Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy

  1. Lorenzo Picchianti
  2. Víctor Sánchez de Medina Hernández
  3. Ni Zhan
  4. Nicholas AT Irwin
  5. Roan Groh
  6. Madlen Stephani
  7. Harald Hornegger
  8. Rebecca Beveridge
  9. Justyna Sawa‐Makarska
  10. Thomas Lendl
  11. Nenad Grujic
  12. Christin Naumann
  13. Sascha Martens
  14. Thomas A Richards
  15. Tim Clausen
  16. Silvia Ramundo
  17. G Elif Karagöz
  18. Yasin Dagdas

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Abstract

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 C53‐mediated autophagy activation.

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  1. Version published to 10.15252/embj.2022112053
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Autophagy of the endoplasmic reticulum (ER-phagy) is a fundamental process that is essential for maintaining cellular homeostasis and quality control. We recently identified a novel mechanism regulating ER-phagy in both plants and animals that is based on the ubiquitin-like protein modifiers ATG8 and UFM1, and the ER-associated protein, C53. Here, we use a combination of evolutionary, biochemical, and physiological experiments to investigate the evolution and regulation of this process. We reveal the dynamic evolution of UFM1 and the ubiquity of C53-mediated autophagy across eukaryotes. Leveraging these results, we then identify an ancestral molecular toggle switch, mediated by shuffled ATG8-interacting motifs (sAIMs), that controls C53-mediated autophagy through competitive binding between UFM1 and ATG8. These findings provide new insights into the evolution of UFM1, reveal a conserved mechanism for the regulation of ER-phagy, and raise new and exciting hypotheses about the diversity and function of the UFMylation pathway. We believe that this work will be of interest to those studying autophagy and cellular stress response but will also serve as an interesting example of the benefits of combining evolutionary analyses with biochemical and cellular experiments.

    Our manuscript has been reviewed by three reviewers through ReviewCommons, whose comments, and our responses, can be found below. Two of the reviewers (Reviewer 1 and 3) were supportive of our work and its significance whereas Reviewer 2 questioned the novelty of our findings.

    Each of the reviewers’ comments can be addressed through a few supporting experiments as well as an improved manuscript which clarifies the novelty and significance of our results. While being supportive of our work, Reviewer 1 requested minor additional experiments to support our mechanistic conclusions and Reviewer 3 suggested that we expand our characterizations of C53 function to additional eukaryotic supergroups. These experiments are straightforward to perform, the materials and protocols to accomplish them are already established, and our overall conclusions are robust to the resulting outcomes.

    In contrast, Reviewer 2 did not suggest any additional experiments but rather challenged the novelty of our results as well as some of our interpretations. In particular, Reviewer 2 was uncertain of how our phylogenomic analyses built upon a previous study, published in 2014, which used comparative genomics to identify ubiquitin-related machinery across eukaryotes. Although it was an oversight to not reference this study (we cited a more recent article showing the same results), we were aware of their conclusions that UFMylation was present in the last eukaryotic common ancestor but absent in Fungi. We now clearly outline, both below and within the manuscript, our key phylogenomic results. These were acquired after implementing more advanced and comprehensive comparative genomic searches which allowed us to identify dynamic patterns in UFMylation evolution and permitted co-evolutionary analyses which were not only important for informing our experimental hypotheses but generated new functional questions. Our phylogenomic analyses are also linked to biochemical and physiological data, providing, for the first time, experimental support for our conclusions regarding UFMylation evolution. Similarly, Reviewer 2 suggested that our mechanistic results were an incremental extension of our previous work. Although our current work does of course build on our initial identification of C53-mediated autophagy, this manuscript provides novel insights into the importance and function of this process by revealing its ubiquity across eukaryotes and by characterizing the mechanistic details of its regulation. Ultimately, we disagree with Reviewer 2 but appreciate that this misunderstanding likely resulted from a lack of context and clarity in our manuscript which we have now resolved.

    As outlined in detail below, we will address the reviewers concerns through additional experiments, analyses, and improvements to the text.

    Thank you for considering our manuscript. We look forward to hearing from you.

    Description of the planned revisions

    We thank the reviewers for carefully evaluating our manuscript and for providing us with an opportunity to respond to their suggestions and criticisms. As you can see below in our pointby-point response, we address each of the points raised by the reviewers through the addition of supporting experiments, analyses, and an improved text. Altogether, we think these additional experiments and textual changes will significantly improve the manuscript. Therefore, we would like to thank all the reviewers and editors for their time and input.

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Picchianti et al. provide novel insights into the interaction of C53 with UFM1 and ATG8. Initially, the authors show that protein modification by UFM1 exists in the unicellular organism Chlamydomonas reinhardtii. To that end they demonstrated that pure Chlamydomonas UBA5, UFC1 and UFM1 proteins, can charge UFC1. Then, they showed that C53 interacts with ATG8 and UFM1. Specifically, they found that the sAIM are essential for the interaction with UFM1, while substituting this motif with canonical AIM prevents the binding of UFM1 but not of ATG8. Since binding of C53 to ATG8 recruits the autophagy machinery, the authors suggest that ufmylation of RPL26 releases UFM1 from C53 which allows the binding of ATG8. Overall, the authors demonstrate that C53 that forms a complex with UFL1 connects between protein ufmylation and autophagy by its ability to bind both UBLs. Here the authors revisited the assumption that only multicellular organisms have the UFM1 system. Using bioinformatic tools they show that it exists also in unicellular organism. Also, they show that in some organisms the E3 complex UFL1, UFBP1 and C53 exist but not UBA5, UFC1 or UFM1. This is a very interesting observation that suggests an additional role for this complex. In Fig 1C the authors show that in Chlamydomonas RPL26 undergoes ufmylation. Please use IP against RPL26 and then a blot with anti UFM1. From the current experiment it is not clear how the authors know that this is indeed RPL26 that undergoes ufmylation

    RPL26 is highly conserved across eukaryotes, so by comparing our western blots with previous studies (Walczak et. al., 2019, Wang et al. 2020), we concluded that these bands corresponded to UFMylated RPL26. However, we agree with the reviewer that we need to confirm the identify of RPL26 with additional assays. Since the submission of the manuscript, we tested RPL26 antibodies in Chlamydomonas and showed that they work well. So, we will update our figure with the confirmation westerns.

