Rabaptin5 targets autophagy to damaged endosomes and SCVs by interaction with FIP200 and ATG16L1

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Reply to the reviewers

Point-by-point response to reviewer comments

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In the current manuscript, Millarte et al reports a novel role of Rabaptin5 in selectively clearing damaged endosomes via canonical autophagy. They have identified FIP200 as a novel interactor of Rabaptin5 under basal conditions using yeast-two hybrid screening and further confirmed the interaction of Rabaptin5 with FIP200 with immunoprecipitation. They next used Chloroquine and monitored colocalization of the Rabaptin5 with WIPI2, ATG16L1 and LC3B to demonstrate the potential interaction of Rabaptin5 with the autophagic machinery. They have primarily used Gal-3 as a marker of membrane damage after 30 minutes of Chloroquine treatment. In order to further elucidate the role of Rabaptin5 in autophagic induction mediated by Chloroquine, they have silenced Rabaptin5, FIP200, ULK1 and ATG13 and observed a decrease in the number of LC3 or WIPI2 autophagosome formation. Based on these observations they tested if Rabaptin5 interacts with ATG16L1 upon Chloroquine treatment and confirmed their interaction with potential interaction sites of both Rabaptin5 with ATG16L1 with IP. The authors confirmed the interaction of Rabaptin5 with ATG16L1 by complementing the KO line with the mutant form of Rabaptin5 containing alanine residues in its consensus motif. Finally, they have used Salmonella and SCV as a model to study the role of Rabaptin5 in endomembrane damage and monitored a 50% decrease in the removal of Salmonella in Rabaptin5 KO or KD cells.

Major concerns One of the major concerns is the membrane damage reported by chloroquine which is known to induce lysosomal swelling and further targeting of the swollen compartments to degradation by direct conjugation of LC3 onto single membrane as a form of non-canonical autophagy. The evidence regarding membrane damage by Gal3 colocalization on the Rabaptin5 vesicles is preliminary. As suggested by the authors the canonical autophagy pathway recognizing damaged membranes recruits also ALIX to the damaged membrane which was not observed in Supplementary Figure 2. The link to membrane damage by chloroquine and monensin with Rabaptin5 is not convincing as there is not sufficient evidence of membrane damage. In relation to this issue authors should consider using other damage markers as Gal8, p62 or NDP52 to provide additional claim with respect to membrane damage induced by chloroquine.

To expand on the question of CQ treatment damaging early endosomes, we also tested for Gal8 on Rabaptin5-positive enlarged endosomes and quantified the fraction of Rabaptin5-positive rings positive for Gal3 and Gal8 after 30 min of CQ treatment. We propose to include this data in Figure 2:

*We have tested the importance of Gal3 and p62 by siRNA-mediated knockdown where we found a robust inhibition of induction of WIPI2 puncta with CQ, but not with Torin1. Formation of LC3 puncta was less reduced, similar to knockdowns of FIP200, ATG13, or Rabaptin5. *

We propose to add these knockdown experiments as a supplementary figure:

One of the main claims here is that Rabaptin5 regulates the targeting of damaged endosomes to autophagy. Clearly, these are early endosomes as stated in the abstract. However, the evidence presented here showing these are early endosomes is not convincing. Analysing Gal3 and Gal8 positive vesicles that are Rabaptin5 positive and an early endosomal marker will be important in this context. For example, there need to be additional evidence showing that early endosomes are targeted to autophagy. Is the degradation of TfR affected by this targeting? Did the authors look at the effect of Bafilomycin A1? If this process affects exclusively early endosomes, it should be BafA1 independent. This will direct more into the cellular function of this process.

Rabaptin5 is a bona fide marker of Rab5-positive early sorting endosomes. As a control, we confirmed colocalization of Rabaptin5 with transferrin receptor, another endosomal marker, on CQ-induced rings (Fig. 2B). We now also analyzed swollen endosomes with triple-staining for Rabaptin5, transferrin receptor, and Gal3 as shown in this gallery (30 min CQ, as in Fig. 2). All Rabaptin5-positive swollen endosomes (rings) were positive for transferrin receptor and ~80% for mCherry-Gal3.

