kDa cross-linkproduct was quantitatively immunoprecipitated with anti-nat1under denaturing conditions. No cross-link products betweenthe nascent polypeptide and Ard1p or Nat5p were observed underthe conditions tested (Fig. 1C and data not shown).

Nat1p and Ard1p are known to form a complex termed NatA (33).Nat5p, like Ard1p, belongs to the GNAT family of N-acetyltransferases(30, 41). The fact that Nat1p, Ard1p, and Nat5p copurified ona variety of chromatography columns combined with the sequencehomology between Ard1p and Nat5p suggested that NatA might containan additional subunit. From a ribosomal salt wash, anti-nat1antibodies immunoprecipitated Nat1p and coimmunoprecipitatedArd1p and Nat5p, with efficiencies of 90 to 100% under nativeconditions (Fig. 2A). The immunoprecipitation efficiency ofArd1p with anti-ard1 was approximately 70%, and Nat1p and Nat5pwere coimmunoprecipitated with similar efficiency (Fig. 2A).As a control, zuotin, a protein also contained in the ribosomalsalt wash, was coimmunoprecipitated neither with anti-nat1 norwith anti-ard1 (Fig. 2A). The combined data suggest that thebulk of Nat1p, Ard1p, and Nat5p forms a complex.

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FIG. 2. Nat5p, a member of the GNAT family of acetyltransferases, is a subunit of NatA. (A) Immunoprecipitation reactions with ribosomal salt-wash as the starting material were performed under native conditions. Aliquots of the ribosomal salt-wash (T), of the material bound to the respective antibody (B), and of the unbound material (U) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunodecoration with antibodies specifically recognizing Nat1p, Ard1p, Nat5p, and zuotin. Protein A-Sepharose beads were coated with preimmune serum (pre), antibody recognizing Nat1p ( ${alpha}$ -nat1), or antibody recognizing Ard1p ( ${alpha}$ -ard1). (B) Separation of yeast cytosol (C) into a ribosomal pellet (P) and a postribosomal supernatant (S) at increasing concentrations of potassium acetate (KAc) as indicated. Aliquots were separated on 10% Tris-Tricine gels, transferred to nitrocellulose, and decorated with antibodies directed against Nat1p, Ard1p, Nat5p, and Rpl16a (ribosomal marker). The band labeled with an asterisk is a polypeptide cross-reacting with anti-nat1 on Western blots.

NatA is quantitatively bound to the ribosome via Nat1p. We had purified NatA from the ribosomal salt wash, suggestingthat a fraction of NatA was bound to ribosomes. To determinethe distribution of NatA, the yeast cytosol was separated intoa ribosomal pellet and a postribosomal supernatant at increasingconcentrations of salt. At concentrations of potassium acetateup to 200 mM, NatA was quantitatively recovered in the ribosomalpellet. At higher potassium acetate concentrations, all threesubunits were simultaneously released from the ribosomal fraction(Fig. 2B). The result suggests that the bulk of NatA is associatedwith ribosomes at physiological salt concentrations.

Efficient cross-linking of Nat1p to the nascent polypeptide(Fig. 1A and C) raised the question of whether binding of NatAto ribosomes was mediated exclusively via the interaction ofNat1p to the nascent polypeptide or whether NatA was also interactingdirectly with the ribosome. To distinguish between these possibilities,we applied two different approaches. First, we tested whetherNatA would bind to nascent chain-free ribosomes in vitro. Tothis end, nascent chains were released by puromycin treatmentand ribosomal subunits were subsequently incubated in the presenceof NatA. Indeed, NatA was recovered in the ribosomal pellet(Fig. 3A). This was not due to aggregation, since NatA remainedin the supernatant fraction when ribosomes were absent (Fig.3A). Second, we made use of a yeast strain (ts-187) expressinga temperature-sensitive version of the ß-subunit ofeIF3, which at 37°C quickly leads to the generation of nontranslatingribosomes in vivo (19, 53). When the cytosol of heat-shockedts-187 cells was separated into a ribosomal pellet and a postribosomalsupernatant, NatA was recovered exclusively in the ribosomalpellet, from which it was released upon treatment with highsalt (Fig. 3B and data not shown). The combination of data indicatesthat NatA binds directly to ribosomes, even in the absence ofnascent polypeptides.