    In the second part of the manuscript the authors characterize the interaction of C53 with ATG8 and UFM1. This is a continuation of their previous published work (Stephani et al, 2020). Here the reviewer thinks that further data on the binding of these proteins to C53 is required. Specifically, defining the Kd of these interactions using ITC or other biophysical method can contribute to the study.

    We agree with the reviewer. To obtain the KD values, we will perform ITC experiments with C53 wild type, a C53 sAIM mutant and a C53 cAIM variant titrated with ATG8 and UFM1.

    Under normal condition the authors suggest that C53 binds UFM1 and this keeps it inactive. The reviewer thinks that this claim needs further support. Using IP (maybe with crosslinker) the author can show that C53, in normal conditions, bind more UFM1 than ATG8. Also, since the interaction of UFM1 to C53 is noncovalent, it will be nice to show how alternations in UFM1 expression levels can affect the activation of C53.

    We thank the reviewer for this suggestion. Since the submission of the manuscript, we have obtained UFM1 overexpression lines. We will pull on C53 using our C53 antibody and check for ATG8 levels in wild type and UFM1 overexpressing lines under normal and stress conditions. We think this will show how alterations in UFM1 levels can affect C53 activation.

    Finally, the authors suggest that ufmylation of RPL26 allows binding of ATG8 to C53 and this, in turn, leads to C53 activation. Can the authors show that in cells lacking UBA5, under normal condition or with Tunicamycin treatment, ATG8 does not activate C53 due to the fact that UFM1 does not leave C53.

    In Stephani et al., we showed that C53-mediated autophagy requires the UFMylation machinery. In ufl1 and ddrgk1 mutants, C53 becomes insensitive to ER stress. However, to supplement these results, we will perform autophagic flux assays using the native C53 antibody to test autophagic degradation of C53 in a uba5 and ufc1 mutant under normal and tunicamycin stress conditions. The uba5 mutant that we have is a knockdown, so that’s why we will include the ufc1 mutant in our experiments.

    Significance

    This manuscript advances our understanding of the connection of ufmylation to autophagy which is mediated by C53.

    Thank you!

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript from Picchianti et al. seeks to define the role of CDK5RAP3 (hereinafter referred as C53) during autophagy and its interplay with UFMylation. Together with UFL1 and DDRGK1, C53 is a component of a trimeric UFM1 E3 ligase complex that modifies the 60S ribosomal protein RPL26 at the endoplasmic reticulum (ER) surface upon ribosomal stalling (among other proposed functions that are not addressed). Several previous studies have implicated the UFMylation pathway in autophagy or ER-phagy although a non-autophagic fate for UFM1- tagged ribosomal subunits has also been reported. A previous study from the same authors (PMID: 32851973) identified an intrinsically disorder region (IDR) in C53 that is necessary and sufficient for interaction between C53 and autophagy receptor, ATG8. They reported that this IDR comprises four non canonical ATG8 interacting motifs (AIM), named shuffled AIMs (sAIMs) and showed that combinatorial mutagenesis of sAIM1, sAIM2, and sAIM3 abrogates ATG8 binding. A similar effect was observed for plant C53, though an additional canonical AIM (cAIM) in the C53 IDR had to be mutated to completely abolish C53 and ATG8 interaction. The earlier study reported that C53 IDR also interacts with UFM1, and this interaction can be disrupted in vitro by adding increasing concentration of ATG8, suggesting that ATG8 and UFM1 may compete with one another for C53 binding. The present paper attempts to build on this previous work by using phylogenomics to infer a coevolutionary relationship between UFMylation machinery and sAIMs in C53, which the authors argue, constitutes further evidence of the primary importance of a role for UFMylation in ER homeostasis. The manuscript includes a lot of biochemical data using variations of in vitro and in vivo pull-down experiments to define the roles of individual AIMs in mediating the binding of C53 to ATG8 and to UFM1. They also use NMR spectroscopy in an attempt to define the structural basis of the UFM1 and ATG8 binding to C53, concluding that plant C53 interacts with UFM1 mainly through sAIM1, while interaction with ATG8 requires cAIM as well as sAIM1 and sAIM2. Finally, the authors attempt to contextualize these findings by conducting studies on Arabidopsis mutants, showing that replacing sAIMs with cAIMs causes increases sensitivity to ER stress and apparently increases formation of C53 intracellular puncta that may colocalize with ATG8. From these data the authors concluded that the dual-ATG8 and UFM1 binding of C53 IDR regulates C53 recruitment to autophagosomes in response to ER stress. Major Issues:

    1. The phylogenomics analysis conclusion that UFM1 is common in unicellular lineages and did not evolve in multicellular eukaryotes is not novel, as another comprehensive analysis of UFM1 phylogeny, published eight years ago - in 2014 - by Grau-Bové et al. (PMID: 25525215), also reported that UFM1, UBA5, UFC1, UFL1 and UFSP2 were likely present in LECA and lost in Fungi. Although the phylogenomic analysis by Picchianti et al. is also extended to DDRGK1 and C53 proteins, and some parasitic and algal lineages, their findings are incremental. Their proposed coevolution of sAIM and UFM1 is based on presence-absence correlation observed within five species (i.e., Albugo candida, Albuco laibachii, Piromyces finnis, Neocallimastix californiae, Anaeromyces robustus). However, this coevolutionary relationship must be further investigated by substantially increasing the taxonomic sampling within the UFM1-lacking group.

