Characterization of the Human Small‐Ribosomal‐Subunit Proteins by N‐Terminal and Internal Sequencing, and Mass Spectrometry

Abstract

Reverse-phase HPLC was used to fractionate 40s ribosomal proteins from human placenta. Application of a C, reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40s human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40s proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.Keywords: human ribosomal proteins ; HPLC ; N-terminal and internal amino acid sequencing ; matrixassisted-laser-desorption ionization MS ; post-translational modifications.Human ribosornd proteins are the main components of the fundamental processes which translate genetic information into proteins at the ribosome. Some ribosomal proteins (rp) are further involved in non-ribosomal functions. Recently, rp L7 was shown to contain the basic-region-leucine-zipper sequence which mediates the stable binding of this protein to DNA [I]. Moreover, rp S3 was found to act as an endonuclease in the DNA-repairing system [2]. Finally, some data indicate that tumor diseases may be accompanied by overexpression of rp genes 13, 4) and by chromosomal rearrangements in regions of rp genes [ S ] . Therefore, structural analysis of these proteins is necessary for complete understanding of their fundamental role in the cell. The task of structure determination of rat rp has been almost fully resolved by the application of recombinant-DNA technology ; details are reviewed in [6]. However, knowledge of the cDNA sequences does not provide information concerning post-translational modifications of the proteins after their biosynthesis and assembly into the ribosomal subunits. Several modifications of rp have been determined in the eubacterial ribosome [7, 81, and the eukaryotic rprotein S6 is known to be phosphorylated 191. This phosphorylation was shown to be connected with cell growth [I 01 and cell cycle control [I I]. At least seven 40s proteins from HeLa cells were found to be methylated, and the level of their methylation was shown to be altered in the Abbreviations. MALDI, matrix-assisted laser-desorption ionization ; Lys-C, endoproteinase Lys-C; RP, reverse-phase; rp, ribosomal protein ; S-proteins, proteins of the small ribosomal subunit; 40S, small ribosomal subunit; TP40, total protein mixture of the small ribosomal subunit; TOF, time-of-flight ; Pth-Xaa, phenylthiohydantoin -amino-acid.course of the cell cycle [12]. It is also known that ribosomal proteins may be acetylated 17, 13, 141. However...

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

“…Interestingly, a comparison of the results indicates that the ribosomal proteins of A. thaliana, are modified to a much larger extent, including for some protein families more than 10 different forms (e.g. for L5 and S3, Table 2), compared to the few modifications observed for each ribosomal protein of the other organism analysed (e.g Escherichia coli, (Wittmann-Liebold, 1986); yeast, (Link et al, 1999;Lee et al, 2002); rat, (Wool et al, 1995;Louie et al, 1996); human, (Vladimirov et al, 1996;Odintsova et al, 2003); spinach, and Chlamydomonas reinhardtii, ).…”

“…This implies that there are multiple different forms for many of the ribosomal proteins present in our ribosome population. Indeed, numerous ribosomal proteins in bacteria, rat, human and yeast have been found to be methylated, acetylated and/ or phosphorylated (Louie et al, 1996;Vladimirov et al, 1996;Arnold and Reilly, 1999;Arnold and Reilly, 2002;Lee et al, 2002;Odintsova et al, 2003). We observe multiple protein spots of varying molecular weights and with different pI values from the 80S fraction derived 2-DE gel (Figure 2), which can also be indicative for several different transcriptional, translational or even post-translational modifications, such as alternative splicing (Smith et al, 1989;Brett et al, 2002), specific chemical amino acid modifications (Krishna and Wold, 1993) as well as directed protein degradation (Callis and Vierstra, 2000;Estelle, 2001).…”

“…Proteomic analyses of ribosomes from higher eukaryotes have been limited to cytoplasmic ribosomes from rat (Wool et al, 1995;Louie et al, 1996), as well as both the small (Vladimirov et al, 1996) and large subunit (Odintsova et al, 2003) from humans. Although studies have addressed the composition of bacteriallike ribosomes, such as the chloroplast 70S ribosome from Spinacea oleracea and Chlamydomonas reinhardtii , to date, little work has addressed the composition of the higher plant cytoplasmic ribosomes.…”

Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7

“…The N-terminal portion of S7 is rich in positively charged residues, a characteristic commonly found in the so-called arginine-rich motif that occurs in several RNA-binding proteins (Tan & Frankel, 1995)+ Deletion of the N-terminal 17 residues of S7 dramatically affected its affinity for rRNA because it caused a complete to near-complete loss of binding, depending upon the ionic strength of the binding buffer+ A similar observation was made by Miyamoto et al+ (1999) with the truncation of the N-terminal 10 residues of B. stearothermophilus S7+ Point mutations within the N-terminal region also significantly affected S7 binding to rRNA, with a fivefold decrease of the affinity for mutants Q8A and F17G+ The S7 terminal portion is an unstructured and flexible region, which has been crosslinked to C1378, the same base that was crosslinked to K75 in the b hairpin (Urlaub et al+, 1995(Urlaub et al+, , 1997see Fig+ 2) and is part of the P site (Green & Noller, 1997)+ It was also crosslinked to puromycin, an antibiotic that binds to the A site (Bischof et al+, 1994)+ Moreover, directed hydroxyl radical probing showed that it is proximal to the loop capping helix 43 (Miyamoto et al+, 1999)+ The results obtained with the mutations in the N-terminal portion indicate that this region of S7 plays a crucial role in the binding of the protein to rRNA, whereas the crosslinking studies and hydroxyl radical probing suggest that this region remains flexible when S7 is bound to rRNA+ To account for the loss of binding in the absence of the N-terminal region, we propose that it makes an initial interaction with the rRNA that is required for the other contacts to occur+ Once S7 is bound to the rRNA, the N-terminal region could disengage and then interact with the A site or with the P site+ The flexibility of the N-terminal region of protein S7 makes it likely that the crosslinks involving this region and K75 can occur simultaneously within the 30S subunit+ Alternatively, each of these crosslinks could correspond to a different conformational state of the 30S subunit+ The N-terminal portion of S7 is not conserved in its eukaryotic homolog (Kuwano et al+, 1992;Vladimirov et al+, 1996;Wimberly et al+, 1997), making this region an interesting potential target for the development of novel antibiotics that interfere with bacterial ribosome assembly+ In various other RNA-binding ribosomal and nonribosomal proteins such as L1 (Eliseikina et al+, 1996), the antitermination protein NusB of E. coli (Huenges et al+, 1998), and the bacteriophage l N protein (Legault et al+, 1998), the flexible and positively charged N-terminal portion has also been shown to play a crucial role in the interaction with the RNA+ When this work was completed, Fredrick et al+ (2000) published a study that examined the effects of various mutations in E. coli S7 on 30S subunit assembly in vivo+ The N-terminal deletion of S7 and mutations at positions 34 and 35 were also investigated by these researchers+ Interestingly, the mutants with the N-terminal deletion or a substitution of K35, which bind weakly to th...…”

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…By means of MALDI-MS [8][9] and ESI-MS/MS n [10][11][12] analysis of protein complexes, e.g. such as ribosomes [13,14] was facilitated, and interesting new proteins involved in the various cell processes and pathways were discovered. A common strategy for performing proteome studies is illustrated in Fig.…”