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FIG. 3. Nat1p anchors Ard1p and Nat5p to the ribosome. (A) Nascent chain-free ribosomes, generated by high-salt-puromycin treatment, were isolated, resuspended, incubated in the absence (-) or presence (+NatA) of partially purified NatA, and subsequently reisolated by centrifugation. As a control, NatA was subjected to centrifugation in the absence of ribosomes. LS-ribos, ribosomes isolated at low-salt conditions; HP-ribos, high-salt-puromycin treated ribosomes; P, ribosomal pellet; S, postribosomal supernatant. (B) The yeast strain ts-187, defective in translationinitiation at elevated temperatures, was incubated at 37°C for 20 min prior to harvest. Subsequently, cytosol (C) was prepared as described in Materials and Methods and separated into the ribosomal pellet (P) and a postribosomal supernatant (S) in the presence of low salt (120 mM potassium acetate) or high salt (700 mM potassium acetate) concentrations. (C) Cytosol (C) from a wild-type yeast strain, a strain lacking Nat1p ( ${Delta}$ nat1), a strain lacking Ard1p and Nat1p but expressing NAT5 under control of a GAL promoter ( ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 + pESC-NAT5), and a strain lacking Nat5p and Ard1p but expressing NAT1 under control of a GAL promoter ( ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 + pESC-NAT1) was separated into a postribosomal supernatant (S) and a ribosomal pellet (P) in the presence of low or high salt concentrations as described above. (A to C) Samples were separated on 10% Tris-Tricine gels, transferred onto nitrocellulose and decorated with antibodies directed against Nat1p, Ard1p, Nat5p, and Rpl16a (ribosomal marker) as indicated.

To determine which of the NatA subunits were responsible forstable association with the ribosome, we tested the ribosomeassociation of Ard1p in ${Delta}$ nat1. A direct comparison between theamount of ribosomes and Ard1p in wild-type and ${Delta}$ nat1 strainsrevealed that Ard1p was significantly reduced in ${Delta}$ nat1 (datanot shown). The remainder, however, did not bind to ribosomesand was recovered in the postribosomal supernatant even underlow-salt conditions (Fig. 3C, Ard1p). Destabilization of Nat1pand Nat5p in ${Delta}$ ard1 was even more severe. In the absence of Ard1p,neither Nat1p nor Nat5p could be detected by immunoblotting(data not shown). To test the localization of Nat1p and Nat5pin the absence of Ard1p, we placed NAT1 and NAT5 under the controlof the strong inducible GAL1-10 promoter (22). When strains ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 plus pESC-NAT1 and ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 plus pESC-NAT5were grown on galactose-containing medium, Nat1p and Nat5p werereadily detectable by immunoblotting (see Fig. 6A, lower panel).Analysis of the localization of Nat5p revealed that in the absenceof Nat1p-Ard1p, the protein was not associated with ribosomes(Fig. 3C, Nat5p). Ribosome association of Nat1p, however, remainedunaltered in the absence Ard1p-Nat5p. As in the wild-type strain,Nat1p was detected in the ribosomal fraction under low-saltconditions and was released from the ribosome by high-salt treatment(Fig. 3C, Nat1p). We conclude that stable association of Ard1pand Nat5p with the ribosome depends on the presence of Nat1pand that Nat1p is able to bind to the ribosome by itself.

