Genome-wide single-cell-level screen for protein abundance and localization changes in response to DNA damage in S. cerevisiae

Abstract

An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.

Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveal chaperone-dependent regulation of ribonucleotide reductase

“…These conditions for MMS treatment were chosen because of their previous use in several proteomic studies examining the global response of cells to DNA damage [1, 2]. This provided us with a way to compare and contrast our data with those already obtained.…”

“…Studies of the global response of cells to a variety of DNA damaging agents have revealed dramatic changes in post-translational modification, sub-cellular localization, expression and degradation of key effector proteins that play a critical role in the DNA damage response (DDR). Indeed, one such study in budding yeast observed 14% of proteins changed localization or abundance in response to DNA damage agents [1, 2]. These and other studies have established a paradigm where DNA damage induces rapid accumulation and modification of DDR proteins that are critical for checkpoint arrest and DNA repair, such as p53.…”

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…glucose) with 500 μg mL -1 G418 to exponential phase (OD 600 = 1.0) and fixed in paraformaldehyde solution as described previously (Mazumder et al, 2013). Nucleic acid was dyed with 4',6-diamidino-2-phenylindole (DAPI).…”

Heritability and genetic basis of protein level variation in an outbred population

“…or a tandem affinity purification tag (Ghaemmaghami et al 2003), ORF-GFP or ORF-TAP, respectively. Previous work on these collections has established the baseline level (Ghaemmaghami et al 2003), localization , sources of individual variation (Newman et al 2006), and response to perturbations (Tkach et al 2012;Breker et al 2013;Denervaud et al 2013;Mazumder et al 2013) for individual fusion proteins. Despite the availability of these resources, the effect of genotype on protein abundance has not been interrogated on a genome scale (Parts 2014).…”