N^α-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

Abstract

N^α-Acetyltransferases (NATs) cause the N^α-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N^α-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N^α-acetylated by NatA, and two by NatB. The N^α-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N^α-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N^α-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N^α-acetylation is necessary to maintain the ribosome's protein synthesis function.

Introduction

Methionine cleavage and N^α-acetylation are two common protein N-terminal modifications [1], [2]. A majority of experimentally characterized eukaryotic proteins are N-terminally acetylated by N^α-acetyltransferases (NATs) during their translation from mRNA [3]. In yeast (Saccharomyces cerevisiae), approximately 57% of proteins are predicted to have an N^α-acetyl group, while the corresponding figure for mammalian proteins is about 84% [4]. The N^α-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 N^α-acetylation, it is unknown how many proteins require N^α-acetylation for function. For instance, the N^α-acetylation of Orc1p and Sir3p was shown to be necessary for transcriptional silencing in yeast [8], [9]. Also, the N^α-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 N^α-acetylation of ribosomal proteins has been known for decades [12], [13], [14], the role of N^α-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 N^α-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 N^α-acetylation of ribosomal proteins, and therefore changes of ribosome function remain unknown.

In this study, we comprehensively analyzed ribosomal protein N^α-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 N^α-acetylation on protein synthesis using the NatA deletion mutant.