    We were aware that previous studies had investigated the distribution of UFMylation proteins across eukaryotes and that these analyses had predicted the presence of UFMylation in LECA and subsequent loss in Fungi. We included a more recent citation noting this (Tsaban et al. 2021) but apologise for not citing Grau-Bové et al. (2014), which we have now included. We must emphasize that our results are not incremental. Although we had made a point of emphasizing the presence of UFM1 in LECA, this was to counter a recent and highly cited paper in the field which claimed that UFMylation evolved in plants and animals (Walczak et al. 2019). Below we note the novel and important results from our phylogenomic analyses:

    1. We used improved taxonomic sampling and more advanced comparative genomics methods to identify UFMylation components sensitively and specifically across eukaryotes. This involved the inclusion of additional eukaryotic genomes, phylogenetic annotation of orthologs, and genomic searches to complement proteome predictions. These methods are essential for accurately identifying UFMylation components and yield more robust results than using sequence similarity clustering (Tsaban et al. 2021) or un-curated Pfam HMMER search results (Grau-Bové et al. 2014).
    2. By placing our UFMylation reconstructions in a modern phylogenetic context we were not only able to support previous observations which noted the presence of UFM1 in LECA and its loss in Fungi (Grau-Bové et al. 2014) and Plasmodium (Tsaban et al. 2021), but also to identify novel patterns in the evolution of UFMylation. This included the observation of recurrent losses in diverse but trophically-related lineages (such as algae and parasites) and revealed the retention of certain UFMylation components in the absence of UFM1. We identified the frequent coretention of UFL1 and DDRGK1 following UFM1 loss in multiple eukaryotic groups, including Fungi, which were previously thought to be devoid of UFMylation machinery. These previously uncharacterized patterns, suggest that these proteins could have alternative functions and may be functionally associated with life history. These results therefore expand on and add complexity to our understanding of the evolution of UFMylation.
    3. By conducting a comprehensive and accurate survey of UFMylation components we were able to use our data to examine co-evolutionary trends between C53 and UFM1, which would have been incomplete and inaccurate using previously curated datasets. As the reviewer noted, only five species were identified that encoded C53 but lacked UFM1. This is not a reflection of insufficient taxon sampling, but rather the strong co-evolution between C53 and UFM1 (i.e., when UFM1 is lost, C53 is almost always lost as well). We attempted to identify additional cases by searching hundreds of fungal and oomycete genomes as well as those from other eukaryotes, but no other species were found. We agree with the reviewer that additional taxa would have made our analyses stronger, but importantly, we do not rely on genomic correlations to infer function. Rather, we use these correlations to generate functional hypotheses which we then tested experimentally. In this way, we do not rely on the strength of our correlations. We have now revised the manuscript to include additional context (including citations) and have improved the clarity of the text to better convey the novelty of our findings.
    1. The manuscript presents an overwhelming amount of biochemical and structural data obtained from a variety of protein binding techniques (i.e., NMR spectroscopy, in vitro GSTpulldown, fluorescence microscopy-based on-bead binding assays, and native massspectrometry). The results are poorly explained and not organized in a logical manner. Moreover, no attempt was made to explain the rationale behind using one technique over the other or how one method complements another to build a stronger conclusion than any individual approach. Given that none of the methods employed report quantitative measurement of binding affinities between C53 IDR and UFM1 or ATG8, it is not clear how the data presented in this manuscript contribute to our understanding of the proposed competition model for UFM1 and ATG8 binding to C53 IDR. To conclude that an interaction is "stronger" or "weaker" it is necessary to measure equilibrium binding constants. Fortunately, there are suitable techniques, including surface plasmon resonance (SPR), microscale thermophoresis (MST), fluorescence anisotropy, or calorimetry that are available to dissect these complex competitive binding interactions and to build models.

    We thank the reviewer for their suggestion. Although we attempted to describe the rationale behind each experiment (please see the line 135-137; on-bead binding assays, line154-157; NMR, 177-181), we agree that the volume of data and variety of techniques warrants additional explanation. We will revise the manuscript to further explain our rationale for using each of the different approaches. As we noted above in our response to reviewer 1, we will also perform relevant ITC binding assays to quantify the interaction between C53, ATG8, and UFM1.

    1. The NMR studies have the potential to dissect the types of dynamic binding inherent in unstructured proteins. However, the abundant NMR data presented combined with the aforementioned binding studies, remarkably, do not seem to significantly advance our understanding of how the system is organized or even how UFM1 and ATG8 bind C53, beyond the rather vague and somewhat circular conclusion stated in the abstract: "...we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound the sAIMs in a different mode." Or on line 165 "Altogether these results suggested that ATG8 and UFM1 bind the sAIMs withn C54 IDR, albeit in a different manner".

