Continuous sensation and reaction to environmental fluctuations is especially critical to the survival of unicellular organisms. Stress response mechanisms are essential for cells during the vegetative and sexual life cycles and quiescence. The Schizosaccharomyces pombe mitotic activator and stress response serine/threonine kinase Ssp1 acts independent of the major fission yeast Spc1 MAP kinase stress response cascade. Ssp1 is required at high temperatures in the presence of other stressors, ensures long-term viability in quiescent cells and allows efficient cell division in low-glucose conditions. Ssp1 is cytoplasmic but briefly localizes to the cell membrane after exposure to extracellular stress. It plays a role in actin depolymerization and is required for the change of growth polarity after cell division. After identifying 14-3-3 proteins Rad24 and Rad25 as putative Ssp1 binding partners, we confirmed the interaction with co-immunoprecipitation. Association of Ssp1 with Rad24 diminishes after 15 minutes of hyperosmotic stress, however Rad25 binding is retained. Loss of the rad24 gene product rescues both ssp1- mitotic delay at elevated temperatures and sensitivity to 0. 6M KCl. Conversely, overexpression of rad24 exacerbates ssp1 stress sensitivity and mitotic delay. Diffuse actin polarity and spheroid morphology in rad24- cells improves in an ssp1- background. Ssp1 localization to the cell membrane is negatively regulated by Rad24. Ssp1 does not co-localize with Arp3C (actin-related protein 3 homologue C) after osmotic stress, but instead appears to form a ring around the cell, suggesting localization to fission scars. Ssp1 is basally phosphorylated and hyperphosphorylated after glucose deprivation. Ssp1 is shuttled in and out of the nucleus and accumulates in the nucleus in an exportin Cmr1 dependent manner. Ssp1-GFP levels are constant in all stages of the vegetative cell cycle and Ssp1-GFP is present in both the sexual life cycle and quiescence. C-terminal and N-terminal truncation of ssp1 alters its subcellular localization. The C-terminal region is the site of hyperphosphorylation following glucose deprivation and is also necessary for membrane localization following osmotic stress. / Thesis (Ph.D, Biology) -- Queen's University, 2012-01-24 09:49:58.225

