Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

  1. Tadayuki Komori
  2. Tomoki Kuwahara
  3. Tetta Fujimoto
  4. Maria Sakurai
  5. Ikuko Koyama-Honda
  6. Mitsunori Fukuda
  7. Takeshi Iwatsubo

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here, we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

Article activity feed

  1. Version published to 10.1242/jcs.261003
  5. Review Commons

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

    Learn more at Review Commons

    Reply to the reviewers

    1. General Statements [optional]

    We would like to thank the reviewers for taking time in reviewing and commenting on our paper. The comments were very constructive and conscientious, thanks to their expertise in the field. These comments and the revisions would surely make this paper a better and more robust finding in the field.

    The comments were about clearer explanations, increasing the quality of the data and additional experiments for a stronger conclusion, all of which we are eager to accomplish. Now we have sorted out the problems and planned the experiments required in the revision, as detailed below.

    2. Description of the planned revisions

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

    Summary In this manuscript, Komori et al. examined the role of the LRRK2 substrate and regulator Rab29 in the lysosomal stress response. Briefly, in chloroquine (CQ)-treated HEK293 cells the authors observed an apparent LRRK2-independent increased in Rab29 phosphorylation which was accompanied by translocation of Rab29 to lysosomes. Intriguingly, the authors detected a similar increase in Rab29 phosphorylation when Rab29 was tethered to lysosomes in the absence of CQ treatment. Using mass spectrometry, mutagenesis and a phospho-specific anti-body, the authors mapped the CQ-induced phosphorylation site to S185 and demonstrated its independence from LRRK2. Next, the authors found that PKCa was the kinase responsible for S185 phosphorylation and lysosomal translocation of Rab29. Lastly, the authors showed that in addition to PKCa the lysosomal translocation of Rab29 was also regulated by LRRK2. Overall, Komori and colleagues provide interesting new insights into the phosphorylation-dependent regulation of Rab29. However, there are. Number of technical and conception concerns which should be addressed.

    Major points

    1. Figure 1F: the localization of Rab29 to lysosomes is not convincing at all. The authors should either provide more representative image examples or image the cells at a higher resolution. The authors should also confirm the CQ-induced lysosomal localization of Rab29 in a different cell type (e.g., HEK293).

    We will replace Fig 1F pictures with slightly more magnified images with higher resolution. We will also include additional cell types (HEK293, and other cells that are predicted to express endogenous Rab29); Reviewer #2 also raised this point (see Reviewer #2 comment on Significance).

    Moreover, the authors should show that prenylation of Rab29 is required for its CQ-induced phosphorylation.

    We will test the effect of lovastatin, a HMG-CoA reductase inhibitor that causes the depletion of the prenylation precursor geranylgeranyl diphosphate from cells (Binnington et al., Glycobiology 2016, Gomez et al, J Cell Biol 2019), or 3-PEHPC, a GGTase II specific inhibitor that also causes the inhibition of Rab prenylation (Coxon FP et al, Bone 2005).

    1. The rapalog-induced increase in Rab29 phosphorylation in Figure 2D is not convincing since there is at least 2-3-fold more Rab29 in FRB-LAMP1 expressing cells compared to their FRB-FIS1 counterparts. An independent loading control is also missing. This is a key experiment and should be properly controlled and quantified. In addition, can CQ treatment drive 2xFKBP GFP-Rab29 from mitochondria to lysosomes (in the presence of rapalog and FRB-Fis1)?

    We will carefully examine another round of rapalog-induced phosphorylation of Rab29, with an independent loading control such as alpha-tubulin. The immunoblot analysis will be made against the intensity of non-p-Rab29. The response to the latter question was described in the section 4 below.

    1. Figure 4A-C: Are these stable Rab29 expressing cells? If not, the quantification of "the size of largest lysosome in EACH cell" becomes very problematic. This analysis should be repeated with stable Rab29 variant cells in a background lacking endogenous Rab29. Furthermore, the LAMP1 signal is too dim to see any convincing colocalization (e.g., with WT) or the lack thereof (e.g., in the case of S185D).

    The cells shown in Figure 4 are HEK293 cells transiently expressing Rab29, and the issue of quantification was described in the section 3 below. We agree that the signal of LAMP1 was dim, and it turned out that the confocal microscope we used had problems with the sensitivity of the red channels. We will be taking another round of these images with a new confocal microscope.

    Lastly, the authors should corroborate their findings with an ultrastructural analysis since the electron microscopy would definitively be more suitable for this type of measurements.

    We are planning to obtain electron microscopic images, according to this reviewer’s request. We plan to invite an expert in electron microscopy analysis as a co-author.

    1. The lysosomal colocalization of Rab29 in Figure 5C is again not convincing. This analysis needs to be repeated with high resolution imaging.

    Again, we will repeat this experiment with a new confocal microscope, with the hope that it would yield better images.

    1. The authors need to show the level of LRRK2 depletion (Figure 6). Given the role of LRRK2 in driving lysosomal Rab29 translocation, the importance of the LRRK2 independent pS185 for this process remains unclear.

    We will add the level of LRRK2 on its knockdown; we have experienced that LRRK2 knockdown usually occurs with more than 50% efficiency every time. The response to the latter comment was described in the section 3 below.

    1. In general, the authors employ an alternative, biochemical assay (e.g., LysoIP) for the lysosomal translocation of Rab29. This would in particular help to clarify the effect of the Rab29 variants and LRRK2 inhibition.

    We have previously shown that the overexpressed Rab29 (and LRRK2) is enriched in the lysosomal fraction from CQ-treated cells, which was performed using dextran-coated magnetite (Eguchi et al, PNAS 2018). Using the same biochemical method, we will show the enrichment of endogenous Rab29 in the lysosomal fraction.

    Minor points

    1. Figure 2C is lacking the control IF staining for mitochondria (to which 2xFKBP-GFP-Rab29 is assumed be recruited upon co-expression with FRB-FIS1).

    We will stain the cells with MitoTracker to ensure that anchoring away of 2xFKBP-GFP-Rab29 by FRB-Fis1 results in mitochondrial localization.

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

    The data in the manuscript convincingly demonstrates that lysosomal overload by Chloroquine treatment induces Rab29 localisation to the lysosomes and that this membrane association is dependent on PKCalpha-dependent phosphorylation at Ser185.

    We have a number of rather minor comments listed below:

    Figure 2

    The increasing levels of non-phosphorylated Rab29 over the indicated time course of AP21967 treatment in Figure 2B are concerning. First, could you provide an explanation for this clear increase in both non-p-Rab29 and p-Rab29 in the phostag but not the normal gel? Second, could all quantifications of p-Rab29 be made relative to the non-p-Rab29?

    We will try another round of rapalog-induced phosphorylation of Rab29, with an independent loading control. The immunoblot analysis will be made against the intensity of non-p-Rab29. Reviewer #1 raised a similar concern on Figure 2D.

    Figure 5

    To further demonstrate that PKCalpha phosphorylates endogenous Rab29 at Ser185, we recommend reperforming the Go3983/PMA treatment in figure B with the anti-p-Ser185 antibody. It may be sufficient to perform the treatment only at 4 or 8 hours, simply to provide stronger evidence regarding the phosphorylation of endogenous Rab29.

    We will give a try, although the anti-phosphorylated protein antibodies that we tried never worked for phos-tag SDS-PAGE. With the conventional western blot, we will be able to try this experiment.

    It is not clear whether the activity of PMA in the assay is due to inhibition of PKCalpha. Are the effects ablated by PKCalpha KD

    We will test the knockdown of PKCalpha, beta, gamma and delta by siRNAs to further narrow down the effects of PKC-dependent phosphorylation of Rab29.

    Reviewer #2 (Significance (Required)):

    These cell biology findings are important in the field as both Rab29 and LRRK2 are implicated in the pathogenesis of Parkinson disease. The phosphorilation of Ser185 of Rab29 by PKCalpha is novel and contributes to our understanding of Rab29 and LKRR2 regulation. One limitation of the study is that is conducted in only two cell types quite unrelated to the disease, so how general and disease relevant are the findings it is not clear. Most of the data are solid. There are two experiments whose results are difficult to interpret and a few controls missing. Also a few issues with quantifications, all of which is described in details above and will need to be fixed prior to publication. My expertise for this paper is in the cell biology of lysosomal function.

    The issue that only two cell types were analyzed was also raised by reviewer #1, so we will examine additional cell types, especially those that are predicted to express endogenous Rab29. Our responses to other issues raised are described elsewhere. Thank you for these insightful comments.

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

    Figure 4A-C: Are these stable Rab29 expressing cells? If not, the quantification of "the size of largest lysosome in EACH cell" becomes very problematic. This analysis should be repeated with stable Rab29 variant cells in a background lacking endogenous Rab29. (Reviewer #1)

    As described in the section 2 above, the cells shown in Figure 4 are HEK293 cells transiently expressing Rab29. We are sorry that the description “the size of largest lysosome in each cell” was misleading. As we analyzed only cells overexpressing GFP-Rab29 that were marked with GFP fluorescence, we believe that transient expression should not be a problem. To avoid any misunderstandings, we have described in Figure 4 legends that only lysosomes in Rab29-positive cells (and all cells expressing Rab29) were included in the analysis of the largest lysosome of each cell.

    Regarding the effect of endogenous Rab29 in Figure 4 experiments, Reviewer #2 similarly raised the issue on whether Rab29 phosphomimetics are acting as dominant active, preventing lysosomal enlargement. On this point, we have previously reported that knockdown of endogenous Rab29 causes the enhancement of lysosomal enlargement upon CQ treatment (Figure 5I,J of Eguchi et al, PNAS 2018), suggesting that the lysosome-deflating effect by phosphomimetics is a dominant active effect rather than dominant negative suppressing endogenous Rab29. This point is considered significant, and thus has been explained in the results section (page 7, lines 168-171).

    Along similar lines: why not all cells in Figure 5E and Figure 5G show Rab29- and LRRK2-positive structures? How do the authors know which of these phenotypes is the prevalent one? (Reviewer #1)

    As for the ratio of cells with Rab29- and LRRK2-positive structures, it seems reasonable given that different cells have different levels of exposure to lysosomal stress and that the response is transient and does not occur simultaneously. The ratio of these positive cells may also vary depending on the cell culture conditions. Since Rab29- and LRRK2-positive structures are rarely seen in control cells, we think this would be a meaningful phenotype even if only 20-30% of cells show such structures. The result that the ratio of localization changes is not 100% is now noted in the results section explaining Figure 1G (page 4-5, lines 108-110) where the immunocytochemical data first appears.

    Given the role of LRRK2 in driving lysosomal Rab29 translocation, the importance of the LRRK2 independent pS185 for this process remains unclear. (Reviewer #1)

    Our data suggested that Rab29 is stabilized on lysosomes only when LRRK2-mediated phosphorylation and S185 phosphorylation both occur on Rab29 molecule (as shown in Figure 7 scheme), so we believe there is no contradiction. We have now described more clearly about this notion at the end of the results section (page 9, lines 235-236).

    It is not clear what the authors mean by "lysosomal overload stress". Since mature lysosomal incoming pathways such as autophagy or endocytosis are disrupted by CQ, it is difficult to picture an overload. Maybe rephrasing would help to clarify this. (Reviewer #1)

    Chloroquine (CQ) is known as a lysosomotropic agent that accumulates within acidic organelles due to its cationic and amphiphilic nature, causing lysosome overload and osmotic pressure elevation, and this is what we call “lysosomal overload stress”. The well-known effects of CQ to disrupt lysosomal incoming pathways are ultimately caused by the above consequences. Also, we have previously reported that lysosomal recruitment of LRRK2 is caused by CQ but not by bafilomycin A1, the latter being an inducer of lysosomal pH elevation, or by vacuolin-1 that enlarges lysosomes without inducing lysosomal overload/pH elevation (Eguchi et al, PNAS 2018), and further found that not only CQ but also other lysosomotropic agents commonly induced LRRK2 recruitment (Kuwahara et al, Neurobiol Dis 2020). We thus have described the effect of CQ as “overload”. However, it is true that we have not provided a clear explanation for readers, so we have added some notes for lysosomal overload stress in the introduction section (page 3, lines 69-71).