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FIG. 6. (A) None of the NatA subunits by itself supports growth at elevated temperature. ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 strains overexpressing NAT1, ARD1, or NAT5 as indicated from a GAL-inducible promoter were grown on galactose as a carbon source. Serial 10-fold dilutions were analyzed on galactose at 30 or 38°C. Aliquots of each strain were also analyzed on immunoblots with antibodies directed against Nat1p, Ard1p, Nat5p, and Ssb1/2p as indicated. (B) Mating efficiency in ${Delta}$ ard1 is increased by high levels of Ssb1/2p. The mating efficiencies of ${Delta}$ ard1 MATa, ${Delta}$ ard1 MATa complemented with ARD1 on a centromeric plasmid ( ${Delta}$ ard1+ CEN-ARD1), and ${Delta}$ ard1 MATa expressing SSB1 on a multicopy plasmid ( ${Delta}$ ard1+ 2µm-SSB1) are shown. The mating efficiency with a MAT ${alpha}$ tester strain was quantified as described in reference 8. The mating efficiency of ${Delta}$ ard1 MATa ± CEN-ARD1 was set at 100%. (C) Simultaneous lack of nat1 and ssb1/2 results in synthetic phenotype enhancement. The wild type, ${Delta}$ ssb1/2, ${Delta}$ nat1, and ${Delta}$ nat1 ${Delta}$ ssb1/2 were grown to early log phase at 30°C on minimal glucose medium. Serial 10-fold dilutions containing the same number of cells were spotted from left to right and incubated as indicated. Note that the weak growth defect of ${Delta}$ nat1 on 1 M NaCl-containing medium is only visible upon shorter incubation times.

NatA is in close proximity to the N termini of a variety of nascent polypeptides. Methionine, the N-terminal amino acid of prepro ${alpha}$ -factor, thenascent chain used for the initial purification of NatA, isnot a substrate for N-acetylation by NatA (40). Nevertheless,Nat1p efficiently cross-links to prepro ${alpha}$ -factor, indicatingthat it contacts a nonsubstrate nascent chain (Fig. 1C and 4A).To test whether NatA interaction was affected by its abilityto recognize the nascent chain as a substrate, RNCs carryingthe first 86 amino acids of the ribosomal protein Rpl4A, anin vivo substrate of NatA (4, 48), were generated and subjectedto cross-linking. During in vitro translation, the initial methioninewas efficiently removed from Rpl4A (data not shown) and Nat1pformed a cross-link to Rpl4A (Fig. 4A). Replacement of serinewith lysine at position 2 changes Rpl4A to a nonsubstrate proteinand, at the same time, generates an additional free amino groupfor the cross-link reaction. Rpl4A-S(2)K efficiently formeda cross-link to Nat1p (Fig. 4A). Nat1p also formed cross-linkproducts to the precursor and the mature form of mitochondrialmalate dehydrogenase (14). We conclude that Nat1p interactswith a variety of ribosome-bound nascent polypeptides, independentlyof their amino acid sequence or their N-terminal residue. Inthis respect, NatA resembles proteins with chaperone-like properties,like NAC and Ssb1/2p. NAC and Ssb1/2p only interact efficientlywith nascent polypeptides in the context of the ribosome (36,37, 56). To test whether NatA would also tightly interact witha polypeptide in the absence of ribosomes, we performed cross-linkreactions with released nascent polypeptides. Nat1p formed across-link only when both the nascent polypeptide and Nat1pwere bound to the ribosome (Fig. 4B). A less stable interactionbetween Nat1p and a polypeptide, however, may not be detectedwith this experimental setup.