We further tested transferrin receptor levels with and without CQ. Since CQ inhibits autophagic flux, this assay may not be very sensitive. Nevertheless, we found a significant reduction of ~15% and ~30% after overnight incubation with CQ in parental HEK293A cells and in Rbpt5-KO cells re-expressing wild-type Rabaptin5, resp., but no reduction in Rbpt5-KO cells expressing the Rabaptin5-AAA mutant defective in binding to ATG16L1:

As to the effect of BafA1, see our general response on top. The osmotic effect of CQ or Mon on endosomes that leads to membrane breakage requires an acidic pH. Preincubation with BafA1 neutralizes the pH, prevents osmotic swelling by CQ/Mon, and was shown to block LC3 lipidation (Florey et al., 2015, Jacquin et al., 2017). When BafA1 was added simultaneously, CQ was found to induce LC3 despite the presence of BafA1 (Mauthe et al., 2018), and Mon was shown to still be able to break endosomal membranes and recruit LC3 to EEA1-positive endosomes (Fraser et al., 2019). However, CQ-induced LC3 recruitment to latex bead-containing phagosomes or entotic vacuoles, i.e. LAP-like autophagy, was blocked (Florey et al., 2015). Consistent with this literature, we found increased LC3B lipidation already within 30 min of CQ treatment independently of BafA1 (no preincubation).

Upon longer incubations, LC3B lipidation is very strong already with BafA1 alone so that the effect of CQ cannot be assessed anymore, since both drugs inhibit autophagic flux.

Furthermore, we found a CQ-dependent increase in WIPI2- and LC3B-positive puncta to be insensitive to BafA1 (panel A below). Colocalization of Rabaptin5 to LC3B and LC3B to Rabaptin5 significantly increased upon CQ treatment independently of the presence of BafA1 (no pretreatment), indicating that at least a large part of CQ-induced LC3B puncta is not due to LAP-like autophagy.

Minor concerns Both for Figure 2 and Supplementary Figure 7 it will be clearer to have the images in colour rather than black and white for better interpretation.

We thought the grayscale images were clearer, but are happy to provide color images.

The interaction of FIP200 and ATG16L1 with Rabaptin5 is well characterized with immunoprecipitation and imaging but the interaction of Rabaptin5 in presence of chloroquine with FIP200 and ATG16L1 DWD are missing and it will be important to include if in the presence of chloroquine these interactions will increase or not.

We can do co-IP experiments also upon CQ treatment.

In order to further support the role of Rabaptin5 for LC3 lipidation upon chloroquine induced membrane damage, western blots of WT, +Rabaptin5, Rabaptin5 KO, Rabaption5 KO +WT or +AAA cell lines were analysed. However, the lysates were collected upon 30 minutes of chloroquine treatment which does not correlate with the imaging performed in Figure 2 as the number of LC3 vesicles did not show an increase upon 30 minutes of chloroquine treatment. The authors should include the 150 minutes time point for the LC3 lipidation in these conditions.

Because CQ inhibits autophagic flux, LC3-II accumulates after longer times in all cell lines. The differences can only be seen early.

The experiments with Salmonella are of great quality. The relationship of Rabaptin5 with SCV and the endomembrane damage induced by Salmonella could be further elucidated with Rabaptin5 positive vesicles at early infection stages. It is not very clear from the text how authors link the endosomal network previously described for chloroquine with infection. It would be important here to show that Salmonella mutants unable to damage endosomal membranes do not have an effect. In Figure 7 panel C, the time points on graphs are in hours but it should be in minutes. corrected.

*Since Salmonella require T3SS for infection of HEK cells and T3SS causes the membrane damage, the proposed experiment is difficult. *

The events of targeting the damaged membranes for degradation was well characterized by the recognition of these membranes by Gal3, Gal8 and recruitment of autophagic receptors to the site of damage (Chauhan et al. 2016; Jia et al. 2019; Thurston et al. 2012; Maejima et al. 2013; Kreibich et al. 2015). This manuscript introduces a new potential platform for the formation of autophagic machinery on endosomes with the interaction of Rabaptin5 with FIP200 and ATG16L1, however more evidence is required to link this to the clearance of damaged membranes. Previously it was shown that endolysosomal compartments that were neutralized and swollen by monensin and chloroquine had been directed to degradation by direct conjugation of LC3 to single membranes via noncanonical autophagy, but here authors propose another mechanism for this event via canonical autophagy.

*As discussed in the general response above, the literature reports CQ and Mon to initiate both canonical autophagy and LAP-like autophagy, the latter particularly on phagosomes containing latex beads or entotic vacuoles. Our results – including the additional data above –concern the effects of CQ and Mon damaging early endosomes and causing recruitment of galectins and ubiquitination, triggering autophagy dependent on the ULK complex and WIPI2 as hallmarks of canonical autophagy, and Rabaptin5. The reviewer comments highlighted the possibility of LAP-like autophagy occurring in parallel, perhaps on endosomes that are not broken, which might explain the relative insensitivity of LC3 puncta induced by CQ and Mon – compared to the strong and robust reduction of WIPI2 puncta – on the knockdown of FIP200, ATG13, or Rabaptin5. In an alternative explanation, inhibition of autophagic flux causes remaining canonical autophagy to accumulate, while WIPI2 puncta are strongly inhibited. **In support of the latter interpretation, ULK inhibition by MRT68921 (Fig. 4C and D) or FIP200 knockout (Fig. 6B and C) abolished CQ-induced LC3 structures, suggesting that – unlike on phagosomes or entotic vacuoles – there is little LAP-like autophagy. *We propose to revise the manuscript to discuss these considerations more clearly.

