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S1 Table. Strains used in this study.

S2 Table. Primers used in this study.

S3 Table. Raw numerical data of all the figures.

S2 Fig. EDEM-1 and EDEM-2 are required for CPL-1* degradation (A) Fluorescence intensities of WT animals carrying the CPL-1* transgene treated with indicated concentrations of kifunensine. Values in scatter gram represent mean fluorescence/µm2 x 1000 in arbitrary units (AU). The red bars indicate the average ±SEM. ****P<0.0001; one way ANOVA with Dunnett post test. (B) Immunoblot analysis of CPL-1* degradation in edem-1 and edem-2 mutants carrying the rescuing EDEM-1::mCherry and EDEM-2::mCherry transgenes, respectively. Total protein lysates derived from edem-1 and edem-2 mutants carrying the CPL-1* and rescuing transgenes were separated by SDS-PAGE and immunoblotted with anti-GFP polyclonal antisera. Tubulin was used as loading control. (C) Histogram showing the densitometry values of the bands presented in (B) normalized to the value of WT condition (n=3 ± SEM, one way ANOVA with Fisher test), *P<0.05; **P<0.01).

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival., [Author summary] ER stress and UPRER malfunctions have been implicated in the pathogenesis of neurodegeneration, metabolic and inflammatory diseases as well as tumor progression and diabetes, whereby disturbed ER homeostasis negatively influences the pathology of the disease. Under ER stress conditions, the cells either activate UPRER-dependent cytoprotective mechanisms when ER stress is at subtoxic levels or, in case of an excessive ER stress, the cytotoxic response stimulates cell death. Here, we used Caenorhabditis elegans to study the cellular responses to ER stress at organismal level. We show that EDEMs respond differently to ER stress stimuli, and moreover, EDEMs deficiencies activate an XBP-1 independent adaptive program to promote organism survival under acute ER stress. Corroborated with the fact that loss of EDEM-2 and EDEM-3 induces resistance to acute ER stress in an intact organism, our data implicate EDEM proteins in a broader response to ER stress than previously established, which opens a new avenue for understanding the regulation of ER stress with implications for clinical and therapeutic investigations.

Leucine-rich-repeats (LRRs) belong to an archaic procaryal protein architecture that is widely involved in protein–protein interactions. In eukaryotes, LRR domains developed into key recognition modules in many innate immune receptor classes. Due to the high sequence variability imposed by recognition specificity, precise repeat delineation is often difficult especially in plant NOD-like Receptors (NLRs) notorious for showing far larger irregularities. To address this problem, we introduce here LRRpredictor, a method based on an ensemble of estimators designed to better identify LRR motifs in general but particularly adapted for handling more irregular LRR environments, thus allowing to compensate for the scarcity of structural data on NLR proteins. The extrapolation capacity tested on a set of annotated LRR domains from six immune receptor classes shows the ability of LRRpredictor to recover all previously defined specific motif consensuses and to extend the LRR motif coverage over annotated LRR domains. This analysis confirms the increased variability of LRR motifs in plant and vertebrate NLRs when compared to extracellular receptors, consistent with previous studies. Hence, LRRpredictor is able to provide novel insights into the diversification of LRR domains and a robust support for structure-informed analyses of LRRs in immune receptor functioning.