NatA forms cross-link products to nascent polypeptides downstream of NAC and Ssb1/2p. Yeast NAC interacts with nascent chains of 72 and 86 amino acids(42). Ssb1/2p was shown to bind to nascent chains of 54 aminoacids; however, it failed to interact with a 34-amino-acid nascentchain (21). We have now, in parallel experiments, determinedthe minimal length requirement for the interaction of yeastmalate dehydrogenase (Mdh1p) to NAC, Ssb1/2p, and NatA (Fig.4C and D). NAC and Ssb1/2p showed interaction with an Mdh1pof at least 45 amino acids. However, Nat1p only formed a cross-linkto an Mdh1p longer than 100 amino acids (Fig. 4C). The cross-linkof Nat1p to Mdh1p required somewhat longer nascent chains thanthe cross-link to Rpl4A or the prepro ${alpha}$ -factor (Fig. 4A and datanot shown). This variability might be due to positioning ofthe primary amino groups or might be caused by conformationaldifferences in the nascent polypeptides. Nascent chains of 50and 70 amino acids, derived from prepro ${alpha}$ -factor or Rpl4A, weretested for cross-link formation to confirm that Nat1p, in general,required longer nascent chains than NAC and Ssb1/2p. In agreementwith the data obtained for Mdh1p, both prepro ${alpha}$ -factor and Rpl4Aof 50 and 70 amino acids formed cross-link products with NACand Ssb1/2p; however, Nat1p did not (Fig. 4C). We conclude thatcross-link formation of different nascent chains with Nat1prequires significantly longer nascent chains than cross-linkformation with either NAC or Ssb1/2p.

Deletion of either NAT1 or ARD1 results in temperature sensitivity. No general growth defect was associated with the deletion ofall three NatA subunits ( ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5) on a variety of mediaat 20 and 30°C (Fig. 5A). Growth rates of the wild typeand ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 were determined in parallel experiments. Bothstrains displayed doubling times of 1.7 h on yeast extract-peptone-dextroseand of 2.6 h on yeast extract-peptone-galactose at 30°C.At 20°C on yeast extract-peptone-dextrose, the wild typeand ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 displayed doubling times of 4.3 and 4.2 h,respectively. At 38°C, however, deletion of either NAT1or ARD1 results in temperature sensitivity (Fig. 5B). The tripledeletion strain ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 also displayed temperature sensitivity(Fig. 5B and Fig. 6A). Growth at 38°C was tested in thetriple deletion strain ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 overexpressing NAT1, ARD1,or NAT5. None of the subunits by itself was able to supportgrowth at 38°C, whereas cooverexpression of Nat1p and Ard1prestored growth at 38°C (Fig. 5B and Fig. 6A). These dataare compatible with a model in which the biological functionof NatA strictly depends on the Nat1p-Ard1p complex, as wassuggested earlier (28). In light of our findings, the simplestexplanation might be that Nat1p in the absence of the catalyticsubunit Ard1p is not an active N-acetyltransferase, whereasArd1p cannot act as an N-acetyltransferase if not anchored tothe ribosome via Nat1p (Fig. 3C and Fig. 7).

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FIG. 5. Absence of either Nat1p or Ard1p causes temperature sensitivity. (A) A haploid wild-type strain and the corresponding mutant strain lacking nat1, ard1, and nat5 ( ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5) were grown to early log phase at 30°C on minimal glucose medium. Serial 10-fold dilutions containing the same number of cells were spotted from left to right onto plates containing different carbon sources and additives and were incubated as indicated. (B) The wild type, ${Delta}$ ard1, ${Delta}$ nat1, and ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 expressing ARD1, NAT1, or SSB1 on multicopy plasmids (all based on pYEPlac195; see Materials and Methods) were grown as described above. (C) Serial dilutions of the wild type and ${Delta}$ nat5 were grown at 38°C. (D) Patch mating assay. A W303 (MATa HML ${alpha}$ ) wild-type strain and its ${Delta}$ nat1, ${Delta}$ ard1, and ${Delta}$ nat5 derivatives were assayed with a MAT ${alpha}$ tester strain for mating efficiency in a patch mating assay.

