Genetic factors that regulate the attenuation of the general stress response of yeast

Abstract

The general stress response of yeast involves the induction of approximately 200 genes in response to any one of several stresses. These genes are activated by Msn2 and repressed by the Srb10 kinase, a member of the mediator complex. Normally, Msn2 is exported from the nucleus, and Srb10 represses STRE gene expression. Under stress, Msn2 relocalizes to the nucleus and, with the relief of Srb10 repression, activates transcription. The stress response is rapid, but quickly attenuated. We show here that this attenuation is due to a nuclear-dependent degradation of Msn2. Msn2 rapidly disappeared from cells after heat or osmotic shock. This disappearance was not due to a change in MSN2 RNA levels, which remain constant during stress. Pulse-chase experiments confirmed the stress-dependent Msn2 degradation. The levels of Msn2 were significantly reduced in msn5 deletion cells that have been shown to constitutively retain Msn2 in the nucleus. The degradation was Srb10-dependent; Msn2 was not degraded in an srb10 deletion mutant. An Msn2 internal deletion mutant was insensitive to Srb10 repression, but was degraded by the Srb10-dependent mechanism. Thus, this mutation uncoupled Srb10 repression from degradation.

Figure 1.—

The induction and attenuation of the STRE gene RNAs in response to heat shock and osmotic shock. Cell cultures of RZ53-6 were grown to midexponential phase in SC medium and then subjected to either heat (A) or osmotic (C) shock as described in materials and methods. Samples were taken at the times designated above the lanes, RNA was prepared, and RNA blots were carried out. The hybridization probes used are listed in materials and methods. The RNA for each gene is indicated at the right of the blot. Because ALD3 and ACT1 and CYC7 and DDR2 RNAs are similar in size, blots were first hybridized with the MSN2, ACT1, and CYC7 probes and then stripped and hybridized with the ALD3 and DDR2 probes. The hybridization bands were visualized and quantitated using a Molecular Dynamics (Sunnyvale, CA) Storm 850, and the analyses are shown in B for heat-shock induction and in D for osmotic-shock induction. The levels of RNA for each STRE gene were normalized to the ACT1 RNA level for each time point and then divided by the peak expression level for that RNA. This allowed the best visualization of the induction and attenuation patterns and placed less emphasis on the barely detectable levels of RNA present at time zero.

Figure 2.—

The disappearance of Msn2 upon heat shock and osmotic shock. Cell cultures of RZ53-6 transformed with either YCp(33)MSN2HA (A and C) or YCp(33)c-MSN2 (B) were grown to midexponential phase in SC minus uracil medium and then subjected to either heat (A and B) or osmotic (C) shock as described in materials and methods. Samples were taken at the times designated above the lanes and prepared for immunoblots as described in materials and methods. The lane designated C represents a sample taken from cells transformed with YCplac33, a plasmid lacking the epitope-tagged MSN2 allele. The immunoblots were first probed with anti-HA (A and C) or anti-c-myc (B) antiserum and then stripped and reprobed with eIF5A antiserum. The visualized bands are identified to the right of each blot. For the graph in D, the blots were quantitated by scanning the X-ray films and analyzing the band densities using the Molecular Dynamics ImageQuant 5.0 program. The Msn2 protein levels were normalized to eIF5A for each time point and then divided by the time zero value.

Figure 3.—

The differential degradation of ³⁵S-Msn2-HA under nonstress and heat shock. (A) MZ171-60 cells transformed with YEp(195)MSN2HA were subjected to heat shock for the designated times, and the protein was subjected to immunoblot as described in materials and methods. The immunoblots were first probed with anti-HA antibody and then stripped and reprobed with eIF5A antiserum. (B) MZ171-60 cells transformed with YEp(195)MSN2HA were labeled with [³⁵S]methionine for 5 min, the label was chased, and growth was continued under nonstress (25°) and heat-shock (37°) conditions as described in materials and methods. Total soluble protein from each time point and the control (C) were size fractionated by SDS polyacrylamide gel electrophoresis. (C) The Msn2-HA in the cleared lysate samples from B was subjected to immunoprecipitation with anti-HA antibody (time points) or anti-c-myc 9E10 antibody (C, control) as described in materials and methods. The proteins in the immunoprecipitates were size fractionated by SDS polyacrylamide gel electrophoresis. The volumes of each sample loaded were normalized to the total trichloroacetic acid insoluble radioactivity in each cleared lysate. (D) The radioactivity in Msn2 in C was quantitated using a PhosphoImager. The values were normalized to radioactivity in total protein and then normalized to time zero.

