UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation independently of the p97 ATPase

  1. Brittany A. Ahlstedt
  2. Rakesh Ganji
  3. Sirisha Mukkavalli
  4. Joao A. Paulo
  5. Steve P. Gygi
  6. Malavika Raman

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Abstract

Endoplasmic reticulum (ER) protein homeostasis (proteostasis) is essential to facilitate proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis due to cell stress or mutations in ER proteins can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 significantly sensitizes cells to ER stress and activates canonical UPR signaling pathways. This in turn leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins belonging to ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.

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    Reply to the reviewers

    Manuscript number: RC-2022-01803

    Corresponding author(s): Brittany A., Ahlstedt, Rakesh, Ganji, Sirisha Mukkavalli, Joao A., Paulo, Steve P., Gygi, Malavika, Raman

    If you wish to submit a full revision, please use our "Full Revision" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We thank the reviewers for their insightful comments and agree that the many of these revision experiments will improve the strength of our manuscript. Some of these we have already completed or are in the process of completing which will be outlined below. In particular, the reviewers asked that we investigate the mechanistic link between increased translation and UPR induction. We have detailed the studies that we will perform to establish this connection in detail below.

    Description of the planned revisions

    __Response to Reviewer 1: __

    1. __The authors need to express UBXN1 and mutants lacking either the UBX or UBA domain in UBXN1 knockout cells to test whether the ER stress phenotype (Figure 1) and the protein upregulation phenotype (Figure 5A-F) can be rescued. This would eliminate the possibility that the reported phenotypes are the off-target effects of CRISPR. __
    • We will express the Myc-tagged wildtype UBXN1 and UBX or UBA point mutants (used in translation rescue studies in Figure 6) into UBXN1 knockout (KO) cells to determine whether the ER stress phenotype can be rescued. We will determine the level of xbp1s by real-time PCR and BiP by immunoblot.
    • The studies in Figure 5 A-F were completed in cells depleted of UBXN1 with siRNA, not the CRISPR knockout cells. Thus, it is unlikely an off-target effect of CRISPR. We will attempt rescue of this phenotype with the wildtype and mutant constructs.

    __For Figure 2, please indicate whether the repeat is a biological replicate or a technical replicate from RT-PCR. __

    • We apologize for the omission. The data from the RT PCR studies in Figure 2 are biological replicates – the figure legend and main text of the manuscript will be edited to clarify this.

    __In Figure 1A, the authors show that the knockout of UBXN1 causes an upregulation of phosphorylated eIF2alpha, which is known to suppress protein translation globally. In this regard, it is surprising to see the authors also concluded from Figure 7 that there is an upregulation of protein translation in UBXN1 knockout cells. The authors do not provide any explanation on how these seemingly contradictory phenotypes could be seen in the same cells. __

    • We will provide a detailed discussion of the apparent paradox between upregulation of phosphorylated eIF2a and increased protein translation. Several prior studies have demonstrated that elevated expression of ATF4 (as we observe in UBXN1 KO cells) activates a transcriptional program that restarts translation. This occurs through the upregulation of the phosphatase PPP1R15a that dephosphorylates eiF2a, as well as aminoacyl tRNA synthetases and ribosomal subunits. We propose that elevated ATF4 levels leads to premature translational restart in UBXN1 KO cells. In addition, our data suggests that UBXN1 represses translation upstream of UPR activation and thus and increase in protein translation dysregulates ER-proteostasis which hyperactivates the UPR.

