A dynamic model of proteome changes reveals new roles for transcript alteration in yeast

Lee, M Violet; Topper, Scott; Hubler, Shane L.; Hose, James; Wenger, Craig D.; Coon, Joshua J.; Gasch, Audrey P.

doi:10.1038/msb.2011.48

Cited by 270 publications

(362 citation statements)

References 69 publications

(120 reference statements)

Supporting

Mentioning

315

Contrasting

Unclassified

Order By: Relevance

“…2B, orange line). A similar result was recently observed for ∼2,500 proteins measured from bulk populations by mass spectrometry in response to osmotic shock (17). At steady state, protein abundance is determined by synthesis, degradation, and dilution rates.…”

Section: Significancesupporting

confidence: 58%

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud

Becker

Delgado-Gonzalo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

124

151

View full text Add to dashboard Cite

Observing cellular responses to perturbations is central to generating and testing hypotheses in biology. We developed a massively parallel microchemostat array capable of growing and observing 1,152 yeast-GFP strains on the single-cell level with 20 min time resolution. We measured protein abundance and localization changes in 4,085 GFP-tagged strains in response to methyl methanesulfonate and analyzed 576 GFP strains in five additional conditions for a total of more than 10,000 unique experiments, providing a systematic view of the yeast proteome in flux. We observed that processing bodies formed rapidly and synchronously in response to UV irradiation, and in conjunction with 506 deletion-GFP strains, identified four gene disruptions leading to abnormal ribonucleotide-diphosphate reductase (Rnr4) localization. Our microchemostat platform enables the large-scale interrogation of proteomes in flux and permits the concurrent observation of protein abundance, localization, cell size, and growth parameters on the single-cell level for thousands of microbial cultures in one experiment.O bserving proteins in the cellular milieu has been a longstanding technical challenge in biology. One major advance was the development of GFP, enabling the visualization of proteins in vivo (1). High-content imaging has been primarily applied to mammalian cells, using either reverse transfection arrays or microtiter-based systems in which the slow doubling time of mammalian cells enables long-term imaging under static conditions (2, 3).The Saccharomyces cerevisiae GFP fusion library covering 4,159 proteins provided the first static view of global protein abundance, localization, and noise (4, 5). This library was recently used to establish the static differences in protein abundance and localization in response to DNA replication stress induced by methyl methanesulfonate (MMS) and hydroxyurea (HU) (6), in response to DTT, H 2 O 2 , and nitrogen starvation (7), and 800 cytoplasmic proteins were analyzed upon entry into stationary phase (8). These three recent large-scale screens all relied on standard microtiter plates for imaging the yeast strains at a single time point before and after perturbation. Meanwhile, microfluidic devices emerged as powerful tools for conducting complex time-lapse experiments on small to medium scales (9, 10), enabling the analysis of cellular network responses (11) and the implementation of synthetically engineered systems (12). However, it has thus far been technically impossible to interrogate thousands of continuously growing microbial strains with high spatiotemporal resolution in a single experiment.Despite the fact that a wealth of systems-level information is available for S. cerevisiae, the single-cell temporal dynamics of protein abundance and localization has not yet been measured on a systems scale. To enable such analyses we developed a microfluidic platform capable of growing and observing 1,152 yeast strains with a temporal resolution of 20 min. We explored the dynamic behavior of ∼2/3 of the...

show abstract

Section: Significancesupporting

confidence: 58%

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud

Becker

Delgado-Gonzalo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

124

151

View full text Add to dashboard Cite

show abstract

“…Use of TMT proteomics to study microbial communities Prior proteomics studies using TMT-or isobaric tags for relative and absolute quantification-based isobaric chemical labeling have been applied exclusively to human tissues, plants and cultured isolates (for example, Dayon et al, 2010;Lee et al, 2011;Li et al, 2012;Westman et al, 2012;Chen et al, 2013;Paulo et al, 2013;Li et al, 2014). Here, we validate the TMT-based quantitative proteomics approach as applied to microbial communities, showing precise quantification of thousands of proteins across a large range of fold changes.…”

Section: Discussionmentioning

confidence: 84%

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

et al. 2014

View full text Add to dashboard Cite

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 1C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.

show abstract

“…However, research in various organisms has also revealed that protein dynamics is often partly decoupled from RNA expression levels (e.g. recent data in (1)(2)(3)(4)(5)(6)). In Arabidopsis, especially diurnal transcript level fluctuations were not matched by corresponding changes in the detected proteome (7).…”

mentioning

confidence: 99%

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

Svozil

Hirsch‐Hoffmann

Dudler

et al. 2014

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra. Molecular & Cellular

show abstract

A dynamic model of proteome changes reveals new roles for transcript alteration in yeast

Abstract: By characterizing dynamic changes in yeast protein abundance following osmotic shock, this study shows that the correlation between protein and mRNA differs for transcripts that increase versus decrease in abundance, and reveals physiological reasons for these differences.

Cited by 270 publications

References 69 publications

A chemostat array enables the spatio-temporal analysis of the yeast proteome

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

Contact Info

Product

Resources

About