Figure 4.—

The effect of msn5Δ and srb10Δ mutations on Msn2 degradation. The yeast strains MZ161-8A (msn2Δ), MZ161-39B (msn2Δ msn5Δ), MZ161-41 (msn2Δsrb10Δ), and MZ161-8D (msn2Δ msn5Δsrb10Δ) were each transformed with YCp(33)MSN2HA. Cell cultures were grown on SC minus uracil medium to midexponential phase, and cell extracts were prepared for immunoblots as described in materials and methods. For each blot, the C lane contained a sample prepared from MZ161-8A cells transformed with the YCplac33 vector. Each immunoblot was probed sequentially with anti-HA and anti-eIF5A antisera. (A) Extracts were prepared from nonstressed cultures of the above cells. The numbers below the lanes represent the fraction of mutant to wild type of Msn2-HA protein after normalization to eIF5A for four experiments. (B) MZ161-39B transformants were subjected to heat shock, and samples were taken at the indicated times. (C) MZ161-41 transformants were subjected to heat shock, and samples were taken at the indicated times.

Figure 5.—

The effect of deletions of MSN2MSN4, MSN5, and SRB10 on heat-shock induction of CYC7-lacZ expression. The MZ171 series of yeast strains all contained the msn2Δmsn4Δ double deletions, an integrated CYC7-lacZ fusion, and the additional relevant markers: MZ171-60 (none), MZ171-48 (srb10Δ), MZ171-15 (msn5Δ), and MZ171-75 (msn5Δsrb10Δ). Each strain was transformed with YCp(33)MSN2HA (A) or YCpMSN2ΔE (B). MZ171-60 was also transformed with the vector YCplac33. Cell cultures were grown to midexponential phase in SC minus uracil medium and then subjected to heat shock as described inmaterials and methods. Samples were taken at various times after stress, and cellular levels of β-galactosidase were determined. The strain designations are as above with MZ171-60 YCplac33 transformants labeled msn2 msn4 and YCp(33)MSN2HA (A) and YCp(33)MSN2ΔE (B) transformants labeled WT. (○) WT; (⋄) msn5; (□) srb10; (♦) srb10 msn5; (•) msn2 msn4.

Figure 6.—

The induction and attenuation of STRE gene RNA in an srb10Δ mutant. Cell cultures of MZ161-41 transformed with YCp(33)MSN2HA were grown to midexponential phase in SC minus uracil medium and then subjected to heat shock as described in materials and methods. Samples were taken at the times designated above the lanes, RNA was prepared, and RNA blots were carried out. The hybridization probes used are listed in materials and methods. The RNA for each gene is indicated at the right of the blot. The hybridization bands were visualized (A) and quantitated (B) as described for Figure 1. The analyses shown in B represent the averages for two independent blots. The plot in C represents a replotting of the data in B (black lines) with that in Figure 1B (gray lines) on a log scale for the y-axis.

Figure 7.—

The effect of the internal ΔE deletion on Msn2 degradation. The yeast strains MZ161-8A (msn2Δ; A) and MZ161-41 (msn2Δsrb10Δ; B) were each transformed with YCp(33)MSN2ΔE. In addition, MZ161-8A was transformed with YCplac33 (for lanes marked C) or YCp(33)MSN2HA (for lanes marked WT). Cell cultures were grown on SC minus uracil medium to midexponential phase, and cell extracts were prepared for immunoblots as described in materials and methods. Each immunoblot was probed sequentially with anti-HA and anti-eIF5A antisera. The positions of the full-length Msn2HA and the ΔE deletion mutant are shown to the right of the blot.