    __Any evidence that UBXN1 is associated with translating ribosomes? __

    • We now have new data that UBXN1 is associated with 40S, 60S, and 80S ribosomal fractions as well as actively translating polysome fractions that we isolated by polysome purification. In agreement with our finding that the role of UBXN1 in repressing translation is independent of p97, p97 appears to associate largely with the 40S, 60S, and 80S ribosomal fractions but not with the actively translating polysomes. This data will be included in the revised manuscript. __Response to Reviewer 2: __
    1. Authors found that significant enrichment of the ER proteins in UBXN1 KO cells, while there is no change in the abundance of proteins in the cytosol or nucleus. Mitochondrial proteins are even down-regulated in UBXN1 KO cells. I found these observations very interesting. However, I was frustrated that authors did not investigated the reason why such differences are associated in UBXN1-suppressed cells. Authors demonstrate that depletion of UBXN1 resulted in suppression of protein synthesis, but did not address whether ER proteins are specifically repressed by UBXN1 or it represses translation globally, as noted in their Discussion section. Do the mRNAs encoding signal sequence at the N-terminus of their products are specifically translated in UBXN1-suppressed cells? Do the translations of mRNAs encoding mitochondria translocation signals are suppressed in UBXN1 KO cells? It should be possible to investigate these issues by using appropriate model ER- or mitochondrial proteins with or without specific signal sequences. Such kind of analysis should be necessary to support the claim of this manuscript.
    • Previous studies by Luke Wiseman’s group showed that PERK activation resulted in hyperfusion of the mitochondria and loss of Tim17 leading to decreased mitochondrial import. We already show that mitochondrial proteins are downregulated (by TMT proteomics and by immunoblotting). We now have preliminary data that mitochondria are more fused in UBXN1 KO cells consistent with data from the Wiseman group. We will include this in the resubmission.
    • In addition, we have re-analyzed our TMT proteomics data to parse out proteins with ER-signal sequences and define the topology of ER proteins (Type 1, 2, multimembrane spanning and luminal proteins) and those with mitochondrial targeting sequences. This data will be included in the revised manuscript.

    __Related to my previous comments, ER-targeted mRNAs are known to be degraded by a process termed RIDD in the case of ER stressed condition. Since the rapid degradation of mRNAs through RIDD functions to alleviate ER stress by preventing the continued influx of new polypeptides into the ER, I wondered why UBXN1 depletion greatly stimulates ER protein synthesis, escaping IRE1-dependent mRNA degradations. Does UBXN1 depletion suppress RIDD? __

    • In the revised manuscript, we will determine the relative mRNA abundance of the bona fide RIDD targets BLOC1S1 and CD59 by quantitative PCR in cells stressed with dithiothreitol (DTT). We will utilize previously published and validated primers for each target to quantify RIDD activity in wildtype and UBXN1 KO cells. These studies will address whether loss of UBXN1 impacts IRE1-dependent RIDD.

    __Authors mentioned that the elevated levels of ER proteins are not due to increased transcription of target genes. However, they only provided the quantification of prp transcript levels, which was unchanged between wildtype and UBXN1 KO cells. To support this important conclusion, it is necessary to provide whole transcriptome data to compare the expression levels of corresponding ER proteins (quantified by their proteomics data) and transcripts (quantified by, for an example, RNA-seq analysis). __

    • We thank the reviewer for this comment. Currently, we show that mRNA levels of Prp do not significantly change between control and siUBXN1 cells (Supplementary Figure 4). For a more comprehensive analysis, we will additionally assess the mRNA levels of the proteins we determinized to be significantly enriched in Figure 5 (AGAL, ALPP2 and TRAPa). RNA sequencing is currently beyond the scope of this study.

    __Authors claimed that UBXN1 loss is detrimental to cell viability and have elevated levels of the apoptosis in the face of ER stress. However, authors did not examine apoptotic cell death in UBXN1 KO cells. They only provided evidence for defective proliferation of cells and transient induction of CHOP expression, but these are not enough to support the ER-stress induced apoptosis. __

    • We will address the levels of apoptotic cell death in wildtype and UBXN1 KO cells by assessing PARP, caspase-3, or caspase-8 cleavage in these cells by immunoblot.

    __Authors showed that UBA domain of UBXN1 is critical for suppressing protein synthesis. Could you provide a bit more detailed discussion how UBA domain modulates protein translational events and promote expressions of ER-related proteins. Have you ever checked whether UBA domain of UBXN1 is necessary for suppressing UPR-specific target gene expressions? __

    • We will express the Myc-tagged wildtype UBXN1 and UBX or UBA point mutants (used in translation rescue studies in Figure 6) into UBXN1 knockout (KO) cells to determine whether the ER stress phenotype can be rescued.
    • We will also include a discussion on how the UBA domain in UBXN1 may recognize distinct ubiquitylation events on ribosomes that modulate their abundance and function. __Response to Reviewer 3: __