    We agree that NMR has the potential to dissect the complex binding interactions between UFM1, ATG8, and C53, but disagree with the reviewer’s interpretation that our NMR data fail to achieve this. To sum up, our NMR data:

    1. Revealed the structural basis of the interaction of C53-IDR with ATG8 and UFM1 at atomic resolution by showing that UFM1 binds preferentially to sAIM1 in the fast-intermediate exchange [Fig.4 and Fig. S7B], instead ATG8 binds cAIM in the slow-intermediate exchange, and once cAIM is occupied, it binds sAIM1,2 with lower affinity in the fast-intermediate exchange (Fig.4 and Fig.S7D).
    2. Determined conformational changes in C53 IDR upon binding of ATG8, but not UFM1 (Fig.S7E), which lead to increased dynamics in distinct regions in C53 IDR. These data could explain how binding of first ATG8 would trigger C53-dependent recruitment of the tripartite complex to autophagosomes.
    3. Identified how UFM1 binds to atypical hydrophobic patch in C53 sAIM, similar to what was shown for the UBA5 LIR/UFIM. To sum up, our results shed light on how both UBLs interact with C53, being sAIM1 the highest affinity binding site for UFM1 while ATG8 binds cAIM preferentially before occupying sAIM1,2. To provide more detailed information on the atomic details of the interaction between C53 and the UBLs, we will perform molecular docking studies by using the restraints obtained from the experimental NMR data.
    1. The functional assays performed in Arabidopsis do not support the competitive model between UFM1 and ATG8 for binding to C53 during C53-mediated autophagy. The fluorescence microscopy images do not provide convincing evidence of colocalization between C53 and ATG8. In fact, in contrast to the claims made in the text or the quantification, mCherry-C53 fluorescence does not seem to localize in discrete puncta and its signal does not seem to overlap with ATG8A.

    We disagree with the reviewer’s interpretation of these results although we acknowledge that there is some subtlety in interpreting the co-localization data. Importantly, Arabidopsis has 9 ATG8 isoforms and C53 can bind to most of them with varying affinities (see Stephani et al). Because of this, we do not expect C53 puncta to fully colocalize with ATG8A puncta. Additionally, the C53 puncta are smaller and more subtle than ATG8 puncta, which label the entire autophagosome. To reconcile this, we will quantify the effect by performing colocalization analyses under normal and stress conditions. We will also upload all the raw images as supporting material, so that anyone can independently assess our images.

    Minor Issues:

    1. The authors might choose to avoid teleological arguments such as (line 135): "As the phylogenomic analysis suggested that eh sAIMs have been retained to mediate C53-UFM1 interaction..."

    We thank the reviewer for this suggestion and will modify the text accordingly.

    1. The authors refer on multiple occasions to C53 "autoactivation" without defining what they mean by this. Do they propose that C53 UFMylates itself?.

    We refer to C53 activity as the ability to recruit the autophagy machinery and initiate cargo sequestration and degradation in the vacuole. We attempted to explain this in lines 57-61 but we will reword it more clearly, as suggested by the reviewer.

    1. The paper might want to avoid preachy philosophical statements like "Our evolutionary analysis also highlights why we should move beyond yeast and metazoans and instead consider the whole tree of life when using evolutionary arguments to guide biological research." (333- 335). While this is indeed a laudable goal, given the rather limited insights from this study, it is unclear how this paper exemplifies the notion.

    We added this statement as we were intrigued by our evolutionary analyses’ ability to link C53 to UFM1 (an association which took years to identify experimentally) and generate useful functional hypotheses about the interaction between C53 sAIMs and UFM1. As we mentioned above, we also wanted to highlight this point in reference to a recent prominent study in the field which drew conclusions after only considering animals, plants, and fungi (Walczak et al., 2019). We believe this point is important and underappreciated by some cell biologists, but we will modify the text to make it more generic: “This work highlights the utility of using evolutionary analyses and eukaryotic diversity to generate mechanistic hypotheses for cellular processes”.

    Significance

    Overall, while the manuscript contains an abundance of new data, the overall conclusion of the work, stated in the title: "Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy" does not constitute a significant advance beyond other published phylogenomic analysis (below) and the two previous papers by the same authors, including the 2020 paper "A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress (PMID: 32851973)" and the 2021 paper "C53 is a cross-kingdom conserved reticulophagy receptor that bridges the gap between selective autophagy and ribosome stalling at the endoplasmic reticulum PMID: 33164651)". While a regulatory interaction between UFMylation and autophagy is of potential importance, the data in this manuscript do not constitute a major advance and fail to provide new mechanistic insight to explain the role of C53 IDR in autophagy and its interplay with UFMylation

    We disagree with the reviewer’s suggestion that our work does not constitute a significant advance. We outlined above in detail the novel insights that were obtained from our phylogenomic analysis which involved using improved methods to reveal a much more dynamic and informative picture of UFMylation evolution than has been described previously. Likewise, this manuscript builds substantially on our previous mechanistic work. In our 2020 paper (which is summarized in the mentioned 2021 review article), we identified C53 as an ER-associated protein that binds ATG8 through sAIMs and interacts with the phagophore after RPL26 UFMylation. This work linked C53 activity to ER-phagy and highlighted its importance in plant and animal stress response. However, key questions remained unanswered prior to our current work such as whether this mechanism is conserved across eukaryotes, especially in unicellular species, how C53 activity is regulated, and how UFM1 and ATG8 interact with C53. Our current manuscript builds on this work with the following key results:

    1. We use a combination of phylogenomic and experimental analyses to demonstrate that C53 function is conserved across eukaryotes.
    2. We reveal a mechanism whereby UFM1 and ATG8 compete for binding at the sAIMs in the C53 IDR and characterize how each of these ubiquitin-like proteins interacts in an alternative way (see the NMR results described above).
    3. We show how the sAIMs are required for the regulation of C53-mediated autophagy and reveal the importance of UFM1-ATG8 competition in preventing C53 autoactivation, which causes unnecessary autophagic degradation and impairs cellular stress responses.

    These insights are fundamental for understanding the mechanisms regulating C53-mediated autophagy which were unknown before this work. We will therefore adjust our manuscript to more clearly and explicitly explain how our data build on previous observations so that the novelty and significance of our results are clearer.

    Referee #3

    Evidence, reproducibility and clarity

    Picchianti and colleagues have investigated a conserved molecular framework that orchestrates ER homeostasis via autophagy. For this, they have carried out phylogenomics and large-scale gene family analyses across eukaryote diversity as well as a barrage of molecular lab work. The amount of work carried out as well as the overall quality of the study is impressive.