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FIG. 7. A model for cotranslational N-acetylation of polypeptides by NatA. Nat1p, the noncatalytic NatA subunit, mediates the stable interaction of the complex with the large ribosomal subunit (LS). In addition, Nat1p contacts the nascent polypeptide when approximately 40 amino acids have emerged from the exit tunnel (i.e., the nascent chain has a total length of approximately 80 amino acids). This interaction, possibly mediated via TPRs contained in Nat1p, may guide the growing polypeptide to the catalytic subunit Ard1p and the putatively catalytic subunit Nat5p, which transfer an acetyl moiety from acetyl-coenzyme A to specific N-terminal amino acids (see the introduction). Ard1p requires the presence of Nat1p for stable association with the ribosome. It is unknown whether Nat5p interacts with the ribosome via Nat1p, Ard1p, or both (indicated by question marks). The catalytic subunits may have little or no affinity for the nascent polypeptide in the absence of Nat1p; hence, a stable complex with Nat1p is essential for N-acetyltransferase activity. Complex formation between Nat1p and Ard1p might occur via predicted coiled coil domains in their C-terminal regions. Two other ribosome-bound factors, NAC and the Hsp70 homolog Ssb1/2p bind to nascent chains between 40 and 50 amino acids in length. Binding of NAC and Ssb1/2p precedes the binding of Nat1p. Whether or not NAC, Ssb1/2p, and NatA simultaneously bind to one and the same ribosome (as suggested here) or independently to different ribosome molecules is currently unknown. For details, see Results and Discussion. Please note that other factors bound to the ribosome or the nascent chain have been omitted for simplicity.

Deletion of NAT5 does not cause temperature sensitivity or HML derepression. In contrast to the ${Delta}$ nat1 and ${Delta}$ ard1 strains, ${Delta}$ nat5 did not displaytemperature sensitivity (Fig. 5C). As ${Delta}$ nat1 (MATa HML ${alpha}$ ) and ${Delta}$ ard1(MATa HML ${alpha}$ ) were previously shown to cause derepression of theHML silent mating-type locus (54), we tested whether ${Delta}$ nat5 woulddisplay the same phenotype. In a patch mating assay, ${Delta}$ nat5 (MATaHML ${alpha}$ ) showed wild-type mating efficiency (Fig. 5D). To examinewhether ${Delta}$ nat5 caused more subtle HML silencing defects, we alsodetermined the response to ${alpha}$ -factor mating pheromone in an ${alpha}$ -factorconfrontation assay (27). Like the wild type, ${Delta}$ nat5 showed 100%shmoo formation, whereas only approximately 10% of ${Delta}$ nat1 and ${Delta}$ ard1 strains formed shmoos (data not shown). The combined dataindicate that HML in the ${Delta}$ nat5 strain was repressed as efficientlyas in the wild-type control. It has been suggested that HMLderepression might be caused by the lack of N-acetylation ofa so far unidentified substrate (40). According to this hypothesis,Ard1p, not Nat5p, would be responsible for that activity.

Sequence-independent interaction with nascent polypeptides,as observed for Nat1p, are properties reminiscent of chaperone-likeproteins (18). To test the possibility that chaperones mightbe able to suppress the temperature-sensitive phenotype of strainslacking functional NatA, we have tested overexpression of theHsp70 homologs SSA1, SSB1, and SSZ1; the Hsp40 homologs YDJ1and ZUO1; HSP26; and HSP104 and co-overexpression of both subunitsof NAC (EGD1 and EGD2). Only a high level of Ssb1/2p resultedin partial suppression of temperature sensitivity in ${Delta}$ nat, ${Delta}$ ard1,and ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5 (Fig. 5B and data not shown). Overexpressionof Ssb1/2p was also tested for its effect on derepression ofthe HML silent mating-type locus caused by lack of NatA. Whencompared to the corresponding wild-type strain, the mating efficiencyof ${Delta}$ ard1 (MATa) with a MAT ${alpha}$ tester strain was reduced to 0.076%. ${Delta}$ ard1 overexpressing Ssb1/2p ( ${Delta}$ ard1 MATa plus 2µm-SSB1)displayed a more-than-10-fold increase in mating efficiency(1.2% of the wild type) (Fig. 6B). We conclude that both temperaturesensitivity and derepression of the HML locus are partly suppressedby overexpression of Ssb1/2p.