    __(Major comments) __

    1. __My main reservation about the current manuscript is whether the UPR activation can be directly ascribed to the loss of UBXN1. The authors do not differentiate between acute depletion (through siRNA in Fig. 5) versus permanent UBXN1 knockout in most of the experiments. The latter may lead to extensive adaptation of the cellular proteome due to chronic stress. Prior studies from the authors have shown that UBXN1 knockout leads to loss of aggreasomes. This raises a major question whether the observed UPR activation can be directly attributed to UBXN1 loss or be an indirect result of adaptation in the knockout cells, for instance due to accumulation of BAG6 substrates in insoluble aggregates as the authors have shown previously (ref. 40). Along those lines, the authors already showed in the same study that UBXN1 knockout cells are more sensitive to proteotoxic stress. __
    • We agree with the reviewer that cells can adapt to CRISPR knockout. However, the IRE1a clustering studies found in Figure 1 were completed in the context of acute depletion of UBXN1 by siRNA and demonstrate a significant increase in IRE1a clustering when UBXN1 is depleted.
    • We now have new data that that acute depletion of UBXN1 with siRNA results in a significant increase in BiP and ATF4 expression as well as ATF6 N-terminal processing.
    • Furthermore, we have new data that acute depletion of UBXN1 with siRNA phenocopies UBXN1 KO in terms of increased puromycin incorporation into newly synthesized proteins.
    • Thus, we will have both genetic knockout as well as siRNA acute depletion for all major studies. We will include these new studies in the revised manuscript.

    __The later results in the study nicely show that the repressed protein translation phenotype is dependent on the ubiquitin binding domain of UBXN1. These segregation-of-function mutants and complementation experiments could be easily used to more clearly distinguish whether the UPR activation can be directly attributed to UBXN1 and the increase in protein translation. For instance, can overexpression of UBXN1 in the knockout background suppress the UPR activation? Is the UBX-domain mutant capable of suppressing the UPR phenotype? These results would provide critical support as to whether the UPR activation is a direct result of the loss of UBXN1. __

    • We will express the Myc-tagged wildtype UBXN1 and UBX or UBA point mutants (used in translation rescue studies in Figure 6) into UBXN1 knockout (KO) cells to determine whether the ER stress phenotype can be rescued. We will determine the level of xbp1s by real-time PCR and BiP by immunoblot.
    • To delineate the relationship between UPR activation and protein translation, we will halt protein synthesis with the translational elongation inhibitor cycloheximide and assess UPR activation in wildtype and UBXN1 KO cells. If increased protein translation in UBXN1 KO cells is what causes UPR activation, we anticipate that cycloheximide will rescue UPR activation in UBXN1 KO cells back to wildtype levels.

    __Similarly, the authors use transient siRNA knockdown of UBXN1 in Fig. 5 and Supp. Fig. 4, but do not reassess the UPR activation under these conditions. It would be important to validate that the acute UBXN1 knockdown can recapitulate the UPR activation phenotype. __

    • Please see comment 1 above.

    __I am puzzled by the interpretation of the AGAL degradation experiments in Supplemental Figure 4F. Clearly, the rate of AGAL degradation is much faster in WT cells than in UBXN1 knockout cells as indicated by the slope of the curves between 2-4 hours. I disagree with the interpretation that UBXN1 knockout does not impact AGAL turnover. It is not valid to make the comparison at 9 hours because hardly any AGAL substrate is remaining. Importantly, this experiment raises a larger question: Are other ER client degradation rates affected by the UBXN1 knockout? And is the UPR activation more generally due to accumulation of misfolded ER proteins? Their prior publication (ref. 40) evaluated several ERAD clients where UBXN1 was dispensable, but it could be possible that UBXN1 has a more specialized client pool. Showing quantification of the PrP CHX chase would also be helpful - from the single replicate it looks like more PrP remaining in the UBXN1 knockout at 8 hours (Supp. Figure 4G). __

    • Our previous ERAD reporter study using three distinct ERAD clients that are routinely used to assess ERAD found no role for UBXN1 in ERAD (Ganji et al MCB 2018). We do agree with the reviewer that UBXN1 may have discrete roles in regulating the degradation of select p97 ER clients. Determining this in an unbiased and comprehensive manner would require pulse chase SILAC proteomics or similar methodologies which are beyond the scope of the current study. We will therefore evaluate whether loss of UBXN1 affects the rate of degradation of additional ER-client proteins that we identified via TMT. Additionally, we will include a quantification of PrP cycloheximide chase.