    Which cell type is used for the IF analysis in Figure 2C? This information is in general quite sparse. The authors should clearly state the cell type for each experiment/Figure. (Reviewer #1)

    We have added cell type information that was missing in several places in the manuscript. We are very sorry for the inconveniences. For clarification, HEK293 cells were used in Figure 2C.

    Are the images in figure 1F representative? i.e. does Rab29 always colocalise to such enlarged lysosomes upon CQ treatment and does CQ treatment always drastically alter the cellular distribution of Rab29? (Reviewer #2)

    The images in Figure 1F are representative of when Rab29 is recruited, but it is not seen in all cells, and the ratio of recruitment (~80%) is shown in Figure 1G. Reviewer #1 also asked why Rab29 recruitment is not seen in all cells, and we gave the same answer above. It may be reasonable to speculate that different cells have different levels of exposure to lysosomal stress and that the response is transient and does not occur simultaneously. The ratio of these positive cells may also vary depending on the cell culture conditions. For the readers’ clarity, we have added that the ratio of localization change of Rab29 is not 100% and is comparable to that of LRRK2 previously reported (page 4-5, lines 108-110).

    Considering that the "forced localisation technique" induces a non-physiological colocalization of non-endogenous Rab29 to lysosomes, it may be an overestimation to conclude just from these data that phosphorylation of Rab29 occurs on the lysosomal surface. This is also quite in contrast with the later finding that phosphorylation by PKCalpha promotes lysosome localization of Rab29. It seems more reasonable to conclude that Rab29 can be phosphorylated when localised at the lysosomes (as opposed to other organelles such as mitochondria). If the authors feel strongly about this point they might need to find a less non-physiological assay. (Reviewer #2)

    Yes, it could be an overestimation, and as we do not have better means to conduct a less non-physiological assay, we have modified the description from “occurred on the lysosomal surface” to “could occur on the lysosomal surface” (page 5, line 112 (subtitle) and line 128).

    Regarding the comparison with the later finding that phosphorylation by PKCalpha promotes lysosome localization of Rab29, these data (Figure 2 and 5) could be explained with a single speculation: phosphorylation of Rab29 on lysosomal membranes could retain Rab29 on the membranes for a longer time. It is not easy to decipher which comes first, association with membranes or phosphorylation of Rab29, in a physiological assay, but considering reports that show PKCalpha activation happens on membranes (Prevostel et al., J Cell Sci 2000), at least the data favor our conclusion over the idea of PKCalpha phosphorylating Rab29 in the cytoplasm and then promoting lysosomal localization. This point is now clearly described in the discussion (page 10, lines 248-251).

    It is not clear how the Rab29 phosphomimetics are acting as dominant active preventing lysosomal enlargement. Authors should speculate or repeat the experiments in absence of endogenous Rab29 to clarify the matter. (Reviewer #2)

    A similar concern about the effect of endogenous Rab29 was also raised by Reviewer #1 (see above). We have previously reported that knockdown of endogenous Rab29 causes the enhancement of lysosomal enlargement upon CQ treatment (Figure 5I,J of Eguchi et al, PNAS 2018), suggesting that the lysosome-deflating effect by phosphomimetics is a dominant active effect rather than dominant negative suppressing endogenous Rab29. This point is considered important and thus has been explained in the results section (page 7, lines 168-171).

    Overall, there is some missing information regarding repeats for Western blots, such as those in figure 3C, 3D and 3E. Please add indications about repeats in the figure legend or methods. (Reviewer #2)

    We have added the repeat information to each figure legend where it was missing. We are very sorry for the inconveniences.

