The C-terminal region of yeast ubiquitin-protein ligase Not4 mediates its cellular localization and stress response

Abstract

Transient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at cytoplasmic foci after stress. This truncated version of Not4 fails to increase the transcription of the stress genes, is more stable and seems to be toxic to cells undergoing oxidative stress.

Keywords: E3 ubiquitin ligase; gene expression; protein aggregation; stress response; yeast.

Figure 1.

Not4 C-terminus analysis. (A) Schematic representation of truncated Not4 proteins used in this study. (B) Complementation analysis by dilution spot assay of not4 strains expressing the entire NOT4-GFP gene fusion (Not4) and its truncated forms (Not4Δ1, Δ2 and Δ3). Wild type strain (BY4741) and Pug35 (vector) were used as a control. (C) Protein extracts from not4 mutant cells expressing GFP (lane 1) and the Not4–GFP fusion proteins (lanes 2–5) were separated on SDS-acrylamyde gel and probed with an anti-GFP antibody. Additional bands of lower intensity and faster migration might represent degradation products of the fusion proteins. (D) Protein stability. Not4Δ cells transformed with the entire and the truncated forms of Not4 fused to GFP were grown exponentially. After blocking translation with cycloheximide (CHX), proteins were extracted at the indicated time, separated on SDS–PAGE and probed with anti-GFP and anti-tubulin antibodies. Ponceau red staining of membranes is also shown for quantization of transferred proteins. In the bottom part is reported the relative quantity of the Not4-GFP proteins compared to tubulin.

Figure 2.

Protein localization (A) Not4 localization in cells not treated (NT) and after 15′ treatment with 3 mM hydrogen peroxide (H₂O₂), 7% ethanol (EtOH) and after 10′ of glucose deprivation. (B) Percentage of not4Δ cells transformed with the NOT4-GFP constructs showing foci under the indicated conditions. (C) Not4 localization in cells expressing the Not4-GFP fusion integrated into the chromosome under the native promoter after 10′ of glucose deprivation (-Glu), 15′ treatment with 7% ethanol (EtOH) or 3 mM hydrogen peroxide (H₂O₂). NT: not treated.

Figure 3.

Effect of the expression of Not4 truncated forms on viability and stress resistance. Viability of the not4Δ strain (A) and BY4741 (B) transformed with NOT4-GFP fusions and its truncated forms was measured after exposure of cells to H₂O₂ at the indicated concentration for 4 h. The BY4741/not4Δ strain expressing the pUG35 vector was used as a control. Average and standard deviations, obtained from three independent experiments, are indicated. (C) Viability of transformants during aging. Viability of BY4741 transformed with Not4 and Not4Δ3 GFP fusions was measured during exponential (day 1) and stationary phase (day 3). BY4741 strain expressing the pUG35 (Vector) was used as a control. Average and standard deviations, obtained from three independent experiments, are indicated. (D) The same strains as in A were tested for acute heat shock stress. Viability is expressed as the fraction of viable cells compared to the corresponding untreated cells (28°C) set to 1.

Figure 4.

Gene expression in yeast cells expressing the entire Not4 protein and its truncated forms Not4Δ1, Not4Δ2 and Not4Δ3 after oxidative stress. Total RNA was isolated before and after 40′ incubation in 0.4 mM H₂O₂ from not4Δ cells expressing the indicated forms of Not4 and reverse-transcribed to cDNA. Real time PCR was used to analyze the expression levels of SOD1, SOD2, MSN2, MSN4 and YAP1 using the specific primers indicated in Table 1. The expression of the TDH3 gene was used as an internal control for normalization of the real time PCR data and mRNA relative quantity are expressed each vs the own untreated strain cells (values are expressed as Log₂). Vector: not4Δ strain expressing pUG35 plasmid. Data represent the mean of three independent experiments.