Reviewer #1 (Significance (Required)):

Overall this work is very novel and shows some evidence of early endosomal autophagy. It could be relevant for some for of receptor-mediated signalling (although it is not discussed by the authors) My experience is in intracellular trafficking of pathogens and membrane damage.

**Referee Cross-commenting**

In my opinion, the only way you can distinguish between double or single membrane is by EM. For me, the important part is to show this is targeting of early endosomes to autophagy, either using other early endosomal markers, analysing by WB some early endosome receptors such as TfR or other inhibitors. If the authors are able to address some these comments, I agree the paper will be in a better position for publication.

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Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Millarte et al study the role of Radaptin-5 (Rbpt5) during early endosome damage recognition by autophagy. The authors focus on using chloroquine (CQ) as an agent to induce endosomal swelling/damage and suggest that Rbpt5 is required for the recruitment of the autophagy machinery to perturbed endosomes. They further use salmonella infection as a model to confirm the role of Rbpt5 in this process. The authors initially show that Rbpt5 binds to FIP200 and subsequently focus on its interaction with ATG16L1 and identify a mutant that is unable to bind ATG16L1 or mediate the recognition of early endosomes by autophagy. Overall, this is an interesting study which provides molecular insights of how early endosomes can be targeted by the autophagy machinery where Rbpt5 may act as an autophagy receptor. Some specific comments are as follows:

Fig.3A: siRbpt5 seems to induce the localization of LC3 to ring-like structures during CQ treatment. Are these LAP-like structures (e.g. sensitive to BafA1 treatment)? And were they included in the quantification in Fig.3C?

Ring-like LC3 structures were also counted.

As discussed in the general remarks above, it is a possibility that knockdown-resistent LC3 recruitment (particularly rings) is due to a CQ-induced LAP-like process. The alternative explanation is that there is residual canonical autophagy upon knockdown of Rabaptin5, ATG13, or FIP200: while WIPI2 puncta are strongly reduced, LC3-positive structures accumulate due to inhibition of autophagic flux. In support of the latter interpretation, ULK inhibition by MRT68921 (Fig. 4C and D) or FIP200 knockout (Fig. 6B and C) abolished CQ-induced LC3 puncta or rings.

We can also test BafA1 treatment. Certainly, we will revise the text to discuss this point in more detail.

Fig.4A&B: Since Rbpt5 KD has a weak effect on LC3 puncta formation (Fig.3) and to distinguish the effects of CQ in inducing LAP, the effects of ATG13 and ULK1 KD should be assessed by localising Rbpt5 with WIPI2 or ATG16L1.

We can do that.

Fig.4: It is not clear why ULK1 KD would affect Torin1-induced autophagy but not LC3/WIPI2 localisation during CQ induced early endosome-damage. As the ULK inhibitors can target other pathways, the authors should confirm this finding in ULK1/2 double KO or KD cells.

*We have used *MRT68921, because it is frequently used in the literature for this purpose with high specificity. It was used for example by Lystad et al. (2019) together with VPS34IN1 to block all canonical autophagy to analyze exclusively noncanonical effects of monensin treatment. We could perform ULK1/2 double knockdowns, but since ULK2 cannot be detected on immunoblots in HEK293 cells, the result would be interpretable only when there is an effect.

Fig.5: The contribution of FIP200 in the interaction between Rbpt5 and ATG16L1 is unclear. Is binding between Rbpt5 and ATG16L1 mediated by ATG16L1's interaction with FIP200? The plasmid details describing the delta-WD40 deletion plasmid used in this study are missing and could be important to confirm that the detla-WD40 still retains binding to FIP200.

We will of course include the details on the deletion plasmid, which were missing by mistake. Our WD deletion construct of ATG16L1 consists of residues 1–319, precisely deleting just the WD40 repeats, but retaining the FIP200 interaction sequence and the second membrane binding segment (b).

We did a co-immunoprecipitation experiment and found both wild-type ATG16L1 and the ∆WD mutant to co-immunoprecipitate with FIP200:

Fig.5E: the authors should test Rbpt5 AAA mutant binding to FIP200. Since the mutant appears to express less, its binding to ATG16L1 should be quantified or repeated with more comparable expression levels.

We will quantify the immunoblots and perhaps attempt getting more equal expression levels.

