Protein misfolding and temperature up-shift cause G ₁ arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Abstract

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2͞4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3-2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3-1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…Temperature-dependent cell cycle inhibition can be mimicked by treatment with low concentrations of the imino acid analog azetidine-2-carboxylic acid (AZC), a compound that causes the misfolding of nascent polypeptides via a substitution for the amino acid proline. AZC is toxic at high concentrations, but at low concentrations (10 mM or lower), it causes both G 1 arrest and the repression of CLN1 and CLN2 (466). These data suggest that G 1 arrest is not necessarily a direct physiological phenomenon but rather a signaled event.…”

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

“…We predicted that constitutive HSR activation resulting in increased HSP abundance should protect against high levels of protein misfolding. To test this hypothesis, we challenged cells with AZC, a proline analog that incorporates into nascent chains causing protein misfolding (41,64,65). As shown in Fig.…”

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality

“…With additional heat treatment vitality was not significantly lower. The light heat shock for 3 h to 35°C was meant to initiate a heat stress response (11,34) and thus expected to produce a more significant loss of vitality. Storage in ethanol affected yeast vitality to a greater extent.…”