    Thank you!

    I have only a few comments that should be very easy to tackle. (1) Maybe I missed it, but please upload all alignments used for phylogenetics and phylogenomics for reproducibility to e.g. Zenodo, Figshare or other suitable OA databases.

    We included the alignments in the supplementary data, but as suggested, we will upload all the source data including the scripts and the alignments to Zenodo.

    (2) "Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown" --> Maybe there is no "why" and these were not selected at all. Could be random... drift, non-adaptive constructive neutral evolution. I am not saying that asking "why" in evolutionary biology is wrong. It, however, often does not yield satisfactory answers--or any answer at all.

    The reviewer is completely right that “why” is not the right way to frame an evolutionary question. Thank you for pointing this out. We will revise the text and make sure that we remove these kinds of deterministic statements.

    (3) The authors make a case for UFMylation in LECA and I am fully sympathetic with this. However, getting rid of misfoled/problematic proteins and subcellular entities is something that prokaryotes also to a certain degree must have (and still do) master. Are inclusion bodies or export their only answers (I don't know)? Of course, in eukaryotes with all their intracellular complexity this is likely more of an issue. Given the scope of this manuscript (i.e. shedding light on that ancient framework, deep evolutionary roots in eukaryote evolution etc. etc.) it would be very interesting to read the authors thoughts on this and also pinpoint the prokaryote/eukaryote divide in light of the machinery discussed here.

    Thank you for this suggestion. We did indeed check whether any of the UFMylation machinery were present in prokaryotes and only found homologs of UFSP2. These results are consistent with Grau-Bové et al. (2014) who conducted an equivalent analysis and concluded that UFMylation machinery were derived during eukaryogenesis. We will make reference to this in the revised manuscript.

    Significance

    This study not only impresses with the volume of experiments and data, but also the courage to show conservation of a molecular framework by working with such a range of distantly-related eukaryotes. The results and conclusions from this study should be interesting to anyone working in the broad fields of cellular stress and/or autophagy--both extremely timely topics.

    We thank the reviewer for understanding our take-home message and the advances made. We especially thank the reviewer for understanding the challenge of connecting in silico genomic data with in vivo and in vitro experiments.

    CROSS-CONSULTATION COMMENTS

    Referee #2 The challenge in providing a fair review of this manuscript is to clearly define what contributions are novel, significant advances. It is difficult to tell the way the manuscript is written, as it is unclear how the new data - which are voluminous- actually advance the model already put forth by the same authors in two previous publications. It is also unfortunate that the authors overlooked the 2004 phylogenomics paper. There clearly are some new pieces of information here, but the overall increment in knowledge is rather minimal. Response from Referee #3 I agree that the authors somehow steamroll the reader with a wealth of data. But I think this can be addressed by the authors by requesting a lot more justification and by giving them the opportunity to put the significant advances into their own words. This is, in my opinion, quite doable in course of a revision. Overall I have to say that I am very sympathetic with the crosseukaryote reactivity approach that the authors have taken. It is quite intriguing.

    We thank the reviewers for this useful exchange. We agree that our manuscript was not clear enough to emphasize the novelty of our results which likely resulted from the volume and diversity of the experiments and analyses that were presented. We have now revised the manuscript to improve the context and rationale for the study, the intent and hypotheses behind each experiment, and the novel results and insights obtained in each section.

    Response from Referee #2 I agree that the cross-eukaryote approach is intriguing. Shouldn't we be concerned that the 2004 publication already made two of their key points (ie present in LECA, loss in Fungi). What is the incremental insight from this paper? I'd appreciate an opinion from an evolutionary biologist as to how strongly one can conclude functional co-evolution from such correlative data, especially given the rather small number of supporting examples. Is it also necessary to consider counter-examples- ie species that have sAIMs but no UFM1 (I believe that they found a few such cases)?

    Importantly, we do not conclude functional co-evolution from our correlative data. Instead, we used these correlations to generate hypotheses that we tested with various experiments in different model systems. For example, the apparent correlation between C53 sAIMs and UFM1 prompted us to test whether or not UFM1 and sAIMs interact. Regardless of sample size or statistical significance, phylogenomic analyses can never demonstrate functional links, only correlations, which is why we combined these two approaches. Although only a few species encoded C53 without UFM1, each of these contained C53 cAIMs and lacked sAIMs (Figure 2c). There are species with UFM1 that lack C53 but this makes sense as UFM1 is used in other processes besides ER-phagy. We have revised the text to make our approach and reliance on certain data clearer.

    Response from Referee #3 Well with these deep evolutionary questions this is always a challenge. Where does one stop to sample more homologs for one's analyses (one from each supergroup [which are no longer recognised by the community])? In that sense, the authors are right to make the parsimonious base assumption that if X and Y interact in species A and B (no matter how distant they are related) then X and Y interacted in the last common ancestor of A and B. That being said, if I would have designed this study, I would have sampled more broadly for my in vitro crosseukaryote approach. But also this, I think, could be carried out by the authors in a reasonable timeframe. Specifically, they have now sampled from Amorphea and Archaeplastida, they should add one from TSAR, one Haptista, one Cryptista, and one CRuM. If they synthesised the proteins via a company, they could have the constructs in a few weeks for about 1K Euro - I do not think that this would be an unreasonable request.

    We agree that testing C53 function in additional species would strengthen our understanding of the conservation of this pathway across eukaryotes, as it cannot be assumed that orthologous proteins will function in the same way across all species. To our knowledge there is no other work showing experimentally that the UFMylation pathway is working in a single-celled organism. We focussed our efforts on the unicellular green alga, Chlamydomonas due to its relative experimental tractability. However, testing this was not trivial as it required us to establish expression and purification protocols, isolate Chlamydomonas mutants, optimize physiological stress assays, and perform the experiments.