Fig.6: CQ treatment can induce various endosomal damage (in addition to early endosomes) and LC3 lipidation processes (e.g. LAP-like). The authors show that Rbpt5 is specifically involved in damaged early endosome autophagy. In this figure, it would be important to distinguish CQ-induced LC3 puncta as a result of early endosome damage or other lipidation processes (e.g. canonical or non-canonical autophagy). The use of FIP200 KO cells shows that LC3 puncta is inhibited. However, here a specific readout to look at early endosome recognition by autophagy is important. The authors can localize early endosome markers (EEA1) with autophagy players (e.g. WIPI2 and LC3). This is also relevant to other figures (e.g. supplementary figure 7E).

Rabaptin5 is a bona fide marker of Rab5-positive early sorting endosomes. As a control, we confirmed colocalization of Rabaptin5 with transferrin receptor, another endosomal marker, on CQ-induced rings (Fig. 2B). We also analyzed swollen endosomes with triple-staining for Rabaptin5/ transferrin receptor/ Gal3 as shown in this gallery (30 min CQ, as in Fig. 2). All Rabaptin5-positive swollen endosomes (rings) were positive for transferrin receptor and ~80% for mCherry-Gal3.

Our results are in agreement with Fraser et al. (2019) where they use EEA1 as an endosomal marker upon monensin treatment.

We also performed a colocalization analysis for Rabaptin5 and LC3B, showing enhanced colocalization after CQ treatment for 150 min: ~20% of LC3B is (still) pos for Rabaptin5 after 150 min of CQ treatment:

Fig.6F&G: the authors should show representative images of these localization images quantified here. These can be added in the supplementary figures.

We are happy to do this.

**Minor comments:**

Fig.2E: FIP200 seems to be highly overexpressed in this image. Commercial antibodies that recognise endogenous FIP200 are widely used and should be tested to confirm the colocalisation between FIP200 and Rbpt5.

We plan to do this.

Fig.7C image: the different setting denoted by +/-, +/+ ..etc are not clearly defined.

We will improve this.

Reviewer #2 (Significance (Required)):

This is a interesting study and provides important mechanistic insights underlying the recognition of perturbed early endosomes by the autophagy machinery. Researchers interested in endosomal trafficking or autophagic substrate recognition are likely to benefit from this study.

**Referee Cross-commenting**

In my opinion, the authors have attempted to distinguish single membrane from double membrane LC3 lipidation by looking at the ULK complex requirement. As other reviewers suggested, this can be further confirmed by using ATG16L1 mutants. It is important however that these experiments are supplemented by co-localising autophagy proteins with alternative early endosome markers when Rbpt5 is inhibited.

I think if the authors are able to address the suggested experiments, this would help improve the manuscript and make it suitable for publication.

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Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Millarte and colleagues find that Rabaptin5, a critical regulator of Rab5 activity, and a protein localized to early endosomes, interacts with FIP200 and ATG16L1. This interaction is confirmed and validated by a number of approaches (yeast 2 H, co-immunoprecipitation) and the binding sites of Rabaptin5 are mapped on FIP200 and ATG16L1. More precisely the binding site for ATG16L1 is nicely mapped on Rabaptin 5 by analogy with other ATG16L1 binders. The authors investigate the significance of this binding of Rabaptin5 to the autophagy proteins by proposing this interaction is required for targeting "autophagy to damaged endosomes". Endosomes are damaged with short treatments of chloroquine, a well studied compound previously shown to inhibit autophagy by disrupting fusion of autophagosomes with lysosomes. They propose the recruitment of autophagy (proteins) to the damaged endosomes may allow them to be eliminated. They use another model (phagocytosis of salmonella) to probe the role for rabaptin5 and its partners FIP200 and ATG16L1 in the well-documented role of autophagy on the elimination of salmonella in SCVs (Salmonella containing vacuole) formed from endosomes. Using short infection time points, and the Rabaptin5 mutants which prevent ATG16L1 binding they suggest Rabaptin5 binding contributes to elimination and killing of Salmonella by recruitment of ATG16L1.

**Major comments:**

1. The authors make an unfortunate and confusing choice of wording in the title and the text of "autophagy being recruited" to damaged early endosomes. A protein can recruit another protein but it can not recruit a process or pathway to a membrane.

In the title we use the term "target". It is OK for us to avoid the expression "recruiting autophagy".

The authors conclude that Rabaptin5 may have a role in autophagy directed to damaged early endosomes. The conclusion that Rabaptin5 binds FIP200 and ATG16L1 are convincing. The main issue is however in identifying what sort of process they are following. They have shown WIPI2 and LC3 can be recruited to early endosomes after 30 min chloroquine treatment but there is no data to explain the consequences of the binding of these proteins. They do not provide proof that canonical autophagosomes are formed which engulf and remove the damaged endosomes, nor do they show that the recruitment of WIPI2 is to a single membrane (presumably damaged early endosomes) which would be a non-canonical pathway. They often use the terminology "chloroquine-induced autophagy" (see Figure 4) but have virtually no proof they have induced either canonical or non-canonical pathways in their experiments. The only evidence they provide that there is some alteration in a membrane-mediated event is increase in lipidation of LC3 in Figure 6. The authors must follow either an early endosome protein or cargo to demonstrate lysosome-mediated degradation indicative of autophagy, or demonstrate the process is a variation on non-canonical autophagy.

