Abstract

1. A B C D Samples Environmental Cues Alter the Protein Composition of Yeast RibosomesParimal Samir1, Christopher M. Browne1, Andrew J. Link1,21Department of Biochemistry, 2Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232 USA Grow Cells with Glucose at 30 0C (3 replicates) Grow Cells with Glucose at 37 0C (3 replicates) Grow Cells with Glycerol at 37 0C (3 replicates) Grow Cells with Glycerol at 30 0C (3 replicates) Protein extraction followed by trypsin digestion Purify Ribosomes Purify Ribosomes Purify Ribosomes Purify Ribosomes Spectrum to Peptide Mapping using Proteome Discoverer iTRAQ Reporter Ion Quantitation using ProteoIQ Statistical Analysis using ProteoIQ iTRAQ label Pool Ribosomes (use as control) iTRAQ 115 iTRAQ 116 iTRAQ 117 iTRAQ 114 Pool Results Results Introduction and Hypothesis Abstract Clustering Analysis of Quantified Proteins Growth Curves for WTand Null Mutants of Control (Δrpl8a andΔrpl8b) and Changing Ribosomal Genes (Δrpl9a andΔrpl9b) Quantitation of Proteins in Purified Ribosomes and Whole Cell Extracts using iTRAQ Intensity Experimental Plan Analysis by LC-MS • Ribosomes are ribonucleoprotein complexes that catalytically drive the process of translation. The currently accepted paradigm suggests that ribosomes are just catalytic engines and have no regulatory functions. Recent studies have challenged this view due to evidence in favor of regulatory function of ribosomes. In this study we tested the following hypothesis. • Heterogeneous ribosomal sub-populations exist in equilibrium in a given growth condition. The equilibrium is shifted towards a new state upon environmental cues. • Modulation of the equilibrium between the sub-populations through dynamic changes in ribosomal protein composition or posttranslational modifications is a means to regulate gene expression. Fold Change (log2) of Ribosomal Proteins in Purified Ribosomes Fold Change (log2) of Ribosomal Proteins in Whole Cell Extracts The ribosome is the catalytic engine that drives translation. It has been assumed to have limited regulatory function or mRNA specificity. Recent studies have suggested that there are, in fact, classes of ribosomes that differ in their ribosomal protein (RP) composition. We hypothesized that sub-populations of ribosomes exist in a state of dynamic equilibrium. This equilibrium would shift towards a new state upon environmental or growth cues. We reasoned that the equilibrium of ribosome populations would be reflected in the RP composition of the purified ribosomes. To test this hypothesis, we quantitated the RP composition of S. cerevisiae in both purified ribosomes and whole cell extracts (WCE) grown in either glucose or glycerol and at either 30 0C or 37 0C. Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) labeling followed by mass spectrometry was used to precisely quantify the RPs. We identified five RPs that are differentially present in ribosomes depending on the carbon source. Interestingly, these RPs have paralogs. Our data suggest that changing the equilibrium of ribosome populations may serve as a translational control mechanism. Overlap Between the Identified Proteins In Three Experiments with Purified Ribosomes Purified Ribosomes Whole Cell Extracts • Cluster analysis in Cluster 3.0 • Hierachical • Complete Linkage • Visualized using Java TreeView m/Z Glycerol@30C Glucose@37C Glycerol@37C Proteins up in growth at higher temperature Proteins up in growth in Gycerol Overlap Between the Identified Proteins in Three Experiments with Whole Cell Extract Classification of Ribosomal Proteins Based Upon iTRAQ Fold Change Glycerol@30C Glucose@37C Glycerol@37C The Equilibrium Composition of Ribosomes in Yeast Grown in Glucose is Different Compared to the Cells Grown in Glycerol iTRAQ Overview MS/MS Spectrum of iTRAQ Labeled Peptide for Rpl9A Reporter Ion Spectrum Summary • The composition of ribosome changes in response to environmental cues. • The changing composition may be an important means of regulating gene expression. Sample Workflow MS/MS Spectrum of iTRAQ Labeled Peptide for Rpp1A Reporter Ion Spectrum Future Directions • Time course experiment to study the kinetics of ribosomal composition changes. • iTRAQ based quantitative proteomics. • Identify proteins whose translation is affected by individual ribosomal gene deletion. • Null mutants, iTRAQ labeling and ribosome profiling. Data Analysis Pipeline