Highlights d Tom70 supports the targeting of a wide range of precursor proteins to mitochondria d In vivo, the main function of Tom70 is to recruit chaperones to the outer membrane d Small inner membrane proteins are highly toxic in the absence of Tom70 d Tom70 protects the cytosol against toxic effects of mitochondrial precursors

Heat stress causes proteins to unfold and lose their function, jeopardizing essential cellular processes. To protect against heat and proteotoxic stress, cells mount a dedicated stress-protective programme, the so-called heat shock response (HSR). Our understanding of the mechanisms that regulate the HSR and their contributions to heat resistance and growth is incomplete. Here we employ CRISPRi/a to down- or upregulate protein kinases and transcription factors in S. cerevisiae. We measure gene functions by quantifying perturbation effects on HSR activity, thermotolerance, and cellular fitness at 23, 30 and 38°C. The integration of these phenotypes allowed us to identify core signalling pathways of heat adaptation and reveal novel functions for the high osmolarity glycerol, unfolded protein response and protein kinase A pathways in adjusting both thermotolerance and chaperone expression. We further provide evidence for unknown cross-talk of the HSR with the cell cycle-dependent kinase Cdc28, the primary regulator of cell cycle progression. Finally, we show that CRISPRi efficiency is temperature-dependent and that different phenotypes vary in their sensitivity to knock-down. In summary, our study quantifies regulatory gene functions in different aspects of heat adaptation and advances our understanding of how eukaryotic cells counteract proteotoxic and other heat-caused damage.

Background Baker’s yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity. Results Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate. Conclusion Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes.