Nα-Acetylation of yeast ribosomal proteins and its effect on protein synthesis
Graphical Abstract
Introduction
Methionine cleavage and Nα-acetylation are two common protein N-terminal modifications [1], [2]. A majority of experimentally characterized eukaryotic proteins are N-terminally acetylated by Nα-acetyltransferases (NATs) during their translation from mRNA [3]. In yeast (Saccharomyces cerevisiae), approximately 57% of proteins are predicted to have an Nα-acetyl group, while the corresponding figure for mammalian proteins is about 84% [4]. The Nα-acetylation is catalyzed by NATs that contain catalytic subunits homologous to the GNAT family of acetyltransferase [5]. In yeast, four NATs have been identified, NatA, NatB, NatC, and NatD, which are composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p for NatA; Nat3p and Mdm20p for NatB; and Mak3p, Mak10p, and Mak31p for NatC [6]. A recent study has shown NatD to consist of only a catalytic subunit: Nat4p [7]. The deletion of NATs induces various phenotypes. The NatA deletion mutant exhibits defects in sporulation, salt sensitivity, mating efficiency, and the ability to enter G0. The NatB deletion mutant shows increased osmotic sensitivity, decreased utilization of non-fermentable carbon sources, reduced mating efficiency, inability to form functional actin filaments, defects in mitochondrial and vacuolar inheritance, random polarity, increased sensitivity to the anti-mitotic drugs, and increased susceptibility to a number of DNA damaging agents. The NatC deletion mutant shows a decreased growth on YPG medium at 37 °C, although growth on YPD medium at 30 °C is nearly normal [6].
Despite the wide occurrence of protein Nα-acetylation, it is unknown how many proteins require Nα-acetylation for function. For instance, the Nα-acetylation of Orc1p and Sir3p was shown to be necessary for transcriptional silencing in yeast [8], [9]. Also, the Nα-acetylation of the killer viral coat protein Gag by NatC is required for assembly and maintenance of the L-A dsRNA viral particle in yeast [10]. Unacetylated actin and tropomyosin have a number of defects in vivo and in vitro, although the mutants are viable [11]. While the Nα-acetylation of ribosomal proteins has been known for decades [12], [13], [14], the role of Nα-acetylation in translation has not been determined.
The ribosome is a large ribonucleoprotein complex that synthesizes proteins in the cytoplasm. The core of the structure, as well as many of the ribosomal functions, is highly conserved between eukaryotes and prokaryotes [15]. In yeast, the ribosome consists of two subunits, the large (60S) and small (40S) subunits. The 60S subunit is composed of three ribosomal RNAs (rRNAs) and 46 ribosomal proteins, whereas the 40S subunit is composed of one rRNA and 32 ribosomal proteins [16], [17]. The ribosome translates mRNA sequences into the corresponding amino acids and links them together to synthesize proteins. There are four stages of protein synthesis: initiation, elongation, termination, and recycling [18]. The 60S subunit polymerizes the polypeptide chain during the elongation phase. The 40S subunit is associated with mRNA tracks, the tRNA binding site, and is instrumental in selecting an aminoacyl-tRNA that complements the bound mRNA codon [16]. Although the rRNAs basically catalyze translation of mRNA and peptide bond formation, ribosomal proteins have been shown to play several important roles in protein synthesis, including determining the conformation of the ribosome structure and binding the various translational factors [19].
The ribosomal proteins undergo a variety of post-translational modifications including phosphorylation, methylation, glycosylation, and Nα-acetylation (co-translational). The post-translational modifications are thought to affect the ribosomal function. For example, Ruvinsky et al. reported that phosphorylation of ribosomal protein S6 controls cell size and glucose homeostasis [20]. Phosphorylation of ribosomal protein P1A exerts an effect on the hetero-oligomerization process [21]. Additionally, it is known that arginine methylation of ribosomal protein S10 regulates ribosome biogenesis [22], and arginine methylation of ribosomal proteins S3 affects ribosome assembly [23]. Glycosylation of ribosomal proteins is required for aggregation of untranslated messenger ribonucleoproteins into stress granules [24]. Clearly, modifications of ribosomal proteins are important for protein synthesis. However, the effect of Nα-acetylation of ribosomal proteins, and therefore changes of ribosome function remain unknown.
In this study, we comprehensively analyzed ribosomal protein Nα-acetylation using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). These analyses led to the identification of 19 ribosomal proteins acetylated by NatA and NatB. Subsequently, we investigated the effect of ribosomal protein Nα-acetylation on protein synthesis using the NatA deletion mutant.
Section snippets
Yeast strains and media
The following strains were used in this study: the normal strain, B-8032 (MATα ura3-52 CYC1-963 cyc7-67 lys5-10); the nat1 mutant, B-8360 (MATα nat1::URA3 ura3-52 CYC1-963 cyc7-67 lys5-10); the mak3 mutant, B-9074 (MATα mak3::URA3 CYC1-963 cyc7-67 lys5-10); and the nat3 mutant, B-11974 (MATα nat3::kanMX2 CYC1-963 cyc7-67 lys5-10).
