Quantitative proteomic analysis of ribosome assembly and turnover in vivo

Abstract

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.

Figure 1

Sucrose gradient ultracentrifugation profiles of cell lysate and gel electrophoresis of rRNA. (a) A dissociating sucrose gradient of log phase E. coli. Circles indicate fractions tested for rRNA by agarose gel electrophoresis. Shaded circles indicate the presence of either 16S or 23S rRNA. Clearly visible in the sucrose gradient trace are prominent peaks due to 30S and 50S subunits. Two small peaks are attributed to cell debris, appear in all gradients and do not contain any rRNA. The 16S rRNA is found only in the 30S peak fractions and the 23S rRNA is found only in the 50S peak fractions. (b) A dissociating sucrose gradient of E. coli treated with neomycin. Additional peaks appear in the sucrose gradient trace at 21S and 70S and the 30S peak broadens. Both 16S and 23S rRNA are found in earlier gradient fractions compared to the log phase unperturbed culture, and persist throughout. Fraction 10 was used as the reference fraction for scaling the 30S subunit protein levels and fraction 16 was used as the reference fraction for scaling the 50S subunit protein levels.

Figure 2

Scaled protein levels for the 30S and 50S subunits and assembly intermediates. (a) Protein levels from a single fraction from the 30S subunit peak (blue, Figure 1b fraction 11) and protein levels from a single fraction from the 21S intermediate peak (orange, Figure 1b fraction 6). A log scale is used for the y-axis to highlight the differences among protein levels in the 21S intermediate, which are less than ∼10% of the protein levels in the intact 30S subunits. Values are scaled to represent the occupancy relative to the amount of rRNA as described in Materials and Methods. Error bars represent the standard deviation of multiple measurements from different peptides or ions of the same protein. In the case where a single measurement was obtained, an open circle is used. (b) Protein levels from a single fraction of the 50S subunit peak (blue, Figure 1b fraction 17) and protein levels from a single fraction of the 30S peak, representing the pre-50S assembly intermediate (orange, Figure 1b fraction 12).

Figure 3

Changes in protein levels across different fractions in the sucrose gradient, and protein levels correlated with assembly maps. (a) Curves for representative proteins from the 30S subunit for each of the three defined groups, early binders (green, S15), moderate binders (orange, S7) and late binders (red, S3). The early binders start with a relatively high protein level and start to increase in protein level in earlier fractions compared to moderate or late binders. Error bars represent the standard deviation of multiple measurements from different peptides or ions of the same protein. In the case where only one measurement was obtained error bars are omitted. Fraction numbers correspond to Figure 1b. (b) A Nomura assembly map color-coded based on the three groups. Early binders are represented with a green background, moderate binders with an orange background and late binders with a red background. (c) Curves for representative proteins from the 50S subunit for each of the three defined groups, early binders (green, L4), moderate binders (orange, L5) and late binders (red, L35). (d) A Nierhaus assembly map color-coded based on the three groups. Early binders are represented with a green background, moderate binders with an orange background and late binders with a red background.

Figure 4

Fraction labeled for the 21S intermediate and 30S subunit. Shown are values for a single sucrose gradient fraction from the 30S peak (blue, Figure 1b fraction 11) and the 21S peak (orange, Figure 1b fraction 6). Fraction labeled values for the 21S intermediate are generally higher than for the 30S, but not 1.0 as expected. Error bars represent the standard deviation of multiple measurements from different peptides or ions of the same protein. In the case where only one measurement was obtained a circle is used.

Figure 5

Different ways to achieve the same bulk protein level. In these examples, consider the four hypothetical proteins SA (protein level 0.78), SB (0.56), SC (0.33) and SD (0.11). (a) Perfect correlation between proteins. SD depends on SC, which depends on SB, which depends on SA. The particles mimic a single assembly trajectory. (b) Imperfect correlation allows for the same bulk protein level but differences in composition at the molecular level. The particles mimic multiple parallel assembly trajectories.

Figure 6

Comparison of the neomycin-induced 21S assembly intermediate and RI. (a) Protein levels are normalized such that the value for S15 is 1.0 in both cases. Values for the neomycin 21S particle are in orange and values for RI are in green. The largest differences occur in the primary and secondary proteins of the 5’ domain (S4, S16, S17 and S20), which are present at relatively high levels in RI. (b) The same values plotted one against another. Data points are labeled by protein name and colored by domain (5’ domain in red, central domain in purple, 3’ domain in blue). Again proteins S4, S6, S17 and S20 are clearly visible as different between the two particles.

Figure 7

Possible origins of the unlabeled proteins from previously assembled 30S subunits appearing in the 21S assembly intermediate. (a) Schematic of the isotope-pulse experiment highlighting when the media is unlabeled and 50% ¹⁵N-labeled, and when the 21S intermediate begins to accumulate. Since the 21S particle only accumulates when the media is ¹⁵N-labeled, it is expected to be composed entirely of ¹⁵N-labeled proteins. (b) An altered schematic showing the observed result. Rather that finding 21S particles containing only 50% ¹⁵N-labeled proteins, 21S particles contain a mixture of unlabeled and 50% ¹⁵N-labeled proteins. There are several possible explanations for the presence of unlabeled proteins in the 21S particle. (c) Proteins could exchange between 30S subunits and 21S particles via the free pool of ribosomal proteins (RP Pool). (d) Previously existing unlabeled 30S subunits could degrade into 21S particles of the same size as the assembly intermediates observed. (e) Unlabeled 30S subunits could be completely dismantled into their component proteins, and these unlabeled proteins made available to assembling 21S intermediates.

Figure 8

Experimental design. (a) Schematic of a double-spike protein inventory experiment for quantifying protein levels. Neomycin is added to rapidly growing E. coli in 50% ¹⁵N-labeled media, particles are purified on a sucrose gradient and combined with both unlabeled and fully ¹⁵N-labeled ribosomes. (b) Schematic of an isotope-pulse labeling experiment to timestamp ribosomal proteins. After pulsing with ¹⁵N-labeled media, neomycin is added to rapidly growing E. coli, particles are purified on a sucrose gradient and combined with fully ¹⁵N-labeled ribosomes as an external standard. (c) The resulting isotope distribution for the double-spike experiment consists of three parts, unlabeled (the first standard, red), 50% ¹⁵N-labeled (the sample, green) and fully ¹⁵N-labeled (the second standard, blue). (d) The resulting isotope distribution for the isotope-pulse experiment consists of three parts, unlabeled (sample before the isotope pulse, red), 50% ¹⁵N-labeled (sample after the isotope pulse, green) and fully ¹⁵N-labeled (standard, blue).