We analyzed transferrin receptor levels with and without CQ to test degradation of an early endosomal marker protein. Since CQ inhibits autophagic flux, this assay may not be very sensitive. Nevertheless, we found a significant reduction of ~15% and ~30% after overnight incubation with CQ in parental HEK293 cells and in Rbpt5-KO cells re-expressing wild-type Rabaptin5, resp., but no reduction in Rbpt5-KO cells expressing the Rabaptin5-AAA mutant defective in binding to ATG16L1:

There are concerns about the replicates done for many experiments in particular the co-immunoprecipitations which are not quantified (Figure 1 and 5).

We will quantify these blots.

The rescue experiments, even if done with stable cells lines made in the parental HEK293 cell line should be viewed with caution because of the very different amounts of Rabaptin5 (see Figure 6A). The overexpression of Rabaptin5 has not been well studied and comparisons with the mutants are therefore preliminary (Figure 6F and G).

Fig 6A shows that Rabaptin5 levels are similar except for +Rbpt, where they are higher, and R-KO, which has none. Additional Rabaptin5 seems not to significantly enhance early WIPI and ATG16L1 colocalization.

Conclusions about the role of the ULK complex, or ULK1 versus ULK2, should be expanded by studying the activity of the complex (phosphorylation of ATG13 for example) in order to make the conclusions more significant.

We consider this to be beyond the scope of this study. Rabaptin5-dependent autophagy depends on the components of the ULK complex.

**Minor comments:**

1. Much of the labelling in the immunofluorescence images is not visible even on the screen version.

We were careful to have the signals within the dynamic range of the image, but we can enhance the signals for better visibility.

The LC3-lipidation experiment (Figure 6D) should be re-analysed by normalization to the loading control. The result may be significantly different and is open to re-interpretation. The quality of this western blot is also very poor.

*Quantitation was based on the ratio between LC3B-I and -II or the *percentage of II of the total, always within the same lane and therefore largely independent of loading.

Reviewer #3 (Significance (Required)):

This manuscript topic fits into the field of study of canonical versus non-canonical autophagy. This literature is best described as "LAP" first discovered by Doug Green, (Sanjuan in 2009) but more recently as a phenomena induced by monesin, and viral infection amongst others. Most relevant to this study are the references (in the text) by Florey (Autophagy 2015), Fletcher (EMBO J, 2018) and others. However, this manuscript fails to cite and consider the critical findings in a key study published by Lystad et al., Nature Cell Biology 2019, which examines the role of ATG16 in both canonical and non-canonical autophagy. The current study if placed into the context of the Lystad study would have significantly more value, and potentially make the findings more significant.

We did not refer to Lystad et al. (2019), because they analyzed different ATG16L1 mutants on their contribution to monensin-induced processes on LC3 lipidation after completely blocking canonical autophagy with the ULK inhibitor MRT68921 and the VPS34 inhibitor VPS34IN1. The Rabaptin5-dependent CQ-induced processes are blocked by MRT68921 (Fig. 4C). We plan to refer to this study in the revision.

Furthermore, the short chloroquine treatments used here could be of interest to the field if using the cited study of Mauthe et al., (which very clearly defines the effect of chloroquine after long (5 hrs treatment)) the authors would to revisit and repeat some of the key experiments in order to demonstrate the effects of 30 minute treatment. Does such short treatment block fusion? Does it affect the pH of the acidic compartments? Does it inactivate the endocytitic pathway? As the manuscript stands the lack of this understanding of the effect of chloroquine at short times, makes the observations difficult to be place into any biological context.

This reviewer has expertise in autophagy, autophagosome formation and is familiar with the areas of endocytosis and infection.

**Referee Cross-commenting**

I think a major concern about the manuscript which is present in all reviews is the lack of clarity about what type of membrane LC3 is added to- is this the damaged endosome or a forming autophagosome? This leads to the question of what type of process is being observed here? non-canonical versus canonical autophagy.