Our data indicated that Ssb1/2p and Nat1p are associated withthe ribosome and localize in close proximity to the nascentpolypeptide chain and that SSB1 acts, albeit weakly, as a multicopysuppressor in ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5. However, the phenotype of ${Delta}$ nat1 ${Delta}$ ard1 ${Delta}$ nat5differs significantly from the phenotype of ${Delta}$ ssb1/2. The absenceof Ssb1/2p results in a complex set of phenotypes includingslow growth, cold sensitivity, and sensitivity to high concentrationsof salt (29). We have generated ${Delta}$ nat1 ${Delta}$ ssb1/2 to test for syntheticeffects between ${Delta}$ ssb1/2 and ${Delta}$ nat1. ${Delta}$ nat1 ${Delta}$ ssb1/2 displayed the combinedset of phenotypes. Like ${Delta}$ ssb1/2, the strain displayed slow growthand cold sensitivity, and like ${Delta}$ nat1, the strain was unable togrow at 38°C (data not shown). ${Delta}$ nat1 and ${Delta}$ ssb1/2 strains sharea moderate salt-sensitive phenotype. ${Delta}$ nat1 ${Delta}$ ssb1/2, however, wasentirely unable to grow in the presence of salt (Fig. 6C). Weconclude that simultaneous lack of nat1 and ssb1/2 results ina synthetic enhancement, compatible with parallel but functionallyrelated roles of these two ribosome-bound factors.

DISCUSSION

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Discussion

A model of NatA interaction with the ribosome and the nascent polypeptide. Nat1p is predicted to contain a minimum of 5 and up to 8 tetratricopeptiderepeat (TPR) motifs (2). Clusters of TPR motifs have been foundin a variety of proteins, including chaperones and proteinsinvolved in protein transport (5). The partial crystal structureof the peroxisomal import receptor Pex5p in complex with a peroxisomaltargeting peptide indicates that multiple TPR motifs form thepeptide binding site (12). By analogy, interaction of Nat1pwith the nascent polypeptide might be mediated by at least partof its TPR motifs. Nat1p also contains a highly charged regionbetween amino acids 620 and 670 with the potential to form acoiled coil (25). This region of Nat1p is poised to interactwith Ard1p, which also contains a potential coiled coil in itsC-terminal domain (amino acids 194 to 224). This idea wouldbe in agreement with earlier data indicating that a C-terminallytruncated version of Ard1p did not stably interact with Nat1p(33). Interestingly, Nat5p lacks the C-terminal charged regiondetected in its homologue Ard1p. Based on a combination of structuralfeatures and biochemical data, we propose a model of subunitinteractions for NatA, its binding to the ribosome, and itsinteraction with the nascent polypeptide (for details, see Fig.7).

Nat1p plays a pivotal role in cotranslational N-terminal acetylation. There is long-standing evidence from the mammalian system suggestingthat N-terminal acetylation occurs cotranslationally (16, 32,35, 51). Our results indicate that NatA indeed acts cotranslationallyand that it is the Nat1p subunit that ensures cotranslationalacetylation by dual means. Nat1p not only anchors the catalyticsubunits to the ribosome but also presents the nascent chainsto Ard1p (and possibly Nat5p), which in turn transfer the acetylmoiety from acetyl coenzyme A to the N terminus of the polypeptidechain (Fig. 7). Ard1p and Nat5p are unable to stably interactwith their substrates by themselves, as indicated by their inabilityto bind to the ribosome. Furthermore, overexpression of Ard1por Nat5p failed to suppress temperature sensitivity in the absenceof Nat1p.

A complex arrangement around the ribosomal tunnel exit. NatA shares the ability to contact nascent chains independentof their amino acid sequence with at least two other ribosome-associatedproteins: NAC and Ssb1/2p. In addition, Srp54p, a subunit ofthe signal recognition particle, specifically interacts withnascent secretory polypeptides of 50 to 70 amino acids in length(23). The presence of these various factors at the ribosomaltunnel exit implies a highly ordered arrangement and sequenceof binding events.