    __It would be helpful for the manuscript to clearly distinguish between 1) upregulation of ER proteostasis factors because of ER stress/UPR, and 2) upregulation of secreted clients (AGAL, PrP) which may be partly due to increased translation rates but could also be due to reduced degradation. Many of the hits from the proteomics experiments are ER proteostasis factors that are part of the adaptive stress response (SEC61B, SEC63, CANX, SSR1/2/3, STT3B, RPN1, RPN2, SEC61A1 - compare to ref 12: most are direct IRE1/XBP1s targets). Their increased expression does not lead to increased ER stress as they are involved in the resolution of ER stress. It appears to be circular logic that increased expression of UPR targets would lead to more UPR activation. Currently, the authors do not clearly disentangle the increased expression of endogenous ER proteins from the proteomics experiment versus overexpression of exogenous secreted clients. __

    • We identify many ER proteins with increased abundance in UBXN1 KO cells that are not transcriptional targets of the IRE1-UPR pathway. We will re-format the TMT data to more comprehensively characterize the proteins that we identify (known UPR transcriptional targets, membrane embedded, soluble clients etc.).
    • We will change the language in the current manuscript to clearly demarcate the difference between an increase of ER proteostasis factors in response to ER stress, and the upregulation of secreted proteins. Additionally, we will emphasize the secretory proteins that are significantly enriched in UBXN1 KO cells in our proteomics figures to demonstrate the increase of non-ER stress responsive clients.

    __The authors should tone down on broad generalizations, for instance in lines 306-309: ER aggregation was only observed for a single client protein (AGAL). Further, only a single mitochondrial protein was observed to be downregulated (TOMM20). __

    • We have included the quantifications of the relative expression levels of three mitochondrial proteins, two of which are significantly reduced (TOMM20 and CYC1).
    • Additionally, we have new data where we immunoblotted for additional mitochondrial import factors and observed significant reduction of the mitochondrial proteins TIMM23 and TOMM70A which will be included in the revised manuscript.
    • We also plan to examine the levels of the TIMM17A subunit of the TIMM23 complex in UBXN1-depleted cells. TIMM17A is degraded in response to ER stress to prevent protein import into the mitochondria. (Rainbolt, T. et al. Cell Metab 2013)
    • The language of the manuscript will be changed to tone down on broad generalizations. __(Minor comments) __

    __Does UBXN1 localization to the ER/microsomes fraction depend on p97? What happens in UBX-domain mutant? __

    • We will isolate ER-microsomes from UBXN1 KO cells where we have expressed wildtype and UBX/UBA domain mutants to address if localization is dependent on ubiquitin or p97 interaction.

    __In Fig. 1A it is surprising that no BiP is detected at 0 hours as BiP is highly expressed even in the absence of ER stress. Can the authors comment on this discrepancy. __

    • We provide low exposures of the immunoblots as the UBXN1 KO cells have very high levels of BiP compared to control. We will provide alternative blots where the BiP levels at t=0 in control cells is more obvious.

    __The authors use different ER stressors interchangeably: DTT, Tunicamycin, Thapsigargin. While all results in UPR activation, they do so through different mechanisms and with slight nuances that may be worth considering for the experiments and interpretations. __

    • We thank the reviewer for this comment and agree that these stressors can impact the ER and UPR activation in distinct ways. Our rationale for using these agents interchangeably was to demonstrate the UPR induction in UBXN1 null cells occurs irrespective of the type of stress.
    • DTT is a severe stressor and we used tunicamycin and thapsigargin in some assays (imaging etc.) as they are less toxic and more amenable to downstream analysis. We will include text that explains our rationale better.

    __Line 198: "Hierarchical clustering analysis demonstrates that the gene expression pattern observed in UBXN1 KO cells more closely resembles wildtype cells stressed with DTT than untreated wildtype cells based on similar log2 fold change values (Figure 2)." Where is this clustering shown? __

    • We apologize that this was not clear in the figure. We will edit the figure to make the clustering more obvious.

