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2nSILAC for the study of mito gene deletion effects

ABSTRACT: Knowledge about the functions of individual proteins on a systems-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker's yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker's yeast using heavy arginine and lysine under fermentable and respiratory growth conditions making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Friedel Drepper

LAB HEAD: Prof. Dr. Bettina Warscheid

PROVIDER: PXD010019 | Pride | 2020-01-24

REPOSITORIES: Pride

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Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies. We examined 50 single-gene loss-of-function strains, comprised of gene deletions (del) and temperature-sensitive (ts) or tetracycline promoter-shutoff (tet) alleles. These genes were selected based on their known or potential role in nucleosome biology and include remodeler ATPases and chaperones, histones and histone modifiers, transcription and elongation factors, and components of RNA polymerase I and II. The compendium also includes 4 compounds targeting transcription and histone deacetylases, as well as a histone depletion time course performed with a strain in which H4 gene expression is exclusively under the control of a GAL1 promoter. Genome-wide nucleosome occupancy profiles were generated using Affymetrix Tiling arrays with probes spaced every 4 bp [PMID:16569694], or next-generation sequencing. Identically prepared samples for each strain and treatment were analyzed on the same tiling arrays for strand-specific expression differences. Each compendium condition was compared to a matched wild-type (WT) reference grown in parallel.

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2nSILAC for the study of mito gene deletion effects

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