    The model in figure 7 however seems to suggest that Rab29 associates to lysosomal membranes independently, and is then stabilised at the membranes by LRRK2 and PKCalpha - a point which is not directly supported by the data. (Reviewer #2)

    As noted earlier, we consider that phosphorylation of Rab29 on lysosomal membranes could retain Rab29 on the membranes for a longer time, given the present data and previous reports that phosphorylation of Rab29 is more likely to happen on the lysosomal membrane than in the cytosol. Also, as inhibition of either of the two phosphorylations ends up in disperse Rab29 localization, we have made this figure as a model of what is plausible right now. This explanation is now added in the discussion (page 10, lines 248-251).

    English proofreading should be improved: "CQ was treated to HEK293" (page 4), "As we assumed that this phosphorylation is independent of LRRK2" as an opening line (page 5) (Reviewer #2)

    Thank you for pointing out these incorrect wordings. They were corrected.

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

    In addition, can CQ treatment drive 2xFKBP GFP-Rab29 from mitochondria to lysosomes (in the presence of rapalog and FRB-Fis1)? (Reviewer #1)

    We do not think that a comparison between the affinities of FKBP-rapalog-FRB and Rab29-[unknown factor that directs Rab29 to lysosomes] is necessary, as the former has a Kd in the single digit nM range (Banaszynski et al, JACS 2005), whereas the latter (based on estimations from related PPIs) is estimated to be in the μM range, which shows a much weaker affinity than the former (McGrath et al, Small GTPases 2019). Furthermore, even if Rab29 appears to have migrated from mitochondria to lysosomes as a result of this experiment, one cannot rule out the possibility that a small portion of the mitochondrial membrane was incorporated into the lysosomal membrane that was enlarged by CQ treatment.

    Molecular weight markes should be provided for all immunoblot experiments. (Reviewer #1)

    The immunoblot pictures without molecular weight markers in our paper are all Phos-tag SDS-PAGE blot analyses. Phos-tag SDS-PAGE results in band shifts of phosphorylated proteins, and writing in markers would be misleading. Moreover, previous representative studies heavily using Phos-tag (e.g., Kinoshita et al, Proteomics 2011, Ito et al, Biochemical Journal 2016) also did not show the molecular weight markers. Here we performed phos-tag SDS-PAGE analysis only to find differences in the phosphorylation state of Rab proteins.

    The use of the quantification ratio of cells with Rab29-positive lysosomes in figure 1G might be slightly misleading as it does not allow the reader to understand to what extent Rab29 localisation at lysosomes upon CQ treatment. We recommend using a simpler quantification, such as by measuring the average colocalisation of Rab29 and LAMP1 per cell. (Reviewer #2)

    For figure 5D and 5F, As with figure 1G, we recommend using a more straightforward and impartial method of quantification such as simply measuring the colocalisation of Rab29 with LAMP1. (Reviewer #2)

    Popular colocalization analyses using Pearson’s or Mander’s coefficients would be a good choice if the amounts of Rab29 varied greatly between lysosomes. However, this may not apply in this case; the amount of Rab29 or LRRK2 on each lysosome is considered to saturate quickly and a relatively low amount of them may not be detected on immunofluorescence observations, whereas the probability of finding these structures has been shown to exhibit a moderate sigmoid curve (as seen in Figure 1E or 2H of Eguchi et al., PNAS 2018). Therefore, the amount of Rab29 or LRRK2 could be approximated to a Bernoulli distribution in terms of colocalization with lysosomes, and this is the reason why we chose to quantify “the ratio of cells with Rab29-positive lysosomes”.

    We recommend using a more transparent and simple quantification method, such as average size of lysosomes per cell. (Reviewer #2)

    As one can see in the inset of Figure 4B, unenlarged lysosomes are unfortunately too small for the quantification of their size, much less tell two small lysosomes apart in our experimental settings and laboratory resources, so we decided to analyze the largest lysosome in each cell as a representative of the cells to minimize measurement errors. This measurement only includes GFP-Rab29 positive cells, and by comparing against CQ-untreated cells we intended to increase the validity of this analysis. This quantification method was also used in our previous report (Eguchi et al, PNAS 2018).