    __What are the downregulated UPR genes in Fig. 2, and may this hold significance? __

    • The reviewer points out an interesting observation. Many of the downregulated transcripts are ERAD components. The significance of this is presently unclear and we would require RNA-seq analysis to make a more educated conclusion. However, this finding may point to an environment that has a greater need to induce folding than degradative components. We will include a discussion of this in the revision.

    3. Description of the revisions that have already been incorporated in the transferred manuscript.

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    __Response to Reviewer 1: __

    1. __Taking the increased protein translation phenotype as an example, does this indicate UBXN1 is a translation suppressor for those ER-associated proteins? __
    • We thank the reviewer for this comment. We are indeed very interested in determining whether UBXN1 represses the translation of ER proteins. We are in the process of identifying proteins that are translated in UBXN1 null cells using O-propargyl-puromycin (OPP) labelling and mass spectrometry. However, given the timeframe for these studies, this cannot be accomplished in this revision.

    __How can UBXN1 selectively inhibit the translation of a subset of proteins? __

    • Recent studies suggest that ribosome populations are quite heterogeneous, and ribosome associated proteins can help tune translation of select proteins. For example, pyruvate kinase muscle (PKM) associates with ER docked ribosomes to regulate the translation of ER proteins in particular. We find that UBXN1 is present on ER membranes and localizes to polysomes and thus may regulate the translation of specific proteins. Studies are underway to test this hypothesis but are beyond the scope for this present study. __Response to Reviewer 2: __
    1. __Authors found that significant enrichment of the ER proteins in UBXN1 KO cells, while there is no change in the abundance of proteins in the cytosol or nucleus. Mitochondrial proteins are even down-regulated in UBXN1 KO cells. I found these observations very interesting. However, I was frustrated that authors did not investigated the reason why such differences are associated in UBXN1-suppressed cells. Authors demonstrate that depletion of UBXN1 resulted in suppression of protein synthesis, but did not address whether ER proteins are specifically repressed by UBXN1 or it represses translation globally, as noted in their Discussion section. Do the mRNAs encoding signal sequence at the N-terminus of their products are specifically translated in UBXN1-suppressed cells? Do the translations of mRNAs encoding mitochondria translocation signals are suppressed in UBXN1 KO cells? It should be possible to investigate these issues by using appropriate model ER- or mitochondrial proteins with or without specific signal sequences. Such kind of analysis should be necessary to support the claim of this manuscript. __
    • Please see comment 1 above.
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Ahlstedt et al. investigate a new role for the p97 adapter protein UBXN1 in negatively regulating the ER unfolded protein response. The study starts from the observations that knockdown of UBXN1 in a previously generated HeLa cell line leads to induction of unfolded protein response markers, and the knockout cells display more pronounced UPR activation upon ER stress. This elevated UPR signaling renders the UBXN1 cells more prone to cell death. Global proteomics experiments similarly show an increased abundance of ER localized proteins, although it is not clearly delineated which of those are the result of UPR activation. The authors then probe the expression of two secretory client proteins, alpha-galactosidase (AGAL) and prion protein (PrP) and find that UBXN1 transient knockdown leads to ER accumulation of the two proteins and increased aggregation upon ER stress. The authors claim that degradation of these ER client proteins in unaffected by the UBXN1 knockdown, but accumulation may instead be due to increased protein translation. Indeed, they surprisingly find that UBXN1 knockout leads to constitutively elevated protein translation. This result points to a previously unknown role of UBXN1 in repressing protein synthesis. Complementation with UBXN1 mutants demonstrate that the translation repression is dependent on the ubiquitin binding activity of UBXN1 but that p97 is dispensable. Further investigation into the molecular mechanism for the translation repression remains reserved for a future manuscript.

