The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides

Abstract

The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.

FIG. 1.

Nat1p, a subunit of yeast NatA interacts with RNCs. (A) RNCs carrying 87 N-terminal amino acids of ³⁵S-labeled prepro α-factor (prepro α 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 α 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 α 87); the signal was linear in relation to the exposure time. The amount of prepro α 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 α 87 as a nascent chain. Aliquots of the cross-link reactions were subjected to immunoprecipitation under denaturing conditions with preimmune serum (PI), anti-nat1 (α-nat1), anti-ard1 (α-ard1), and anti-nat5 (α-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).

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 (α-nat1), or antibody recognizing Ard1p (α-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.

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 (Δnat1), a strain lacking Ard1p and Nat1p but expressing NAT5 under control of a GAL promoter (Δnat1Δard1Δnat5 + pESC-NAT5), and a strain lacking Nat5p and Ard1p but expressing NAT1 under control of a GAL promoter (Δnat1Δard1Δnat5 + 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.

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 α-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 (α-ssb1), anti-nat1 (α-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 α-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.

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 (Δnat1Δard1Δnat5) 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, Δard1, Δnat1, and Δnat1Δard1Δnat5 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 Δnat5 were grown at 38°C. (D) Patch mating assay. A W303 (MATa HMLα) wild-type strain and its Δnat1, Δard1, and Δnat5 derivatives were assayed with a MATα tester strain for mating efficiency in a patch mating assay.

FIG. 6.

(A) None of the NatA subunits by itself supports growth at elevated temperature. Δnat1Δard1Δnat5 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 Δard1 is increased by high levels of Ssb1/2p. The mating efficiencies of Δard1 MATa, Δard1 MATa complemented with ARD1 on a centromeric plasmid (Δard1+ CEN-ARD1), and Δard1 MATa expressing SSB1 on a multicopy plasmid (Δard1+ 2μm-SSB1) are shown. The mating efficiency with a MATα tester strain was quantified as described in reference . The mating efficiency of Δard1 MATa ± CEN-ARD1 was set at 100%. (C) Simultaneous lack of nat1 and ssb1/2 results in synthetic phenotype enhancement. The wild type, Δssb1/2, Δnat1, and Δnat1Δssb1/2 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 Δnat1 on 1 M NaCl-containing medium is only visible upon shorter incubation times.

FIG. 7.

A model for cotranslational N^α-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^α-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.