Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques

Abstract

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

FIG. 1.

Relative ratios obtained using isobaric tags are reproducible in a double duplex labeling experiment. (A) Representative ribosomal profile trace of E. coli MG1655 grown at 16°C. Fractions representing 30S and 70S ribosomal particles (shown in boxes) were pooled and labeled with isobaric tags 114/116 and 115/117, respectively. (B) Relative distribution of peptides quantified with isobaric tags. Shown are the distributions of the peptide ratios for isobaric tags 114/116 (duplicate 30S), 115/117 (duplicate 70S), and (114 + 116)/(115 + 117) (30S/70S). For each plot, the mean and standard deviation (SD) are given. (C and D) The relative levels of small ribosomal proteins (C) and key associated proteins (D) are shown, expressed as the average ratio of proteins in 30S/70S particles plus standard error of the mean (error bars).

FIG. 2.

iTRAQ analysis of ribosomal particles isolated from E. coli MG1655 cells grown at 37°C. (A) Representative polysome trace of E. coli MG1655 grown at 37°C. Fractions representing polysomes and 30S, 50S, and 70S ribosomal particles (shown in boxes) were pooled and labeled with isobaric tags 114, 115, 116, and 117, respectively. (B) The relative ratios (30S/polysome, [black], 50S/polysome [dark gray], and 70S/polysome [light gray]) of select proteins are shown. Error bars show standard errors of the means.

FIG. 3.

iTRAQ analysis of ribosomal particles isolated from E. coli MG1655 cells grown at 16°C. (A) Representative ribosomal profile trace of E. coli MG1655 grown at 16°C. Fractions representing 30S, 50S, and 70S ribosomal particles (shown in boxes) were pooled and labeled with isobaric tags 114, 116, and 117, respectively. Tag 115 was left blank. (B and C) Ratios of small ribosomal proteins (30S/70S) (B) and large ribosomal proteins (50S/70S) (C) are shown. (D) The ratios (30S/70S [black] or 50S/70S [dark gray]) of certain proteins are given. Error bars show standard errors of the means.

FIG. 4.

Ribosome association of select E. coli proteins. Extracts from cells expressing the indicated tagged proteins were separated by sucrose density centrifugation, and fractions were probed with relevant antibodies. The proteins were tagged with the SPA or M2 tag. The positions of the 30S, 50S, 70S, and polysome fractions, as determined by absorbance at 254 nm, are shown. The leftmost lane is a loading control (1/100 of the extract loaded).

FIG. 5.

Deletion of potential ribosome-associated proteins results in cold sensitivity in E. coli. The isogenic wild-type BW25113 and deletion strains of nonessential candidate proteins were grown to saturation, diluted into fresh medium to an OD₆₀₀ of ∼0.1, and grown for 2 or 3 generations. Equivalent serial dilutions for each strain were plated onto LB and grown overnight at 37°C or for 3 days at 18°C.

FIG. 6.

Polysome profiles of select potential ribosome assembly factor mutants. Extracts from isogenic wild-type BW25113 and select mutants were separated by sucrose density centrifugation, and the positions of the 30S, 50S, 70S, and polysome fractions were monitored by UV absorbance (254 nm). (A) Polysome profiles of the wild type (BW25113) and ΔnudH, ΔrluB, and ΔyhbY mutants. (B) The average relative peak height ratios of 30S/50S, 30S/70S, and 50S/70S (black, dark gray, and light gray, respectively) from the indicated strains are shown. Error bars show standard errors of the means.