Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl

Abstract

Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.

Figure 1. Schematic overview of the experimental approach.

Yeast cells were grown in media containing either normal (“light”) or labeled (“heavy”) amino acids. Cells were lysed and “light” and “heavy” 80S ribosomes were collected by sucrose gradient centrifugation. After incubation of “light” 80S ribosomes with different concentrations of LiCl, core and supernatant fractions were separated by centrifugation and mixed with ”heavy” ribosomes. Ribosomal proteins were hydrolysed with Lys-C or with Lys-C + trypsin and peptides were separated by HPLC. “Heavy” to “light” ratio of peptides was determined by MS/MS for each protein.

Figure 2. Splitting of ribosomal proteins from yeast ribosomes by LiCl.

“Light” yeast 80S ribosomes were incubated with 0 M, 0.5 M, 1 M or 2 M LiCl, whereafter core and supernatant fractions were separated by centrifugation and “heavy” ribosomes were added to each fraction. Ribosomal proteins were precipitated with TCA, hydrolysed with Lys-C or with Lys-C + trypsin and peptides were separated by HPLC. “Heavy” to “light” ratio of peptides was determined by MS/MS for each protein. Proteins are grouped according to their amount in the core fraction at 0.5 M LiCl. Group A (blue) – 75–100%, B (red) – 48–75%, C (green) – 22–48% and D (black) – 0–22% in the core.

Figure 3. Modeling of split and core proteins in the 3D structure of yeast ribosome subunits.

The structures were generated by PyMol using co-ordinates from (A) 40S subunit, interface view; (B) 40S subunit, solvent side view; (C) 60S subunit, interface view; (D) 60S subunit, solvent side view. rRNA is shown in gray. Proteins are grouped according to their amount in the core fraction at 0.5 M LiCl. Group A (blue) – 75–100%, B (red) – 48–75%, C (green) – 22–48% and D (black) – 0–22% in the core.