    Nevertheless, we agree that we could expand our in vitro assays with C53 orthologs from additional species. As suggested by reviewer 3, we will now synthesize 6 more C53 isoforms from two TSAR representatives (the alveolate, Tetrahymena thermophila, and the stramenopile, Phytophthora sojae), as well as a representative from Haptista (Emiliania), Cryptista (Guillardia), Diplomonada (Trypanosoma), and CRuMs (Rigifila). We will test their interaction with human and plant ATG8 and UFM1 proteins. We have also added two species from CRuMs into our phylogenomic analysis.

    The list of experiments that we can do to address the reviewer’s concerns:

    1. Repeat experiment in Figure 1C probing with �-RPL26.
    2. To calculate KD values, perform ITC experiments with C53 wild-type, C53 sAIM mutant and C53 cAIM variant titrated with ATG8 and UFM1.
    3. Perform CoIP experiments using C53 antibody in wild type and UFM1 overexpressing lines and detect for ATG8 association, under normal and stress conditions.
    4. We will test autophagic degradation of C53 in uba5 and ufc1 mutants under normal and tunicamycin stress conditions by performing autophagic flux assays using the native C53 antibody
    5. Molecular docking studies to see C53’s structural rearrangements leading to ATG8 and UFM1 binding.
    6. Figures from co-localization experiments in Figure 5G will be revisited and we will perform additional co-localization analyses such as Pearson coefficient under normal and stress conditions. We will also upload all the raw images as supporting material, so that anyone can independently assess our images.
    7. We will upload all the source data for phylogenomic analyses, including scripts and alignments to Zenodo.
    8. Test the interaction of 6 newly synthesised C53 isoforms from: (1) an alveolate (tsAr, Ciliate), (2) a stramenopile (tSar, Phaeodactylum), (3) a haptophyte (Emiliania), (4) a cryptophyte (Guillardia), (5) a diplomonad (Trypanosoma) and (6) a CrRuM with human and plant ATG8 and UFM1 proteins.
  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Picchianti and colleagues have investigated a conserved molecular framework that orchestrates ER homeostasis via autophagy. For this, they have carried out phylogenomics and large-scale gene family analyses across eukaryote diversity as well as a barrage of molecular lab work. The amount of work carried out as well as the overall quality of the study is impressive. I have only a few comments that should be very easy to tackle.

    1. Maybe I missed it, but please upload all alignments used for phylogenetics and phylogenomics for reproducibility to e.g. Zenodo, Figshare or other suitable OA databases.
    2. "Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown" --> Maybe there is no "why" and these were not selected at all. Could be random... drift, non-adaptive constructive neutral evolution. I am not saying that asking "why" in evolutionary biology is wrong. It, however, often does not yield satisfactory answers--or any answer at all.
    3. The authors make a case for UFMylation in LECA and I am fully sympathetic with this. However, getting rid of misfoled/problematic proteins and subcellular entities is something that prokaryotes also to a certain degree must have (and still do) master. Are inclusion bodies or export their only answers (I don't know)? Of course, in eukaryotes with all their intracellular complexity this is likely more of an issue. Given the scope of this manuscript (i.e. shedding light on that ancient framework, deep evolutionary roots in eukaryote evolution etc. etc.) it would be very interesting to read the authors thoughts on this and also pinpoint the prokaryote/eukaryote divide in light of the machinery discussed here.

    Referees cross-commenting

    Referee #2

    The challenge in providing a fair review of this manuscript is to clearly define what contributions are novel, significant advances. It is difficult to tell the way the manuscript is written, as it is unclear how the new data - which are voluminous- actually advance the model already put forth by the same authors in two previous publications. It is also unfortunate that the authors overlooked the 2004 phylogenomics paper. There clearly are some new pieces of information here, but the overall increment in knowledge is rather minimal.

    Response from Referee #3

    I agree that the authors somehow steamroll the reader with a wealth of data. But I think this can be addressed by the authors by requesting a lot more justification and by giving them the opportunity to put the significant advances into their own words. This is, in my opinion, quite doable in course of a revision. Overall I have to say that I am very sympathetic with the cross-eukaryote reactivity approach that the authors have taken. It is quite intriguing.

    Response from Referee #2

    I agree that the cross-eukaryote approach is intriguing. Shouldn't we be concerned that the 2004 publication already made two of their key points (ie present in LECA, loss in Fungi). What is the incremental insight from this paper?

    I'd appreciate an opinion from an evolutionary biologist as to how strongly one can conclude functional co-evolution from such correlative data, especially given the rather small number of supporting examples. Is it also necessary to consider counter-examples- ie species that have sAIMs but no UFM1 (I believe that they found a few such cases)?

    Response from Referee #3

    Well with these deep evolutionary questions this is always a challenge. Where does one stop to sample more homologs for one's analyses (one from each supergroup [which are no longer recognised by the community])? In that sense, the authors are right to make the parsimonious base assumption that if X and Y interact in species A and B (no matter how distant they are related) then X and Y interacted in the last common ancestor of A and B. That being said, if I would have designed this study, I would have sampled more broadly for my in vitro cross-eukaryote approach. But also this, I think, could be carried out by the authors in a reasonable timeframe. Specifically, they have now sampled from Amorphea and Archaeplastida, they should add one from TSAR, one Haptista, one Cryptista, and one CRuM. If they synthesised the proteins via a company, they could have the constructs in a few weeks for about 1K Euro - I do not think that this would be an unreasonable request.

