Structural and genetic variability of the proteostasis network in Saccharomyces cerevisiae.

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Homeostasis of the cellular ensemble of proteins (proteostasis) is a basic requirement for fitness. Sudden or progressive decline of proteostasis can be pathologic but is also commonly observed in aging. We have not identified the exact causes of this deterioration nor do we understand its link to the accumulation of toxic protein species in disease. Proteostasis decline in aging and disease depends on events within the cellular network of mechanisms that affect proteostasis. Moreover, individual aging trajectories likely depend on combined effects of multiple genetic variants on this network.Proteome-wide measurements are rapidly changing the paradigms of how we study complex traits such as proteostasis. Here, we propose to apply modern mass spectrometry-based methods that are able to detect protein functional state changes on a proteome-wide scale to study the robustness of the model organism Saccharomyces cerevisiae to proteotoxic stress caused by heat. Application of a recently developed structural proteomics method based on limited proteolysis (“LiP-MS”) will allow us to study proteostasis mechanisms and heat stress robustness as a network by simultaneous monitoring of multiple cellular processes. We aim to detemine the genetic foundation of robustness to heat stress based on a panel of genetically different yeast strains. Phenotypic measurements will be combined with information on the individual genetic, transcriptional and proteomic make-up. These data will allow us to relate individual genetic determinants of heat stress robustness to the functional state of regulatory modules. By means of induction, we will chart the network of mechanisms that underlies heat stress robustness, whereas mechanistic hypotheses will be deduced from network information and molecular data.In addition to the investigation of inter-individual differences in thermotolerance traits, we will also determine the influence of prior stress experience. Yeast can acquire thermotolerance following mild stress pre-treatment. We will assess the overlap of changes due to experience with inter-individual determinants of heat stress robustness. Furthermore, we will study the genetic requirements for the establishment of heat stress memory.In sum, this study will take a network-oriented approach combined with encompassing proteomics methods to elucidate basic processes underlying failure and defense of cellular proteostasis. In-depth investigation of mechanistic hypotheses deduced from proteome-wide measurements will be carried out using detailed molecular biology experiments. Thereby, the ability of our approach to identify inter-connected determinants of stress robustness will be demonstrated.