Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5

Abstract

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.

Figure 1.

SDS-PAGE Profiles of Chlamydomonas Ribosomal Proteins. Total proteins extracted from cytosolic ribosomes (80S), chloroplast ribosomes (70S), and chloroplast subunits (50S and 30S) (10 pmol each of TP80, TP70, TP50, and TP30) were subjected to SDS-PAGE. Proteins were stained with Coomassie blue. Two bands (41 and 38 kD) present in TP70 but absent in TP30 and TP50 are indicated by arrows. The TP30 lane was sectioned into 24 pieces for in-gel digestion, as indicated by the dotted lines.

Figure 2.

Collision-Induced Dissociation Spectra of Tryptic Peptides Derived from the 84- and 57-kD Proteins of the Small Subunit. The SEQUEST algorithm assigned b-ion and y-ion series to the observed mass spectra based on sequence databases. (A) Fragmentation of the doubly charged precursor ion at an m/z of 735.8 yielded detectable singly charged b-ion species (b-2 to b-9) and y-ion species (y-2 to y-8) for which the sequence is indicated. The peptide sequence obtained from the 84-kD protein belonged to a nucleus-encoded protein (PRP S5). (B) Fragmentation of the doubly charged precursor ion at an m/z of 565.6 yielded detectable singly charged b-ion species (b-3 to b-11) and y-ion species (y-2 to y-11) for which the sequence is indicated. The peptide sequence obtained from the 57-kD protein belonged to a plastid-encoded protein (PRP S2).

Figure 3.

Two S1 Domain–Containing Proteins Are Present in the Chlamydomonas Chloroplast 30S Ribosomal Subunit: An Ortholog of E. coli S1 and a Novel S1 Domain–Containing Protein (PSRP-7). (A) Schemes of the E. coli S1 protein (EcoS1), spinach PRP S1 (SolS1), Synechococcus S1 (ScoS1), Chlamydomonas PRP S1 (CreS1), Bordetella pertussis TEX protein (BpeTEX), and Chlamydomonas PSRP-7 (CrePSRP-7). The ribosome binding domains of the S1 proteins are indicated by striped boxes. The RNA binding domains of the S1 proteins are indicated by closed boxes. (B) Phylogenetic tree of the relationships among S1 domains from EcoS1, SolS1, ScoS1, CreS1, BpeTEX, and CrePSRP-7. The tree was created using CLUSTAL W (Thompson et al., 1994). The S1 domains indicated by closed boxes in (A) were grouped in branch b, except d6 of E. coli. These domains contain conserved aromatic residues believed to be involved in RNA binding, based on the NMR structure of the S1 domain of E. coli polynucleotide phosphorylase (Bycroft et al., 1997). The first two domains (d1 and d2) of the S1 proteins, indicated by striped boxes in (A), were grouped in branches a and c. Domains grouped in branches a, c, and d (d6 of E. coli S1) lack the aromatic residues required for mRNA binding. The first two domains in E. coli S1 do not interact with mRNA, nor does the last domain (d6) of E. coli S1, which is required for the autoregulation of its own mRNA but not for overall mRNA recognition (Boni et al., 2000).

Figure 4.

Chlamydomonas 30S PRPs S2, S3, and S5 Are Two to Four Times Larger Than Their Counterparts in Spinach, Synechocystis, and E. coli as a Result of the Existence of Additional Domains in the Chlamydomonas Proteins. Mature polypeptides (N to C terminus) are represented schematically and aligned. Closed boxes represent regions homologous with E. coli proteins. Open boxes represent nonhomologous sequences. The locations of tryptic fragments (black boxes) and Lys-C fragments (gray boxes), which were identified by MS/MS, are indicated. These NTE (PRP S2 and PRP S5) and INS (PRP S3) sequences have no similarity to those of characterized proteins from other organisms. AA, amino acids.

Figure 5.

Comparison of the Molecular Masses of the 30S Proteins from Chlamydomonas Chloroplast, Spinach Chloroplast, and E. coli. Molecular masses of spinach 30S PRPs (Yamaguchi et al., 2000) and E. coli 30S r-proteins (Arnold and Reilly, 1999) were compared with those in Chlamydomonas (see Table 3). The apparent size of PRP S5 is 17 kD larger (striped bar) than that of the predicted mature protein from the sequence (see text).

Figure 6.

Localization of the Chlamydomonas PRPs S2, S3, and S5 and Predicted Locations of Their Extra Domains on the 30S Subunit, Based on T. thermophilus 30S Subunit Structure. The three-dimensional structure of the T. thermophilus 30S subunit (Wimberly et al., 2000) was represented using the Cn3D program provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). (A) Front view showing all of the 30S proteins and 16S rRNA. (B) Side view showing only the S2, S3, and S5 proteins and 16S rRNA. (C) Bottom view showing only the S2, S3, and S5 proteins and 16S rRNA. Probable localizations of the S2 NTE, S3 INS, and S5 NTE in the Chlamydomonas 30S PRPs are indicated by dotted circles. The N termini of T. thermophilus S2 and S5 proteins, and a loop region of the S3 protein (where the Chlamydomonas S2 NTE, S3 INS, and S5 NTE are connected in the Chlamydomonas counterpart), are exposed on the solvent side of the subunit (arrows).