The YPD medium [2% (w/v) glucose, 2% (w/v) pepton, and 1% (w/v) yeast extract] was used for growing yeast. To purify 80S ribosomes, the yeast cells were cultured in
Ribosome purification and ribosomal protein identification by two-dimensional electrophoresis
In this study, we purified the 80S ribosomes from the normal yeast strain and the NAT mutants. To check the quality of the purified 80S ribosomes, rRNAs and ribosomal proteins were separated by standard gel electrophoresis (Fig. 1A). As shown in Fig. 1A, sharp bands of 25S and 18S rRNAs were detected, while no smeared band was detected; this shows that intact 80S ribosomes were purified, with no degradation incurred during purification. Simultaneously, the ribosomal proteins of the 80S
Discussion
Genome sequencing has revealed that yeast contains 137 ribosomal protein genes, encoding 78 unique ribosomal proteins with 59 encoded by duplicate genes [27]. Takakura et al. detected a total of 44 ribosomal proteins in the yeast 80S ribosome by 2-DE using acid-urea gel electrophoresis in the first dimension and SDS-PAGE in the second dimension, and identified 14 ribosomal proteins which were Nα-acetylated by NatA using Edman degradation [12]. Arnold et al. found that 30 of the identified 68
Acknowledgements
This work was supported in part by Special Coordination Funds for Promoting Science and Technology “Creation of Innovation Centers for Advanced Interdisciplinary Research Areas” (to H.H.) and in part by the National Institutes of Health Grant R01 GM12702 (to F.S.).
References (38)
- et al.
The proteomics of N-terminal methionine cleavage
Mol Cell Proteomics
(2006) - et al.
N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families
Trends Biochem Sci
(1998) - et al.
N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins
J Mol Biol
(2003) - et al.
Composition and function of the eukaryotic N-terminal acetyltransferase subunits
Biochem Biophys Res Commun
(2003) - et al.
MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly
J Biol Chem
(1992) - et al.
Nat3p and Mdm20p are required for function of yeast NatB Nα-terminal acetyltransferase and of actin and tropomyosin
J Biol Chem
(2003) - et al.
NH2-terminal acetylation of ribosomal proteins of Saccharomyces cerevisiae
J Biol Chem
(1992) - et al.
The action of N-terminal acetyltransferases on yeast ribosomal proteins
J Biol Chem
(1999) - et al.
Comparison of fungal 80S ribosomes by cryo-EM reveals diversity in structure and conformation of rRNA expansion segments
J Mol Biol
(2007) - et al.
Probing translation with small-molecule inhibitors
Chem Biol
(2010)
Oligomerization properties of the acidic ribosomal P-proteins from Saccharomyces cerevisiae: effect of P1A protein phosphorylation on the formation of the P1A-P2B hetero-complex
Biochim Biophys Acta
Methylation of ribosomal protein S10 by protein–arginine methyltransferase 5 regulates ribosome biogenesis
J Biol Chem
Arginine methylation of ribosomal protein S3 affects ribosome assembly
Biochem Biophys Res Commun
rRNA modifications in an intersubunit bridge of the ribosome strongly affect both ribosome biogenesis and activity
Mol Cell
Selection of tRNA by the ribosome requires a transition from an open to a closed form
Cell
Decoding errors and the involvement of the E-site
Biochimie
The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides
Mol Cell Biol
Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans
Proc Natl Acad Sci USA
Properties of Nat4, an N(alpha)-acetyltransferase of Saccharomyces cerevisiae that modifies N termini of histones H2A and H4
Mol Cell Biol
Cited by (35)
N-terminal acetylation levels are maintained during acetyl-CoA deficiency in saccharomyces cerevisiae
2018, Molecular and Cellular ProteomicsEstablishment of dimethyl labeling-based quantitative acetylproteomics in Arabidopsis
2018, Molecular and Cellular ProteomicsCitation Excerpt :Previous studies have found that various organisms utilize specialized PTMs on ribosomal proteins (121–123). These PTMs help guide nuclear events, expand molecular structures, and facilitate activity regulation (124–129). Yang et al. (127) found that mitochondrial protein synthesis is enhanced by the reversible acetylation of mitochondrial ribosomal protein L10.
Protein post-translational modifications: In silico prediction tools and molecular modeling
2017, Computational and Structural Biotechnology JournalCitation Excerpt :Although Nα-acetylation is more common (roughly 85% in eukaryotic proteins), Nε-lysine acetylation is more biologically important [156–163]. Indeed Nε-acetylation on internal lysines is a reversible post-translational modification involved in several biological processes, such as transcription regulation [159,161], protein expression and stability [153,164–167], DNA repair [162], apoptosis [160,163] and nuclear import [158] (Table 2). Aberrant lysine acetylation is linked with cancer [157,168–170], neurodegenerative disorders [171–173] and cardiovascular diseases [174–178].
The biological functions of Naa10 - From amino-terminal acetylation to human disease
2015, GeneCitation Excerpt :To analyze the impact of Nα-terminal acetylation on translational activity, Kamita and colleagues performed a polyU-dependent poly-(Phe) synthesis assay with purified ribosomes from wild type and NAA15Δ strains (Kamita et al., 2011). The authors detected a decrease of the ribosomal protein synthesis activity by 27% in the mutant compared to wild type ribosomes (Kamita et al., 2011). In dilution spot assays, increased sensitivity of the NAA15Δ mutant cells towards the antibiotic paromomycin and hygromycin was observed and since both antibiotics induce errors during translation, the authors speculate that NTA of ribosomal proteins by NatA may be required to maintain proper translational fidelity (Kamita et al., 2011).
Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions
2014, Biochemical and Biophysical Research Communications