    Major comments:

    1. My main reservation about the current manuscript is whether the UPR activation can be directly ascribed to the loss of UBXN1. The authors do not differentiate between acute depletion (through siRNA in Fig. 5) versus permanent UBXN1 knockout in most of the experiments. The latter may lead to extensive adaptation of the cellular proteome due to chronic stress. Prior studies from the authors have shown that UBXN1 knockout leads to loss of aggreasomes. This raises a major question whether the observed UPR activation can be directly attributed to UBXN1 loss or be an indirect result of adaptation in the knockout cells, for instance due to accumulation of BAG6 substrates in insoluble aggregates as the authors have shown previously (ref. 40). Along those lines, the authors already showed in the same study that UBXN1 knockout cells are more sensitive to proteotoxic stress.
    2. The later results in the study nicely show that the repressed protein translation phenotype is dependent on the ubiquitin binding domain of UBXN1. These segregation-of-function mutants and complementation experiments could be easily used to more clearly distinguish whether the UPR activation can be directly attributed to UBXN1 and the increase in protein translation. For instance, can overexpression of UBXN1 in the knockout background suppress the UPR activation? Is the UBX-domain mutant capable of suppressing the UPR phenotype? These results would provide critical support as to whether the UPR activation is a direct result of the loss of UBXN1.
    3. Similarly, the authors use transient siRNA knockdown of UBXN1 in Fig. 5 and Supp. Fig. 4, but do not reassess the UPR activation under these conditions. It would be important to validate that the acute UBXN1 knockdown can recapitulate the UPR activation phenotype.
    4. I am puzzled by the interpretation of the AGAL degradation experiments in Supplemental Figure 4F. Clearly, the rate of AGAL degradation is much faster in WT cells than in UBXN1 knockout cells as indicated by the slope of the curves between 2-4 hours. I disagree with the interpretation that UBXN1 knockout does not impact AGAL turnover. It is not valid to make the comparison at 9 hours because hardly any AGAL substrate is remaining. Importantly, this experiment raises a larger question: Are other ER client degradation rates affected by the UBXN1 knockout? And is the UPR activation more generally due to accumulation of misfolded ER proteins? Their prior publication (ref. 40) evaluated several ERAD clients where UBXN1 was dispensable, but it could be possible that UBXN1 has a more specialized client pool. Showing quantification of the PrP CHX chase would also be helpful - from the single replicate it looks like more PrP remaining in the UBXN1 knockout at 8 hours (Supp. Figure 4G).
    5. It would be helpful for the manuscript to clearly distinguish between 1) upregulation of ER proteostasis factors because of ER stress/UPR, and 2) upregulation of secreted clients (AGAL, PrP) which may be partly due to increased translation rates but could also be due to reduced degradation. Many of the hits from the proteomics experiments are ER proteostasis factors that are part of the adaptive stress response (SEC61B, SEC63, CANX, SSR1/2/3, STT3B, RPN1, RPN2, SEC61A1 - compare to ref 12: most are direct IRE1/XBP1s targets). Their increased expression does not lead to increased ER stress as they are involved in the resolution of ER stress. It appears to be circular logic that increased expression of UPR targets would lead to more UPR activation. Currently, the authors do not clearly disentangle the increased expression of endogenous ER proteins from the proteomics experiment versus overexpression of exogenous secreted clients.
    6. The authors should tone down on broad generalizations, for instance in lines 306-309: ER aggregation was only observed for a single client protein (AGAL). Further, only a single mitochondrial protein was observed to be downregulated (TOMM20).

    Minor comments

    • Does UBXN1 localization to the ER/microsomes fraction depend on p97? What happens in UBX-domain mutant?
    • In Fig. 1A it is surprising that no BiP is detected at 0 hours as BiP is highly expressed even in the absence of ER stress. Can the authors comment on this discrepancy.
    • The authors use different ER stressors interchangeably: DTT, Tunicamycin, Thapsigargin. While all results in UPR activation, they do so through different mechanisms and with slight nuances that may be worth considering for the experiments and interpretations.
    • Line 198: "Hierarchical clustering analysis demonstrates that the gene expression pattern observed in UBXN1 KO cells more closely resembles wildtype cells stressed with DTT than untreated wildtype cells based on similar log2 fold change values (Figure 2)." Where is this clustering shown?
    • What are the downregulated UPR genes in Fig. 2, and may this hold significance?

    Significance

    General assessment: The authors broadly characterize the UPR activation in the UBXN1 knockout cells, looking both at gene targets by Western blot and qPCR, and characterize the activation of individual sensors (ATF6 cleavage and IRE1alpha clustering). Proteomics results further corroborate the upregulation of ER-localized proteins, although the robustness of the findings is surprising considering that only 2 replicates were included in the mass spectrometry experiment. Most other experiments are technically sound, for instance the puromycilation translation assays. One of the key limitations of the is that the authors fail to make use of their extensive prior toolset on UBXN1, particularly the segregation-of-function mutations for p97 and ubiquitin binding, as well as the knockdown cell lines with inducible overexpression of UBXN1 to rescue the phenotypes. These tools could probe a direct involvement of UBXN1 in the UPR repression, and whether this activity is truly independent of p97. A related limitation is that results are often over-interpreted and too far generalized (see examples above), or wrongly interpreted (see AGAL degradation rates).

