A conserved acidic residue at position 540 is important for Ire1 function

Sequence alignments utilizing bioinformatics tools revealed the existence of an acidic residue at the transition of Ire1’s juxta-membrane AH and its TMH. The acidic character of the glutamate residue at position 540 in yeast Ire1 is conserved from yeast to man. In Ire1 from H. sapiens, an aspartate residue can be found at the equivalent position, as indicated by sequence alignments of various fungal and mammal Ire1 species (Fig. 28).

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Figure 28 | The negative charge of E540 in Ire1 is conserved among species.

(A) HeliQuest analysis of Ire1 orthologs from mammals and yeast strains highlight the conservation of the negative residue in the hydrophilic phase of the AH. (B) Sequence alignment of Ire1's AH and TMH from indicated species. The sequences of the AH is indicated in blue, while the predicted TMH is shown in black. Negatively charged residues localized at a similar localization as the E540 in S. cerevisiae are highlighted in red.

To investigate the role of E540 for Ire1 function, it was substituted by a variety of other amino acids and the sensitivity to ER-stress of cells expressing these mutants was investigated using DTT as stressor (Fig. 29). Cells expressing the E540A mutant was markedly more sensitive to DTT compared to the wild type. Neither the substitution of E540 to glutamine (Q), which structurally rather similar to glutamate (E), nor a mutation to a positively charged lysine (K) rescued this growth phenotype. Substituting E540 by aspartate (D) resulted in a wild type-like growth phenotype, suggesting that the E540D variant of Ire1 is functional. Strikingly, the structurally related asparagine (N) residue at the position of E540 did not rescue the previously described growth defect. Of note, the protein levels of all mutants were comparable to wild type Ire1 levels. These data highlight that a conserved, negatively charged residue at the position E540 is required for normal UPR activation.

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Figure 29 | Substitution of the negative charge at position 540 leads to a functional defect in Ire1.

(A – E) Growth assay of Ire13xHA-GFP, mutations of the glutamate at position 540 to alanine (A, turquoise), glutamine (Q, blue), aspartate (D, purple), asparagine (N, orange) or lysine (K, green) and an IRE1 strain. Cells were cultivated in minimal medium (SCD) supplemented with DTT at 30°C for 18 h and the density of the culture was determined using the OD620. The error bars represent the mean ± SEM of n = 2 biological replicates. (F) Lysates from the indicated, exponentially growing cells were immunoblotted using anti-HA antibodies for comparing the level of Ire13xHA-GFP wild type and its mutant variants. An immunoblot with anti-Pgk1 antibody served as loading control. (G) Quantification of protein levels shown in (F) were normalized to Ire1 wild type and Pgk1. The error bars represent the mean ± SEM of 3 independent experiments.

In order to investigate the impact of the E540A mutation on Ire1 function in the context of different kinds of stress more thoroughly, the potential of this mutant to form high-order oligomers was investigated by confocal fluorescence microscopy on live cells. The formation of Ire1 clusters was induced either by the addition of the ER-stressor DTT to the cultivation medium or by inositol depletion (Fig. 30).

Ire1 clusters were identified and counted using an automated Fiji script and revealed that the occurrence of punctate structures was reduced to ≈ 60% for the E540A mutant compared to the wildtype. This defect in cluster formation was comparable to the defect observed for the F531R and V535R mutants that disrupt the amphipathic character of the ER (Fig. 26).

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Figure 30 | The E540A mutation affects the formation of Ire1 clusters in ER-stress conditions.

Live cell confocal microscopy of yeast strains expressing Ire13xHA-GFP wild type or the E540A mutant during acute ER stress. Cells were treated with DTT (1.5h, 2 mM, SCD media) or cultured in inositol-depleted medium (3h). Quantification of high and low intensity clusters of Ire1 based on a semi-automated script. The error bars represent the mean ± SEM. n = 3 for DTT treated cells, n = 4 for wild type inositol depletion, n = 3 for E540A in inositol depletion experiments. A minimum of 40 cells was analyzed for each replicate. Statistical significance was tested by an unpaired, two-tailed student’s t-test. ***p<0.001, **p<0.01. Scale bar

= 5 µm.

Not surprisingly, unconventional splicing of HAC1 mRNA in response to both types of ER stress was impaired for the E540A mutant compared to the wildtype (Fig. 31). Consequently, also the induction of the UPR target gene PDI1 was reduced in this mutant compared to the wild type version. These data, together with indications of a functional defect by increased sensitivity to DTT stress in the growth assay (Fig. 29) and reduced formation of Ire1 clusters (Fig. 30) highlight the functional relevance of the E540 in Ire1.

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Figure 31 | HAC1 splicing and upregulation of PDI1 mRNA levels is reduced in the E540A mutant.

Levels of spliced HAC1 mRNA (A) and PDI1 mRNA (B). The indicated cells were stressed by a treatment with DTT (1h, 4 mM, YPD media) or inositol depletion (3h), respectively, and analyzed by qRT-PCR. The data were normalized in (A) to the HAC1 mRNA level in stressed wild type cells and in (B) to the steady-state level of the PDI1 mRNA in unstressed cells. The data were normalized as in Fig. 27. All graphs show means ± SEM for three independent experiments, and statistical significance was tested by an unpaired, two-tailed student’s t-test. **p<0.01, *p<0.05.

Intriguingly, the glutamate residue at position 540 is located at the interface between Ire1’s AH and its TMH. Mutation of the R537 residue, which is located only one helical turn away from the conserved glutamate 540, to alanine (R537A) or glutamate (R537E) did not result in an increased sensitivity of yeast to DTT, indicating the lack of a functional role of this residue in Ire1 (Halbleib et al., 2017).

5.4. Functionally impaired AH mutant variants are capable to