  6. Review Commons

    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

    The data in the manuscript convincingly demonstrates that lysosomal overload by Chloroquine treatment induces Rab29 localisation to the lysosomes and that this membrane association is dependent on PKCalpha-dependent phosphorylation at Ser185.

    We have a number of rather minor comments listed below:

    Figure 1

    • Are the images in figure 1F representative? i.e. does Rab29 always colocalise to such enlarged lysosomes upon CQ treatment and does CQ treatment always drastically alter the cellular distribution of Rab29?
    • The use of the quantification ratio of cells with Rab29-positive lysosomes in figure 1G might be slightly misleading as it does not allow the reader to understand to what extent Rab29 localisation at lysosomes upon CQ treatment. We recommend using a simpler quantification, such as by measuring the average colocalisation of Rab29 and LAMP1 per cell.

    Figure 2

    • Considering that the "forced localisation technique" induces a non-physiological colocalization of non-endogenous Rab29 to lysosomes, it may be an overestimation to conclude just from these data that phosphorylation of Rab29 occurs on the lysosomal surface. This is also quite in contrast with the later finding that phosphorylation by PKCalpha promotes lysosome localization of Rab29. It seems more reasonable to conclude that Rab29 can be phosphorylated when localised at the lysosomes (as opposed to other organelles such as mitochondria). If the authors feel strongly about this point they might need to find a less non-physiological assay.
    • The increasing levels of non-phosphorylated Rab29 over the indicated time course of AP21967 treatment in Figure 2B are concerning. First, could you provide an explanation for this clear increase in both non-p-Rab29 and p-Rab29 in the phostag but not the normal gel? Second, could all quantifications of p-Rab29 be made relative to the non-p-Rab29?

    Figure 3

    • It is not clear how the Rab29 phosphomimetics are acting as dominant active preventing lysosomal enlargement. Authors should speculate or repeat the experiments in absence of endogenous Rab29 to clarify the matter.
    • Overall, there is some missing information regarding repeats for Western blots, such as those in figure 3C, 3D and 3E. Please add indications about repeats in the figure legend or methods.

    Figure 4

    • We recommend using a more transparent and simple quantification method, such as average size of lysosomes per cell.

    Figure 5

    • To further demonstrate that PKCalpha phosphorylates endogenous Rab29 at Ser185, we recommend reperforming the Go3983/PMA treatment in figure B with the anti-p-Ser185 antibody. It may be sufficient to perform the treatment only at 4 or 8 hours, simply to provide stronger evidence regarding the phosphorylation of endogenous Rab29.
    • It is not clear whether the activity of PMA in the assay is due to inhibition of PKCalpha. Are the effects ablated by PKCalpha KD
    • For figure 5D and 5F, As with figure 1G, we recommend using a more straightforward and impartial method of quantification such as simply measuring the colocalisation of Rab29 with LAMP1.

    Figure 6

    • Again, we recommend altering the methods of quantification

    Figure 7

    • The model in figure 7 however seems to suggest that Rab29 associates to lysosomal membranes independently, and is then stabilised at the membranes by LRRK2 and PKCalpha - a point which is not directly supported by the data.

    English proofreading should be improved: "CQ was treated to HEK293" (page 4), "As we assumed that this phosphorylation is independent of LRRK2" as an opening line (page 5),

    Significance

    These cell biology findings are important in the field as both Rab29 and LRRK2 are implicated in the pathogenesis of Parkinson disease. The phosphorilation of Ser185 of Rab29 by PKCalpha is novel and contributes to our understanding of Rab29 and LKRR2 regulation. One limitation of the study is that is conducted in only two cell types quite unrelated to the disease, so how general and disease relevant are the findings it is not clear. Most of the data are solid. There are two experiments whose results are difficult to interpret and a few controls missing. Also a few issues with quantifications, all of which is described in details above and will need to be fixed prior to publication. My expertise for this paper is in the cell biology of lysosomal function.