NAC is a heterodimeric complex which can reversibly bind toeukaryotic ribosomes. NAC is located in direct proximity tonewly synthesized polypeptide chains as they emerge from theribosome and is known to interact with nascent chains at a veryearly stage (43). A direct comparison between NAC and Ssb1/2pin our experimental setup revealed that both components bindto nascent chains as short as 45 to 50 amino acids. NAC andSsb1/2p might coreside on the ribosome; however, our experimentscannot distinguish between simultaneous binding of both proteinsto a single nascent chain or mutually exclusive binding to differentnascent chains of equal length. Simultaneous binding of NatAand Ssb1/2p or NAC seems sterically possible, as NatA interactswith more-mature nascent chains (Fig. 4). Our future goal willbe to determine the arrangement of NAC, Ssb1/2p, NatA and theirdynamic interaction with the nascent polypeptide.

Nat5p, a subunit of NatA without function? We found that Nat5p, a predicted member of the GNAT family ofN-acetyltransferases, is bound to the functional NatA complex,consisting of Nat1p and Ard1p, in stoichiometric amounts. Incontrast to Nat1p and Ard1p, which are essential for NatA function,the absence of Nat5p causes neither a growth defect at 38°Cnor HML derepression. Attempts to detect Nat5p substrates inyeast by two-dimensional gel electrophoresis have failed sofar (41). Possibly, Nat5p is responsible for the N-acetylationof a small subset of proteins that are not involved in matingtype silencing or affected at elevated temperature. Resultsfrom a deletion suppressor screen hint at a putative functionfor Nat5p (1). Temperature sensitivity of two different doublenull mutant strains involved in ubiquitin-dependent proteindegradation (mck1 mds1 and bul1 bul2) was suppressed by thedeletion of NAT5 (termed ROG2 in reference 1).

The in vivo function of NatA is unknown. As previously discussed by others, it is quite surprising that,although Ard1p and Nat1p are responsible for the N-acetylationof a large number of proteins, only a few specific phenotypesare observed in ${Delta}$ nat1 and ${Delta}$ ard1 mutant strains at normal growthtemperatures (33). However, ${Delta}$ nat1 and ${Delta}$ ard1 strains displayeda general growth defect at 38°C.

Two different scenarios might account for temperature sensitivityin the absence of functional NatA. In the first hypothesis,temperature sensitivity would be caused by the lack of N-terminalacetylation of one or more proteins. De novo folding, thermalstability, or protease resistance might be impaired if proteinscarry an unprotected N-amino group. However, up to now, experimentalevidence in support of such direct effects is rare. Degradationby the proteasome is not prevented by N-terminal acetylation(26). Purified protein pairs identical but for N-acetylationare not easily obtained, and as a consequence, biochemical andbiophysical properties of such protein pairs have not been studied(40). In the second scenario, the lack of N-acetylation wouldnot be the sole reason for the defects observed in the absenceof NatA. The ability of Nat1p to interact with nascent polypeptidesis a property shared with chaperones. In this respect, it isinteresting that the ribosome-bound chaperone Ssb1/2p can partlysuppress temperature sensitivity and derepression of the HMLlocus. It seems unlikely that high levels of Ssb1/2p would restoreN-acetylation of NatA substrates. Suppression of both temperaturesensitivity and silencing defect suggests that Ssb1/2p is ableto complement for a general function of NatA. Whether Ssb1/2pcan partly substitute for Nat1p function on the ribosome orwhether it suppresses defects caused by specific proteins thatlack N-terminal acetylation awaits further investigation.

ACKNOWLEDGMENTS

This work was supported by the Fonds der Chemischen Industrie(to S.R.) and by SFB 610 (to S.R.).

We thank Thierry Mini and Paul Jenö for the initial identificationof Nat1p, Hans Trachsel for strain ts-187 (originally describedin reference 19), and Hauke Lilie and members of the lab forcritically reading of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Institut für Biochemie undMolekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany. Phone: 49-761-203-5224. Fax: 49-761-203-5261. E-mail: Sabine.Rospert{at}biochemie.uni-freiburg.de.

Present address: Swiss Federal Institute of Technology Zürich,ETH Hönggerberg, CH-8093 Zürich, Switzerland.

REFERENCES

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