Mapping ultrastructural effects of proteotoxic stress on S. cerevisiae

Abstract

Cells are the basic building blocks of any living being, whether the organism consists of only one, or many cells. The different cellular components interact with one another to maintain the viability of the cell. An essential element of cells are proteins – peptide chains folded into a specific conformation unique to every protein. The structure of the protein is important for its function. Proteins execute a range of tasks across the cell, but can misfold during stress, which can lead to inactivity or even toxicity. Stressors, such as heat, are often used to induce proteotoxic stress and simulate certain aspects of ageing-related disease. Upon exposure to stress, genetic programmes, as well as biophysical and biochemical effects contribute to change and adaptation of the cell. This has far-reaching consequences on the cell, which is reflected in numerous ways. The articles discussed in this thesis examine the effect of different stres- sors, primarily heat-stress on the budding yeast (Saccharomyces cerevisiae) and other organisms. The articles reveal that heat-stress leads to large changes in organellar conformation. In addition, vacuolar pH as well as contact sites with the nucleus increase. Aggregates of misfolded proteins are sequestered in the cytoplasm and within organelles, to then be re-folded correctly or be degraded. Our results indicate that there are differences in aggregation and clearance of proteins, even when they are mixed in the same aggregate. We have also observed new structures, such as electron-translucent clusters that form near the plasma membrane, however their contents and role in the heat-shock response are still undetermined. An unexpected result was that virus-like particles, that contain viral capsid proteins but no viral nucleic acids, become less clustered as heat-shock progresses. In two of the papers we focus on material accumulating in the nuclear envelope, which then causes the outer nuclear membrane to bud out toward the cytoplasm. We show that it occurs under normal physiological conditions, but also more frequently in several stress conditions which all cause the increase of misfolded proteins in the cell. The results indicate that budding of the nuclear envelope is used for protein transport between the nucleus and the cytoplasm. The last paper is a review that summarises scattered publications of observations of nuclear envelope budding from the last 70 odd years and points out gaps of knowledge in this novel research field. Overall, this thesis creates a wide, yet detailed overview of how the cell responds to stress, specifically addressing its proteotoxic aspect. This will contribute to the understanding of cellular mechanisms in ageing-related disease.