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Referee #3

Evidence, reproducibility and clarity

Millarte and colleagues find that Rabaptin5, a critical regulator of Rab5 activity, and a protein localized to early endosomes, interacts with FIP200 and ATG16L1. This interaction is confirmed and validated by a number of approaches (yeast 2 H, co-immunoprecipitation) and the binding sites of Rabaptin5 are mapped on FIP200 and ATG16L1. More precisely the binding site for ATG16L1 is nicely mapped on Rabaptin 5 by analogy with other ATG16L1 binders. The authors investigate the significance of this binding of Rabaptin5 to the autophagy proteins by proposing this interaction is required for targeting "autophagy to damaged endosomes". Endosomes are damaged with short treatments of chloroquine, a well studied compound previously shown to inhibit autophagy by disrupting fusion of autophagosomes with lysosomes. They propose the recruitment of autophagy (proteins) to the damaged endosomes may allow them to be eliminated. They use another model (phagocytosis of salmonella) to probe the role for rabaptin5 and its partners FIP200 and ATG16L1 in the well-documented role of autophagy on the elimination of salmonella in SCVs (Salmonella containing vacuole) formed from endosomes. Using short infection time points, and the Rabaptin5 mutants which prevent ATG16L1 binding they suggest Rabaptin5 binding contributes to elimination and killing of Salmonella by recruitment of ATG16L1.

Major comments:

1. The authors make an unfortunate and confusing choice of wording in the title and the text of "autophagy being recruited" to damaged early endosomes. A protein can recruit another protein but it can not recruit a process or pathway to a membrane.
2. The authors conclude that Rabaptin5 may have a role in autophagy directed to damaged early endosomes. The conclusion that Rabaptin5 binds FIP200 and ATG16L1 are convincing. The main issue is however in identifying what sort of process they are following. They have shown WIPI2 and LC3 can be recruited to early endosomes after 30 min chloroquine treatment but there is no data to explain the consequences of the binding of these proteins. They do not provide proof that canonical autophagosomes are formed which engulf and remove the damaged endosomes, nor do they show that the recruitment of WIPI2 is to a single membrane (presumably damaged early endosomes) which would be a non-canonical pathway. They often use the terminology "chloroquine-induced autophagy" (see Figure 4) but have virtually no proof they have induced either canonical or non-canonical pathways in their experiments. The only evidence they provide that there is some alteration in a membrane-mediated event is increase in lipidation of LC3 in Figure 6. The authors must follow either an early endosome protein or cargo to demonstrate lysosome-mediated degradation indicative of autophagy, or demonstrate the process is a variation on non-canonical autophagy.
3. There are concerns about the replicates done for many experiments in particular the co-immunoprecipitations which are not quantified (Figure 1 and 5).
4. The rescue experiments, even if done with stable cells lines made in the parental HEK293 cell line should be viewed with caution because of the very different amounts of Rabaptin5 (see Figure 6A). The overexpression of Rabaptin5 has not been well studied and comparisons with the mutants are therefore preliminary (Figure 6F and G).
5. Conclusions about the role of the ULK complex, or ULK1 versus ULK2, should be expanded by studying the activity of the complex (phosphorylation of ATG13 for example) in order to make the conclusions more significant.

Minor comments:

1. Much of the labelling in the immunofluorescence images is not visible even on the screen version.
2. The LC3-lipidation experiment (Figure 6D) should be re-analysed by normalization to the loading control. The result may be significantly different and is open to re-interpretation. The quality of this western blot is also very poor.

Significance

This manuscript topic fits into the field of study of canonical versus non-canonical autophagy. This literature is best described as "LAP" first discovered by Doug Green, (Sanjuan in 2009) but more recently as a phenomena induced by monesin, and viral infection amongst others. Most relevant to this study are the references (in the text) by Florey (Autophagy 2015), Fletcher (EMBO J, 2018) and others. However, this manuscript fails to cite and consider the critical findings in a key study published by Lystad et al., Nature Cell Biology 2019, which examines the role of ATG16 in both canonical and non-canonical autophagy. The current study if placed into the context of the Lystad study would have significantly more value, and potentially make the findings more significant.

Furthermore, the short chloroquine treatments used here could be of interest to the field if using the cited study of Mauthe et al., (which very clearly defines the effect of chloroquine after long (5 hrs treatment)) the authors would to revisit and repeat some of the key experiments in order to demonstrate the effects of 30 minute treatment. Does such short treatment block fusion? Does it affect the pH of the acidic compartments? Does it inactivate the endocytitic pathway? As the manuscript stands the lack of this understanding of the effect of chloroquine at short times, makes the observations difficult to be place into any biological context.

This reviewer has expertise in autophagy, autophagosome formation and is familiar with the areas of endocytosis and infection.

Referee Cross-commenting

I think a major concern about the manuscript which is present in all reviews is the lack of clarity about what type of membrane LC3 is added to- is this the damaged endosome or a forming autophagosome? This leads to the question of what type of process is being observed here? non-canonical versus canonical autophagy.