    Advance: The AAA+ ATPase VCP/p97 has many divergent cellular roles that are in part mediated by a variety of different adaptor proteins. The authors have previously discovered the important role for UBXN1 in recruiting p97 to mislocalized cytosolic proteins targeted to the BAG6 complex. The current study now aims to establish a new role for UBXN1 in regulating the unfolded protein response. As it stands, the findings that UBXN1 knockdown results in UPR activation and impacts translation rates are solid but largely descriptive in nature. These findings merit reporting but require that the authors tone down their conclusions about a direct role for UBXN1 as a regulator of the UPR. Alternatively, if the authors choose to stick with their current model for a direct involvement of UBXN1, they need to establish the mechanistic link more clearly.

    Audience: In the current form, the manuscript should appeal to a broad biochemistry and cell biology readership interested in topics related to proteostasis, protein quality control, and stress signaling.

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    Referee #2

    Evidence, reproducibility and clarity

    RC-2022-01803 "UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation independently of the p97 ATPase" By Ahlstedt et al.

    Comments to the Author

    UBXN1 is a VCP adaptor UBX domain protein which is known to be involved in elimination of ubiquitylated cytosolic proteins bound to the BAG6 complex. In this study, authors demonstrated that cells depleted of UBXN1 have elevated UPR activation, even without external ER stresses. Cells devoid of UBXN1 have significant and global up-regulation of UPR-specific target genes, and these cells are more sensitive to ER stress than their wildtype counterparts. Using quantitative tandem mass tag proteomics of UBXN1 deleted cells, authors found that significant enrichment of the abundance of ER proteins involved in protein translocation, protein folding, quality control, and the ER stress response in an ERAD-independent manner. Notably, they observed no change in the abundance of proteins in the cytosol or nucleus, and significant decrease in the expression of several mitochondrial proteins when UBXN1 was depleted. Authors further demonstrate that UBXN1 is a translation repressor, and its UBA domain is critical for suppressing protein synthesis. Thus, increased influx of proteins into the ER in UBXN1 KO cells causes UPR activation. Authors concluded that they have identified a new regulator of protein translation and ER proteostasis.

    My specific comments were provided as follows.

    Comments

    1. Authors found that significant enrichment of the ER proteins in UBXN1 KO cells, while there is no change in the abundance of proteins in the cytosol or nucleus. Mitochondrial proteins are even down-regulated in UBXN1 KO cells. I found these observations very interesting. However, I was frustrated that authors did not investigated the reason why such differences are associated in UBXN1-suppressed cells. Authors demonstrate that depletion of UBXN1 resulted in suppression of protein synthesis, but did not address whether ER proteins are specifically repressed by UBXN1 or it represses translation globally, as noted in their Discussion section. Do the mRNAs encoding signal sequence at the N-terminus of their products are specifically translated in UBXN1-suppressed cells? Do the translations of mRNAs encoding mitochondria translocation signals are suppressed in UBXN1 KO cells? It should be possible to investigate these issues by using appropriate model ER- or mitochondrial proteins with or without specific signal sequences. Such kind of analysis should be necessary to support the claim of this manuscript.
    2. Related to my previous comments, ER-targeted mRNAs are known to be degraded by a process termed RIDD in the case of ER stressed condition. Since the rapid degradation of mRNAs through RIDD functions to alleviate ER stress by preventing the continued influx of new polypeptides into the ER, I wondered why UBXN1 depletion greatly stimulates ER protein synthesis, escaping IRE1-dependent mRNA degradations. Does UBXN1 depletion suppress RIDD?
    3. Authors mentioned that the elevated levels of ER proteins are not due to increased transcription of target genes. However, they only provided the quantification of prp transcript levels, which was unchanged between wildtype and UBXN1 KO cells. To support this important conclusion, it is necessary to provide whole transcriptome data to compare the expression levels of corresponding ER proteins (quantified by their proteomics data) and transcripts (quantified by, for an example, RNA-seq analysis).
    4. Authors claimed that UBXN1 loss is detrimental to cell viability and have elevated levels of the apoptosis in the face of ER stress. However, authors did not examine apoptotic cell death in UBXN1 KO cells. They only provided evidence for defective proliferation of cells and transient induction of CHOP expression, but these are not enough to support the ER-stress induced apoptosis.
    5. Authors showed that UBA domain of UBXN1 is critical for suppressing protein synthesis. Could you provide a bit more detailed discussion how UBA domain modulates protein translational events and promote expressions of ER-related proteins. Have you ever checked whether UBA domain of UBXN1 is necessary for suppressing UPR-specific target gene expressions?