    Significance

    This study not only impresses with the volume of experiments and data, but also the courage to show conservation of a molecular framework by working with such a range of distantly-related eukaryotes. The results and conclusions from this study should be interesting to anyone working in the broad fields of cellular stress and/or autophagy--both extremely timely topics.

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript from Picchianti et al. seeks to define the role of CDK5RAP3 (hereinafter referred as C53) during autophagy and its interplay with UFMylation. Together with UFL1 and DDRGK1, C53 is a component of a trimeric UFM1 E3 ligase complex that modifies the 60S ribosomal protein RPL26 at the endoplasmic reticulum (ER) surface upon ribosomal stalling (among other proposed functions that are not addressed). Several previous studies have implicated the UFMylation pathway in autophagy or ER-phagy although a non-autophagic fate for UFM1-tagged ribosomal subunits has also been reported.

    A previous study from the same authors (PMID: 32851973) identified an intrinsically disorder region (IDR) in C53 that is necessary and sufficient for interaction between C53 and autophagy receptor, ATG8. They reported that this IDR comprises four non canonical ATG8 interacting motifs (AIM), named shuffled AIMs (sAIMs) and showed that combinatorial mutagenesis of sAIM1, sAIM2, and sAIM3 abrogates ATG8 binding. A similar effect was observed for plant C53, though an additional canonical AIM (cAIM) in the C53 IDR had to be mutated to completely abolish C53 and ATG8 interaction. The earlier study reported that C53 IDR also interacts with UFM1, and this interaction can be disrupted in vitro by adding increasing concentration of ATG8, suggesting that ATG8 and UFM1 may compete with one another for C53 binding.

    The present paper attempts to build on this previous work by using phylogenomics to infer a co-evolutionary relationship between UFMylation machinery and sAIMs in C53, which the authors argue, constitutes further evidence of the primary importance of a role for UFMylation in ER homeostasis. The manuscript includes a lot of biochemical data using variations of in vitro and in vivo pull-down experiments to define the roles of individual AIMs in mediating the binding of C53 to ATG8 and to UFM1. They also use NMR spectroscopy in an attempt to define the structural basis of the UFM1 and ATG8 binding to C53, concluding that plant C53 interacts with UFM1 mainly through sAIM1, while interaction with ATG8 requires cAIM as well as sAIM1 and sAIM2. Finally, the authors attempt to contextualize these findings by conducting studies on Arabidopsis mutants, showing that replacing sAIMs with cAIMs causes increases sensitivity to ER stress and apparently increases formation of C53 intracellular puncta that may colocalize with ATG8.

    From these data the authors concluded that the dual-ATG8 and UFM1 binding of C53 IDR regulates C53 recruitment to autophagosomes in response to ER stress.

    Major Issues:

    1. The phylogenomics analysis conclusion that UFM1 is common in unicellular lineages and did not evolve in multicellular eukaryotes is not novel, as another comprehensive analysis of UFM1 phylogeny, published eight years ago - in 2014 - by Grau-Bové et al. (PMID: 25525215), also reported that UFM1, UBA5, UFC1, UFL1 and UFSP2 were likely present in LECA and lost in Fungi. Although the phylogenomic analysis by Picchianti et al. is also extended to DDRGK1 and C53 proteins, and some parasitic and algal lineages, their findings are incremental. Their proposed coevolution of sAIM and UFM1 is based on presence-absence correlation observed within five species (i.e., Albugo candida, Albuco laibachii, Piromyces finnis, Neocallimastix californiae, Anaeromyces robustus). However, this coevolutionary relationship must be further investigated by substantially increasing the taxonomic sampling within the UFM1-lacking group.
    2. The manuscript presents an overwhelming amount of biochemical and structural data obtained from a variety of protein binding techniques (i.e., NMR spectroscopy, in vitro GST-pulldown, fluorescence microscopy-based on-bead binding assays, and native mass-spectrometry). The results are poorly explained and not organized in a logical manner. Moreover, no attempt was made to explain the rationale behind using one technique over the other or how one method complements another to build a stronger conclusion than any individual approach. Given that none of the methods employed report quantitative measurement of binding affinities between C53 IDR and UFM1 or ATG8, it is not clear how the data presented in this manuscript contribute to our understanding of the proposed competition model for UFM1 and ATG8 binding to C53 IDR. To conclude that an interaction is "stronger" or "weaker" it is necessary to measure equilibrium binding constants. Fortunately, there are suitable techniques, including surface plasmon resonance (SPR), microscale thermophoresis (MST), fluorescence anisotropy, or calorimetry that are available to dissect these complex competitive binding interactions and to build models.
    3. The NMR studies have the potential to dissect the types of dynamic binding inherent in unstructured proteins. However, the abundant NMR data presented combined with the aforementioned binding studies, remarkably, do not seem to significantly advance our understanding of how the system is organized or even how UFM1 and ATG8 bind C53, beyond the rather vague and somewhat circular conclusion stated in the abstract: "...we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound the sAIMs in a different mode." Or on line 165 "Altogether these results suggested that ATG8 and UFM1 bbind the sAIMs withn C54 IDR, albeit in a different manner".
    4. The functional assays performed in Arabidopsis do not support the competitive model between UFM1 and ATG8 for binding to C53 during C53-mediated autophagy. The fluorescence microscopy images do not provide convincing evidence of colocalization between C53 and ATG8. In fact, in contrast to the claims made in the text or the quantification, mCherry-C53 fluorescence does not seem to localize in discrete puncta and its signal does not seem to overlap with ATG8A.