Read the original source

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Millarte et al study the role of Radaptin-5 (Rbpt5) during early endosome damage recognition by autophagy. The authors focus on using chloroquine (CQ) as an agent to induce endosomal swelling/damage and suggest that Rbpt5 is required for the recruitment of the autophagy machinery to perturbed endosomes. They further use salmonella infection as a model to confirm the role of Rbpt5 in this process. The authors initially show that Rbpt5 binds to FIP200 and subsequently focus on its interaction with ATG16L1 and identify a mutant that is unable to bind ATG16L1 or mediate the recognition of early endosomes by autophagy. Overall, this is an interesting study which provides molecular insights of how early endosomes can be targeted by the autophagy machinery where Rbpt5 may act as an autophagy receptor. Some specific comments are as follows:

Fig.3A: siRbpt5 seems to induce the localization of LC3 to ring-like structures during CQ treatment. Are these LAP-like structures (e.g. sensitive to BafA1 treatment)? And were they included in the quantification in Fig.3C?

Fig.4A&B: Since Rbpt5 KD has a weak effect on LC3 puncta formation (Fig.3) and to distinguish the effects of CQ in inducing LAP, the effects of ATG13 and ULK1 KD should be assessed by localising Rbpt5 with WIPI2 or ATG16L1.

Fig.4: It is not clear why ULK1 KD would affect Torin1-induced autophagy but not LC3/WIPI2 localisation during CQ induced early endosome-damage. As the ULK inhibitors can target other pathways, the authors should confirm this finding in ULK1/2 double KO or KD cells.

Fig.5: The contribution of FIP200 in the interaction between Rbpt5 and ATG16L1 is unclear. Is binding between Rbpt5 and ATG16L1 mediated by ATG16L1's interaction with FIP200? The plasmid details describing the delta-WD40 deletion plasmid used in this study are missing and could be important to confirm that the detla-WD40 still retains binding to FIP200.

Fig.5E: the authors should test Rbpt5 AAA mutant binding to FIP200. Since the mutant appears to express less, its binding to ATG16L1 should be quantified or repeated with more comparable expression levels.

Fig.6: CQ treatment can induce various endosomal damage (in addition to early endosomes) and LC3 lipidation processes (e.g. LAP-like). The authors show that Rbpt5 is specifically involved in damaged early endosome autophagy. In this figure, it would be important to distinguish CQ-induced LC3 puncta as a result of early endosome damage or other lipidation processes (e.g. canonical or non-canonical autophagy). The use of FIP200 KO cells shows that LC3 puncta is inhibited. However, here a specific readout to look at early endosome recognition by autophagy is important. The authors can localize early endosome markers (EEA1) with autophagy players (e.g. WIPI2 and LC3). This is also relevant to other figures (e.g. supplementary figure 7E).

Fig.6F&G: the authors should show representative images of these localization images quantified here. These can be added in the supplementary figures.

Minor comments:

Fig.2E: FIP200 seems to be highly overexpressed in this image. Commercial antibodies that recognise endogenous FIP200 are widely used and should be tested to confirm the colocalisation between FIP200 and Rbpt5.

Fig.7C image: the different setting denoted by +/-, +/+ ..etc are not clearly defined.

Significance

This is a interesting study and provides important mechanistic insights underlying the recognition of perturbed early endosomes by the autophagy machinery. Researchers interested in endosomal trafficking or autophagic substrate recognition are likely to benefit from this study.

Referee Cross-commenting

In my opinion, the authors have attempted to distinguish single membrane from double membrane LC3 lipidation by looking at the ULK complex requirement. As other reviewers suggested, this can be further confirmed by using ATG16L1 mutants. It is important however that these experiments are supplemented by co-localising autophagy proteins with alternative early endosome markers when Rbpt5 is inhibited.

I think if the authors are able to address the suggested experiments, this would help improve the manuscript and make it suitable for publication.

Referee Cross-commenting

In my opinion, the authors have attempted to distinguish single membrane from double membrane LC3 lipidation by looking at the ULK complex requirement. As other reviewers suggested, this can be further confirmed by using ATG16L1 mutants. It is important however that these experiments are supplemented by co-localising autophagy proteins with alternative early endosome markers when Rbpt5 is inhibited.

I think if the authors are able to address the suggested experiments, this would help improve the manuscript and make it suitable for publication.

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Referee #1

Evidence, reproducibility and clarity

In the current manuscript, Millarte et al reports a novel role of Rabaptin5 in selectively clearing damaged endosomes via canonical autophagy. They have identified FIP200 as a novel interactor of Rabaptin5 under basal conditions using yeast-two hybrid screening and further confirmed the interaction of Rabaptin5 with FIP200 with immunoprecipitation. They next used Chloroquine and monitored colocalization of the Rabaptin5 with WIPI2, ATG16L1 and LC3B to demonstrate the potential interaction of Rabaptin5 with the autophagic machinery. They have primarily used Gal-3 as a marker of membrane damage after 30 minutes of Chloroquine treatment. In order to further elucidate the role of Rabaptin5 in autophagic induction mediated by Chloroquine, they have silenced Rabaptin5, FIP200, ULK1 and ATG13 and observed a decrease in the number of LC3 or WIPI2 autophagosome formation. Based on these observations they tested if Rabaptin5 interacts with ATG16L1 upon Chloroquine treatment and confirmed their interaction with potential interaction sites of both Rabaptin5 with ATG16L1 with IP. The authors confirmed the interaction of Rabaptin5 with ATG16L1 by complementing the KO line with the mutant form of Rabaptin5 containing alanine residues in its consensus motif. Finally, they have used Salmonella and SCV as a model to study the role of Rabaptin5 in endomembrane damage and monitored a 50% decrease in the removal of Salmonella in Rabaptin5 KO or KD cells.

