Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

2Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER.3 Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a 4 major quality control mechanism. However, the degree to which ER-phagy is employed by other 5 branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, 6 that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. 7C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif 8 (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with 9 the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The 10 C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation 11 of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation 12 mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that 13 bridges selective autophagy with ribosome-associated quality control at the ER. 14 15 65 stress, we performed an immunoprecipitation coupled to mass spectrometry (IP-MS) screen to identify 66 AIM-dependent ATG8 interactions triggered by ER stress. We hypothesized that a synthetic AIM peptide 67 that has higher affinity for ATG8 can outcompete, and thus reveal, AIM-dependent ATG8 interactors. To 68 identify this synthetic peptide, we performed a peptide array analysis that revealed the AIM wt peptide 69 (Figure S1a, b; Table S1). Using isothermal titration calorimetry (ITC), we showed that the AIM wt binds 70 4 ATG8 with nanomolar affinity (KD=~700 nM), in contrast to the AIM mutant peptide (AIM mut), which 71 does not show any binding ( Figure S1c-f) or the low micromolar-range affinities measured for most cargo 72 receptors (Zaffagnini and Martens, 2016). As plants have an expanded set of ATG8 proteins, we first tested 73 if any of the ATG8 isoforms specifically responded to ER stress induced by tunicamycin (Kellner et al., 74 2016). Tunicamycin inhibits glycosylation and leads to proteotoxic stress at the ER (Bernales et al., 2006). 75 Quantification of ATG8 puncta in transgenic seedlings expressing GFP-ATG8A-I revealed that 76 tunicamycin treatment significantly induced all nine ATG8 isoforms (Figure S2). Since all ATG8 isoforms 77 were induced, we chose ATG8A, and performed peptide competition coupled IP-MS analysis (See methods 78 for detailed description). In addition to well-known AIM dependent ATG8 interactors such as ATG4 79 (Autophagy related gene 4) and NBR1 (Neighbour of BRCA1) (Wild et al., 2014), our analyses revealed 80 that the highly conserved cytosolic protein C53 (aliases: CDK5RAP3, LZAP, IC53, HSF-27) is an AIM-81 dependent ATG8 interactor (Figure 1a, Table S2, Figure S3). 83To confirm our IP-MS results, we performed in vitro pull-down experiments. Arabidopsis thaliana (At) 84 C53 specifically interacted with GST-ATG8A, and this interac...

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Dagdas (2020): C53 is a crosskingdom conserved reticulophagy receptor that bridges the gap betweenselective autophagy and ribosome stalling at the endoplasmic reticulum, Autophagy,

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53mediated autophagy activation.

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