    Significance

    Although the discovery in this manuscript might be potentially interesting for broad audience, the presented study did not provide enough mechanistic insights and their data lacks vital evidences to support their conclusion. I found that the data are preliminary to discuss the validity of this finding. The inadequacy of these points makes this manuscript unsuitable for publication at this stage.

    My expertise is cell biology and biochemistry for protein quality control.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Ahlstedt et al. study UBXN1, an adaptor of the p97/VCP AAA ATPase, using a cell line deficient for UBXN1. They found that the knockout of UBXN1 activates ER stress and sensitizes cells to ER stress-induced cell death. They used a proteomic approach to analyze the change in the global proteome in UBXN1 knockout cells. Interestingly, they found many proteins are upregulated in UBXN1 knockout cells, which appears to be regulated at a post-transcriptional level. Using puromycin labeling, they found that protein translation appears to be upregulated in UBXN1 knockout cells.

    Major comments:

    The conclusions of the manuscript are generally well supported by experimental data, which are of high quality. The presentation is clear. In my opinion, a few issues need to be addressed to further strengthen their conclusions.

    1. The authors need to express UBXN1 and mutants lacking either the UBX or UBA domain in UBXN1 knockout cells to test whether the ER stress phenotype (Figure 1) and the protein upregulation phenotype (Figure 5A-F) can be rescued. This would eliminate the possibility that the reported phenotypes are the off-target effects of CRISPR.
    2. For Figure 2, please indicate whether the repeat is a biological replicate or a technical replicate from RT-PCR.
    3. In Figure 1A, the authors show that the knockout of UBXN1 causes an upregulation of phosphorylated eIF2alpha, which is known to suppress protein translation globally. In this regard, it is surprising to see the authors also concluded from Figure 7 that there is an upregulation of protein translation in UBXN1 knockout cells. The authors do not provide any explanation on how these seemingly contradictory phenotypes could be seen in the same cells.

    Significance

    p97/VCP is an important member of the AAA ATPase family that has a variety of functions. It interacts with a collection of adaptor proteins that all contain a UBX domain. These adaptors help to link the ATPase to the correct substrate in cells. The best-established function of p97/VCP is its role in ERAD, in which it acts together with its adaptors Ufd1-Npl4 and UBXD8 to extract retrotranslocated proteins from the ER for proteasomal degradation. UBXN1 is not required for ERAD. Instead, it appears to be a negative regulator of ERAD. Previous studies have also implicated it in mitophagy (Mengus C., Autophagy, 2022) and aggresome formation (from this group). Overall, the published studies did not pinpoint the precise cellular function of UBXN1.

    This work characterizes the cellular phenotypes associated with UBXN1 loss of function. The information reported here is important, but the biological significance is limited. This is mainly because the authors entirely rely on a genetic approach. While the reported phenotypes associated with UBXN1 deficiency is solid, it is unclear what the underlying mechanisms are. It is not clear whether or not these phenotypes are interconnected, nor is it clear whether UBXN1 is a direct regulator of these processes. Taking the increased protein translation phenotype as an example, does this indicate UBXN1 is a translation suppressor for those ER-associated proteins? How can UBXN1 selectively inhibit the translation of a subset of proteins? Any evidence that UBXN1 is associated with translating ribosomes?

    In summary, because of the limited mechanistic insights on UBXN1 function, the study may only be interesting to a specialized audience.

  5. Version published to 10.1101/2022.12.16.520763 on bioRxiv