Cells grow in environments that can change suddenly. To cope with unpredictable perturbations, they have evolved mechanisms to adjust their metabolism according to the various types of environmental stress. Cells experiencing starvation, for example, have low energy levels and are forced to lower their metabolism and enter a protective quiescent state to survive until nutrients become available again. Recently, it has been shown that starved yeast cells experience a marked acidification of the cytoplasm, due to a passive influx of protons. This pH drop causes multiple rearrangements in the cytoplasm: increased crowding, reduced mobility of intracellular components and formation of stress-induced non-membrane bound compartments of specific metabolic enzymes. Cytoplasm rearrangements are required for cell survival and can be reversed upon replenishment of energy. However, there is little understanding of how cytoplasmic components reorganize in stressed quiescent cells. Using high-pressure freezing, correlative light and electron microscopy (CLEM) and electron tomography, coupled to high-resolution 3D-reconstruction techniques, I investigate the structural modi cations that happen in situ in yeast cells undergoing quiescence. I observe that the cytoplasm becomes increasingly crowded, due to a massive rearrangement of membranous structures, including accumulation of intracellular vesicles and pronounced invaginations in the plasma membrane. This is proved by quantification of the difference in ribosome densities between stressed and not stressed cells. The increased crowding, coupled to cytoplasm acidification, leads to the formation of non-membrane bound enzyme compartments, that appear as foci and elongated structures of fluorescently tagged enzymes. I prove that the fluorescent structures correspond to bundles of filaments. Among many essential enzymes, known to form mesoscale structure in stressed yeast, I demonstrate that the eukaryotic translation initiation factor 2B (eIF2B) forms bundles of filaments in situ, and the evolutionary conserved glutamine synthetase (Gln1) self-assembles into filaments in vitro. The present study on the energy depleted cytoplasm and the structural analysis of filament-forming enzymes provides insights into an unexplored survival strategy that is used by yeast, as well as other organisms, to cope with extreme environmental conditions and stress.:1 Introduction 1 Stress, survival and quiescence 2 1.1 Cytoplasm and cellular compartments 2 1.2 Membraneless compartmentalization in the cell 3 1.3 Stress-induced non-membrane bound assemblies 4 The quiescent sleeping yeast 6 1.4 The yeast S.cerevisiae as model organism 7 1.5 Growth and metabolism of yeast 9 1.5.1 Yeast eukaryotic translation initiation factor 2B: eIF2B 11 1.5.2 Yeast glutamine synthetase: Gln1 12 3D electron microscopy 14 Aims of the Thesis 18 2 Materials and methods 21 Room temperature electron microscopy (EM) 21 2.1 Yeast strains, media and energy depletion 21 2.2 High-pressure freezing of yeast cells 22 2.2.1 EM sample preparation for untagged eIF2B yeast strains 22 2.2.2 EM sample preparation for GFP-tagged eIF2B yeast strains 23 2.3 Electron tomography 23 2.4 Subtomogram averaging 24 2.5 Fiji script for automated ribosome counting 25 2.6 Immunofluorescence of eIF2B in yeast 26 2.7 Western-blot on yeast ribosomes 27 Single particle procedures 30 2.8 Protein purification protocols 30 2.8.1 Baculovirus-insect cell expression and purification of eIF2B 30 2.8.2 Gradient of fixation for fragile complexes 31 2.8.3 Yeast expression and purification of Gln1 33 2.9 Negative staining 34 2.9.1 Image acquisition and analysis—eIF2B 35 2.9.2 Image acquisition and analysis—Gln1 36 Cryo-electronmicroscopy(cryo-EM) 37 2.10 Plunge freezing 37 2.11 Image acquisition and 3D reconstruction 37 3 Results Visualizing yeast’s cytoplasmic reorganization 39 3.1 Quiescence is accompanied by reorganization of the cytoplasm 40 3.2 Ribosome density proves cytoplasmic crowding in starved cells 42 3.3 eIF2B organizes in bundles of filaments in energy-depleted cells 45 3.4 eIF2B filaments are polymers of the eIF2B complex 47 3.5 Filaments are found in wild-type energy-depleted cells 49 Structural analysis of filament forming enzymes 51 3.6 Purification of eIF2B complexes 51 3.7 Single particle analysis of eIF2B 53 3.8 Purification of Gln1 complexes 55 3.9 Single particle analysis of Gln1 56 3.10 Gln1 forms filaments in vitro 58 4 Discussion and Outlook 59 4.1 Yeast cytoplasm reorganizes in response of stress 59 4.2 Ribosomes density is a measure of increased macromolecular crowding 60 4.3 eIF2B forms filaments as a survival strategy 62 4.4 Molecular analysis of filament forming enzymes 64 4.5 Outlook 65 Appendix 67 Bibliography 83

Department of Food Science and Technology / MSCPNT

The presented work was focused on the comparison between the production of selected metabolites by carotenogenic yeasts and microalgae cultivated under conditions of external stress. The main metabolites of interest were carotenoids, further lipophilic substances and lipids. Biotechnological overproduction of these metabolites could serve as a source of potentially beneficial substances not only for the pharmaceutical, cosmetic and food industries, but also for the production of third generation biofuels. Recently, there has been a growing interest in biofuels primarily from microalgae, which have a high potential in biofuel production and seem to be a promising source. The theoretical part deals with the description of individual genera of carotenogenic yeasts, microalgae, cyanobacteria, chemical composition of produced metabolites and brief biosynthesis. In addition, individual methods for analyzing the production of the metabolites of interest were described. The experimental part is focused on the comparison of production of carotenoids, coenzyme Q, ergosterols (phytosterols) and lipids by yeasts, microalgae and cyanobacteria. As a source of external stress, temperature, salt and light stress were chosen. The strains of Rhodotorula glutinis, Rhodotorula mucilaginosa, Sporidiobolus pararoseus and Cystofilobasidium macerans were studied from the yeast strains. Microalgae and cyanobacteria were Scenedesmus obliqus, Scenedesmus dimorphus, Chlorella sorokiniana, Chlorella saccharophila, Botryococcus brauni, Synechococcus nidulans and Arthrospira maxima. The yeast and algal strains were optimized for growth, carotenoid and lipid production. Applied salt stress showed a significant liquidation effect on algal and cyanobacterial strains. The thesis also monitored the biological stress, so-called co-cultivation of microalgae and yeasts. Further experiments will be the subject of future work.