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

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.

Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen1

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10−3 s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azz measurements can provide an estimate of the optimum storage conditions.

Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.

The localization, trafficking, and fluorescence of Aequorea green fluorescent protein (GFP) in cultured vertebrate cells transiently transfected with GFP cDNA were studied. Fluorescence of GFP in UV light was found to be strongest when cells were incubated at 30 degrees C but was barely visible at an incubation temperature of 37 degrees C. COS-1 cells, primary chicken embryonic retina cells, and carp epithelial cells were fluorescently labeled under these conditions. GFP was distributed uniformly throughout the cytoplasm and nucleus independent of cell type examined. When GFP was fused to PML protooncogene product, fluorescence was detected in a unique nuclear organelle pattern indistinguishable from that of PML protein, showing the potential use of GFP as a fluorescent tag. To analyze both function and intracellular trafficking of proteins fused to GFP, a GFP-human glucocorticoid receptor fusion construct was prepared. The GFP-human glucocorticoid receptor efficiently transactivated the mouse mammary tumor virus promoter in response to dexamethasone at 30 degrees C but not at 37 degrees C, indicating that temperature is important, even for function of the GFP fusion protein. The dexamethasone-induced translocation of GFP-human glucocorticoid receptor from cytoplasm to nucleus was complete within 15 min; the translocation could be monitored in a single living cell in real time.