  7. Review Commons

    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 #1

    Evidence, reproducibility and clarity

    Summary

    In this manuscript, Komori et al. examined the role of the LRRK2 substrate and regulator Rab29 in the lysosomal stress response. Briefly, in chloroquine (CQ)-treated HEK293 cells the authors observed an apparent LRRK2-independent increased in Rab29 phosphorylation which was accompanied by translocation of Rab29 to lysosomes. Intriguingly, the authors detected a similar increase in Rab29 phosphorylation when Rab29 was tethered to lysosomes in the absence of CQ treatment. Using mass spectrometry, mutagenesis and a phospho-specific anti-body, the authors mapped the CQ-induced phosphorylation site to S185 and demonstrated its independence from LRRK2. Next, the authors found that PKCa was the kinase responsible for S185 phosphorylation and lysosomal translocation of Rab29. Lastly, the authors showed that in addition to PKCa the lysosomal translocation of Rab29 was also regulated by LRRK2. Overall, Komori and colleagues provide interesting new insights into the phosphorylation-dependent regulation of Rab29. However, there are. Number of technical and conception concerns which should be addressed.

    Major points

    1. Figure 1F: the localization of Rab29 to lysosomes is not convincing at all. The authors should either provide more representative image examples or image the cells at a higher resolution. The authors should also confirm the CQ-induced lysosomal localization of Rab29 in a different cell type (e.g., HEK293). Moreover, the authors should show that prenylation of Rab29 is required for its CQ-induced phosphorylation.
    2. The rapalog-induced increase in Rab29 phosphorylation in Figure 2D is not convincing since there is at least 2-3-fold more Rab29 in FRB-LAMP1 expressing cells compared to their FRB-FIS1 counterparts. An independent loading control is also missing. This is a key experiment and should be properly controlled and quantified. In addition, can CQ treatment drive 2xFKBP GFP-Rab29 from mitochondria to lysosomes (in the presence of rapalog and FRB-Fis1)?
    3. Figure 4A-C: Are these stable Rab29 expressing cells? If not, the quantification of "the size of largest lysosome in EACH cell" becomes very problematic. This analysis should be repeated with stable Rab29 variant cells in a background lacking endogenous Rab29. Furthermore, the LAMP1 signal is too dim to see any convincing colocalization (e.g., with WT) or the lack thereof (e.g., in the case of S185D). Lastly, the authors should corroborate their findings with an ultrastructural analysis since the electron microscopy would definitively be more suitable for this type of measurements.
    4. The lysosomal colocalization of Rab29 in Figure 5C is again not convincing. This analysis needs to be repeated with high resolution imaging. Along similar lines: why not all cells in Figure 5E and Figure 5G show Rab29- and LRRK2-positive structures? How do the authors know which of these phenotypes is the prevalent one?
    5. The authors need to show the level of LRRK2 depletion (Figure 6). Given the role of LRRK2 in driving lysosomal Rab29 translocation, the importance of the LRRK2 independent pS185 for this process remains unclear.
    6. In general, the authors employ an alternative, biochemical assay (e.g., LysoIP) for the lysosomal translocation of Rab29. This would in particular help to clarify the effect of the Rab29 variants and LRRK2 inhibition.

    Minor points

    1. It is not clear what the authors mean by "lysosomal overload stress". Since mature lysosomal incoming pathways such as autophagy or endocytosis are disrupted by CQ, it is difficult to picture an overload. Maybe rephrasing would help to clarify this.
    2. Which cell type is used for the IF analysis in Figure 2C? This information is in general quite sparse. The authors should clearly state the cell type for each experiment/Figure.
    3. Figure 2C is lacking the control IF staining for mitochondria (to which 2xFKBP-GFP-Rab29 is assumed be recruited upon co-expression with FRB-FIS1).
    4. Molecular weight markes should be provided for all immunoblot experiments.

    Significance

    Please see above.

  8. Version published to 10.1101/2022.09.26.509472v2 on bioRxiv
  9. Version published to 10.1101/2022.09.26.509472v1 on bioRxiv