The unfolded protein response supports cellular robustness as a broad-spectrum compensatory pathway

Abstract

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the unfolded protein response (UPR), is responsible for maintaining endoplasmic reticulum (ER) homeostasis. The highly conserved Ire1 branch regulates hundreds of gene targets by activating a UPR-specific transcription factor. To understand how the UPR manages ER stress, a unique genetic approach was applied to reveal how the system corrects disequilibria. The data show that the UPR can address a wide range of dysfunctions that are otherwise lethal if not for its intervention. Transcriptional profiling of stress-alleviated cells shows that the program can be modulated, not just in signal amplitude, but also through differential target gene expression depending on the stress. The breadth of the functions mitigated by the UPR further supports its role as a major mechanism maintaining systems robustness.

Fig. 1.

Δlhs1, Δscj1, and Δalg5 single deletion mutants exhibit mild phenotypes. (A) CPY and Gas1p biosynthesis was examined in wild-type (WT) and Δlhs1 strains by pulse-chase analysis. Positions of the nontranslocated, ER, Golgi, and mature forms of CPY are indicated by pre-CPY, p1, p2, and m, respectively. (B) The degradation of ERAD substrate, CPY*-HA, was monitored in WT and Δscj1 cells by a pulse-chase experiment. The graph shown is the mean ± SD of three independent experiments. (C) CPY biogenesis was examined in WT and Δalg5 strains as described in A. The underglycosylated ER/Golgi forms are indicated with ● and ○, and the underglycosylated mature CPY species are labeled −1 and −2. Treatment with Endoglycosidase H (Endo H) was performed after immunoprecipitation for the relevant samples. (Right) Immature CPY was expressed as a percentage of total CPY and is indicated below each section.

Fig. 2.

The UPR is required for viability of LHS1-, SCJ1-, and ALG5-deficient cells. (A) UPR induction was measured for the indicated cells using a β-galactosidase reporter assay. Data shown are the mean ± SD of three independent experiments. (B) The strains were grown at 23 °C and serial dilutions of the culture were spotted onto plates. These plates were incubated at the indicated temperature until the appearance of colonies.

Fig. 3.

Maintaining the UPR resting state reveals severe defects in LHS1-, SCJ1-, and ALG5-deficient strains. (A) CPY and Gas1p biogenesis was analyzed in lhs1-1Δire1 and sec63-1 strains at 23 °C and 37 °C as described in Fig. 1A. (B) Pulse-chase analysis was performed at 37 °C to examine the degradation of CPY*-HA in WT, Δire1, scj1-1, and scj1-1Δire1 cells. The graph represents the mean ± SD of three independent experiments. (C) The biosynthesis of CPY was monitored in alg5-2Δire1 as described in Fig. 1C. rft1-2 cells were included to indicate positions of underglycosylated mCPY, which are denoted as “−1”, “−2”, “−3”, and “−4”, representing triply-, doubly-, singly-, and nonglycosylated species, respectively (11). “Mx” denotes the portion of the gel composed of p1, p2, and mCPY forms that are not easily differentiated. The other labels are described as in Fig. 1C. The immature form of CPY after Endo H digestion was expressed as a percentage of the total and is indicated.

Fig. 4.

The constitutive UPR activator HAC1ⁱ alleviates defects in the ts strains. (A) Cells with or without HAC1ⁱ-bearing plasmid were grown at 23 °C and serial dilutions of the culture were spotted onto duplicate selective SC plates. These plates were incubated at the indicated temperature until the appearance of colonies. (B) The synthesis of CPY and Gas1p was examined in lhs1-1Δire1 containing a HAC1ⁱ-bearing plasmid as described in Fig. 1A. (C) The degradation of CPY*-HA was analyzed in scj1-1, scj1-1Δire1, and scj1-1Δire1 with a HAC1ⁱ-containing plasmid at 37 °C. The graph was obtained from the mean ± SD of three independent experiments. (D) CPY biogenesis was monitored in Δalg5, alg5-2Δire1, alg5-2Δire1, and rft1-2 carrying HAC1ⁱ-bearing plasmid at 37 °C and labeled as described in Figs. 1C and 3C. The percentage of immature CPY compared to the total is shown below each section.

Fig. 5.

Up-regulated genes by the UPR in Δalg5, Δlhs1, and Δscj1 differ from genes by chemically induced UPR. Shown is a heat map of DNA microarray data for three biological replicates of WT+D (WT cells preincubated 1 h with 2 mM DTT before RNA extraction), Δalg5, Δlhs1, and Δscj1 cells. (Right) Inset assigns heat map colors to the gene expression fold change (FC) compared with WT cells on a log₂ scale. The genes shown were selected from previously reported UPR-regulated genes (8).