Major concerns One of the major concerns is the membrane damage reported by chloroquine which is known to induce lysosomal swelling and further targeting of the swollen compartments to degradation by direct conjugation of LC3 onto single membrane as a form of non-canonical autophagy. The evidence regarding membrane damage by Gal3 colocalization on the Rabaptin5 vesicles is preliminary. As suggested by the authors the canonical autophagy pathway recognizing damaged membranes recruits also ALIX to the damaged membrane which was not observed in Supplementary Figure 2. The link to membrane damage by chloroquine and monensin with Rabaptin5 is not convincing as there is not sufficient evidence of membrane damage. In relation to this issue authors should consider using other damage markers as Gal8, p62 or NDP52 to provide additional claim with respect to membrane damage induced by chloroquine.

One of the main claims here is that Rabaptin5 regulates the targeting of damaged endosomes to autophagy. Clearly, these are early endosomes as stated in the abstract. However, the evidence presented here showing these are early endosomes is not convincing. Analysing Gal3 and Gal8 positive vesicles that are Rabaptin5 positive and an early endosomal marker will be important in this context. For example, there need to be additional evidence showing that early endosomes are targeted to autophagy. Is the degradation of TfR affected by this targeting? Did the authors look at the effect of Bafilomycin A1? If this process affects exclusively early endosomes, it should be BafA1 independent. This will direct more into the cellular function of this process.

Minor concerns Both for Figure 2 and Supplementary Figure 7 it will be clearer to have the images in colour rather than black and white for better interpretation.

The interaction of FIP200 and ATG16L1 with Rabaptin5 is well characterized with immunoprecipitation and imaging but the interaction of Rabaptin5 in presence of chloroquine with FIP200 and ATG16L1 WD are missing and it will be important to include if in the presence of chloroquine these interactions will increase or not.

In order to further support the role of Rabaptin5 for LC3 lipidation upon chloroquine induced membrane damage, western blots of WT, +Rabaptin5, Rabaptin5 KO, Rabaption5 KO +WT or +AAA cell lines were analysed. However, the lysates were collected upon 30 minutes of chloroquine treatment which does not correlate with the imaging performed in Figure 2 as the number of LC3 vesicles did not show an increase upon 30 minutes of chloroquine treatment. The authors should include the 150 minutes time point for the LC3 lipidation in these conditions.

The experiments with Salmonella are of great quality. The relationship of Rabaptin5 with SCV and the endomembrane damage induced by Salmonella could be further elucidated with Rabaptin5 positive vesicles at early infection stages. It is not very clear from the text how authors link the endosomal network previously described for chloroquine with infection. It would be important here to show that Salmonella mutants unable to damage endosomal membranes do not have an effect. In Figure 7 panel C, the time points on graphs are in hours but it should be in minutes.

The events of targeting the damaged membranes for degradation was well characterized by the recognition of these membranes by Gal3, Gal8 and recruitment of autophagic receptors to the site of damage (Chauhan et al. 2016; Jia et al. 2019; Thurston et al. 2012; Maejima et al. 2013; Kreibich et al. 2015). This manuscript introduces a new potential platform for the formation of autophagic machinery on endosomes with the interaction of Rabaptin5 with FIP200 and ATG16L1, however more evidence is required to link this to the clearance of damaged membranes. Previously it was shown that endolysosomal compartments that were neutralized and swollen by monensin and chloroquine had been directed to degradation by direct conjugation of LC3 to single membranes via noncanonical autophagy, but here authors propose another mechanism for this event via canonical autophagy.

Significance

Overall this work is very novel and shows some evidence of early endosomal autophagy. It could be relevant for some for of receptor-mediated signalling (although it is not discussed by the authors) My experience is in intracellular trafficking of pathogens and membrane damage.

Referee Cross-commenting

In my opinion, the only way you can distinguish between double or single membrane is by EM. For me, the important part is to show this is targeting of early endosomes to autophagy, either using other early endosomal markers, analysing by WB some early endosome receptors such as TfR or other inhibitors. If the authors are able to address some these comments, I agree the paper will be in a better position for publication.

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