    Minor Issues:

    1. The authors might choose to avoid teleological arguments such as (line 135): "As the phylogenomic analysis suggested that eh sAIMs have been retained to mediate C53-UFM1 interaction..."
    2. The authors refer on multiple occasions to C53 "autoactivation" without defining what they mean by this. Do they propose that C53 UFMylates itself?.
    3. The paper might want to avoid preachy philosophical statements like "Our evolutionary analysis also highlights why we should move beyond yeast and metazoans and instead consider the whole tree of life when using evolutionary arguments to guide biological research." (333-335). While this is indeed a laudable goal, given the rather limited insights from this study, it is unclear how this paper exemplifies the notion.

    Referees cross-commenting

    Referee #2

    The challenge in providing a fair review of this manuscript is to clearly define what contributions are novel, significant advances. It is difficult to tell the way the manuscript is written, as it is unclear how the new data - which are voluminous- actually advance the model already put forth by the same authors in two previous publications. It is also unfortunate that the authors overlooked the 2004 phylogenomics paper. There clearly are some new pieces of information here, but the overall increment in knowledge is rather minimal.

    Response from Referee #3

    I agree that the authors somehow steamroll the reader with a wealth of data. But I think this can be addressed by the authors by requesting a lot more justification and by giving them the opportunity to put the significant advances into their own words. This is, in my opinion, quite doable in course of a revision. Overall I have to say that I am very sympathetic with the cross-eukaryote reactivity approach that the authors have taken. It is quite intriguing.

    Response from Referee #2

    I agree that the cross-eukaryote approach is intriguing. Shouldn't we be concerned that the 2004 publication already made two of their key points (ie present in LECA, loss in Fungi). What is the incremental insight from this paper?

    I'd appreciate an opinion from an evolutionary biologist as to how strongly one can conclude functional co-evolution from such correlative data, especially given the rather small number of supporting examples. Is it also necessary to consider counter-examples- ie species that have sAIMs but no UFM1 (I believe that they found a few such cases)?

    Response from Referee #3

    Well with these deep evolutionary questions this is always a challenge. Where does one stop to sample more homologs for one's analyses (one from each supergroup [which are no longer recognised by the community])? In that sense, the authors are right to make the parsimonious base assumption that if X and Y interact in species A and B (no matter how distant they are related) then X and Y interacted in the last common ancestor of A and B. That being said, if I would have designed this study, I would have sampled more broadly for my in vitro cross-eukaryote approach. But also this, I think, could be carried out by the authors in a reasonable timeframe. Specifically, they have now sampled from Amorphea and Archaeplastida, they should add one from TSAR, one Haptista, one Cryptista, and one CRuM. If they synthesised the proteins via a company, they could have the constructs in a few weeks for about 1K Euro - I do not think that this would be an unreasonable request.

    Significance

    Overall, while the manuscript contains an abundance of new data, the overall conclusion of the work, stated in the title: "Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy" does not constitute a significant advance beyond other published phylogenomic analysis (below) and the two previous papers by the same authors, including the 2020 paper "A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress (PMID: 32851973)" and the 2021 paper "C53 is a cross-kingdom conserved reticulophagy receptor that bridges the gap between selective autophagy and ribosome stalling at the endoplasmic reticulum PMID: 33164651)". While a regulatory interaction between UFMylation and autophagy is of potential importance, the data in this manuscript do not constitute a major advance and fail to provide new mechanistic insight to explain the role of C53 IDR in autophagy and its interplay with UFMylation

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Picchianti et al. provide novel insights into the interaction of C53 with UFM1 and ATG8. Initially, the authors show that protein modification by UFM1 exists in the unicellular organism Chlamydomonas reinhardtii. To that end they demonstrated that pure Chlamydomonas UBA5, UFC1 and UFM1 proteins, can charge UFC1. Then, they showed that C53 interacts with ATG8 and UFM1. Specifically, they found that the sAIM are essential for the interaction with UFM1, while substituting this motif with canonical AIM prevents the binding of UFM1 but not of ATG8. Since binding of C53 to ATG8 recruits the autophagy machinery, the authors suggest that ufmylation of RPL26 releases UFM1 from C53 which allows the binding of ATG8. Overall, the authors demonstrate that C53 that forms a complex with UFL1 connects between protein ufmylation and autophagy by its ability to bind both UBLs.

    Here the authors revisited the assumption that only multicellular organisms have the UFM1 system. Using bioinformatic tools they show that it exists also in unicellular organism. Also, they show that in some organisms the E3 complex UFL1, UFBP1 and C53 exist but not UBA5, UFC1 or UFM1. This is a very interesting observation that suggests an additional role for this complex. In Fig 1C the authors show that in Chlamydomonas RPL26 undergoes ufmylation. Please use IP against RPL26 and then a blot with anti UFM1. From the current experiment it is not clear how the authors know that this is indeed RPL26 that undergoes ufmylation

    In the second part of the manuscript the authors characterize the interaction of C53 with ATG8 and UFM1. This is a continuation of their previous published work (Stephani et al, 2020) . Here the reviewer thinks that further data on the binding of these proteins to C53 is required. Specifically, defining the Kd of these interactions using ITC or other biophysical method can contribute to the study.

    Under normal condition the authors suggest that C53 binds UFM1 and this keeps it inactive. The reviewer thinks that this claim needs further support. Using IP (maybe with crosslinker) the author can show that C53, in normal conditions, bind more UFM1 than ATG8. Also, since the interaction of UFM1 to C53 is noncovalent, it will be nice to show how alternations in UFM1 expression levels can affect the activation of C53. Finally, the authors suggest that ufmylation of RPL26 allows binding of ATG8 to C53 and this, in turn, leads to C53 activation. Can the authors show that in cells lacking UBA5, under normal condition or with Tunicamycin treatment, ATG8 does not activate C53 due to the fact that UFM1 does not leave C53.

    Significance

    This manuscript advances our understanding of the connection of ufmylation to autophagy which is mediated by C53.

  6. Version published to 10.1101/2022.04.26.489478v1 on bioRxiv