Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomycescerevisiae

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 (Msn2/4-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.

Figure 1

AZC stops proliferation via incorporation into protein and may act by the same mechanism as mild heat shock. (A) JVG718 (EG123 WT) was streaked on YPD plates or on YPD plates containing 7 mM AZC. Plates were incubated at either 28°C or 37°C for 3 days. (B) GAP510 (WT) and a congenic ubc4Δ.ubc5Δ mutant (GAP512) were streaked on YPD plates or on YPD plates containing AZC (3 mM). Plates were incubated at 23°C for 3 days.

Figure 2

AZC treatment causes G₁ arrest and repression of CLN1 and CLN2. (A) A midlogarithmic culture of JVG961 (S288C WT) growing in YPD at 30°C was treated with AZC (10 mM) at 0 h. Samples were stained with propidium iodide and analyzed by flow cytometry. Cells with unduplicated DNA or with duplicated DNA are indicated by the 1N and 2N labels, respectively. (B) A midlogarithmic culture of JVG961 (S288C WT) growing in YPD at 30°C was treated with AZC (10 mM) at 0 min. Total RNA was prepared from samples taken at the times indicated. Samples (50 μg RNA) were subjected to Northern analysis. Blots were probed for CLN1, CLN2, CLN3, and ACT1.

Figure 3

AZC treatment limits G₁ cyclin activity. (A) Strains JVG961 (WT) and JVG1052 (cln1Δ.cln2Δ) were streaked on YPD plates containing AZC (7 mM) or no AZC and incubated at 30°C for 3 days. (B) Strains JVG961 (WT), JVG1022 (cdc28–1), JVG1023 (cdc28–9), and JVG1041 (cdc28–1N) were streaked on plates containing AZC (5 mM) or no AZC and were incubated at 23°C for 4 days. (C) Midlogarithmic cultures of JVG961 (S288C WT) transformants containing pCLN3–2 (pJO96) or a vector control (YCp50) growing in SD-URA at 23°C or 30°C were subjected to a temperature up-shift (TU: 23°C to 37°C) or were treated with AZC (10 mM, 30°C), respectively. The percentage of unbudded cells was determined for each culture as a function of time after treatment was initiated.

Figure 4

The EXA3–1 mutation in the gene encoding HSF prevents G₁ arrest upon both temperature up-shift and AZC treatment. Midlogarithmic cultures of DS10 (WT) and the congenic EXA3–1 mutant (MH297) growing in YPD at 23°C were subjected to a temperature up-shift (TU: 23°C to 37°C). Midlogarithmic cultures of the same strains growing in YPD at 30°C were treated with AZC (50 mM) at t = 0 without a temperature change. The percentage of unbudded cells was determined for each culture as a function of time after treatment was initiated.

Figure 5

Model for cellular response to temperature up-shift and AZC treatment. AZC misfolds cellular protein into which it is incorporated. Misfolded protein specifically activates HSF, causing induction of HSE-driven transcripts and (by unknown mechanisms) subsequent repression of the ribosomal protein (rp) genes and G₁ arrest. Temperature up-shift causes protein misfolding and thereby triggers the HSF-dependent events. The thermal trigger for the STRE regulon is unknown (?) but is independent of protein misfolding. The Pkc1 pathway is activated by thermal stress to the cell surface independently of misfolded proteins.