Proteome wide analysis of granular protein complexes evoked by differential heat stress stringency in Saccharomyces cerevisiae

SILVIA MORENO DE COLONNA; CARLA BARRAZA; MARIA PIA VALACCO; JORGE GERMAN FERNANDEZ; PAULA PORTELA

Potsdam

Congreso; Annual meeting of the German Society for Proteome Research-Proteomic Forum 2017; 2017

German Society for Proteome Research

Cellular responses to stress comprise a variety of different mechanisms, including translation arrest and relocation of the translationallyrepressed mRNAs to ribonucleic particles (mRNPs) such as stress granules (SGs) and processing bodies (PBs). Given recent evidence on therole of liquid phase transition in signalling and cytosolic mRNPs formation, it is possible that SGs might represent regions where certainprocesses or the activity of enzymes are concentrated. Our results from biochemical and microscopy approaches show that under mild heatstress the catalytic subunit of PKA isoform Tpk3 aggregates and promotes transfer of closed mRNA loop complexes into SGs, whereas severeheat stress leads to the formation of PBs and SGs that also contain the Tpk2 isoform and a larger 48S translation initiation complex (1,2 andunpublished). PKA affects the translational response to heat stress, where each Tpk catalytic isoform appears to have a different role, with Tpk2and Tpk3 playing negative and positive roles in the translational response, respectively. We favour a model where depending on the severity ofan external stimulus, such as heat stress, each catalytic isoform of PKA interacts with a complex network of distinct protein factors and potentialsubstrates.Our ongoing studies are focused on a global characterization of protein complexes under different heat stress conditions. To this end, weperformed a proteomic analysis of the granular enriched fraction from mild and severe heat stress by two approaches: I) Differential 2D gelelectrophoresis and mass spectrometry identification by MALDI-TOF-TOF and II) Label-free quantitative proteomics using a QExactive. Weidentified proteins differentially enriched in mild and severe heat stress, as well as proteins common to both groups. Gene ontology analysis ofthe enriched proteins showed annotations associated with several GO biological processes, such as structural constituent of ribosome, RNAbinding, translation factor activity, ATPase activity and unfolded protein binding. Networks built from these results will let us start defining howdifferent degrees of severity of the same stress evoke a specific response on RNP assembly.1- Eur J Cell Biol. 2010 89:339-482- J Cell Sci. 2012 125:5221-32