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    Referee #1

    Evidence, reproducibility and clarity

    This study seeks to define how human lysosomes selectively downregulate membrane proteins and identify the machinery involved in this process. To this end, the authors screened a set of 30 lysosome membrane proteins (LMPs) in a cycloheximide chase assay in a human cell line which led to the identification of RNF152 (an E3 ligase) as a particularly short lived LMP. Further experiments demonstrate that RNF152 degradation is ubiquitin, ESCRT and lysosome dependent. They also show that the E3 ubiquitin ligase activity of RNF152 is critical for its turnover. The overall technical quality of the experiments is high and conclusions about the degradation of RNF152 are mostly reasonable. My most significant concern is that while compelling data is provided for RNF152 turnover, the authors over-reach in their efforts to generalize their findings to other LMPs. Given that the E3 ligase activity of RNF152 is so important for its turnover, RNF152 might be a special case. Consistent with this, the authors did not characterize other LMPs with similarly high rates of turnover. Although it would be interesting if RNF152 regulates the stability of other LMPs, until such proteins are identified, the authors should be more cautious in their interpretation. Speculation on this matter is reasonable so long as it is labeled as such. Even with respect to RNF152 turnover mechanisms, the overall conclusions would be significantly strengthened by a demonstration that the endogenously expressed, untagged protein behaves in a similar manner to what was described for the GFP-tagged transgene. With respect to the question about how long it would take for the authors to address these concerns, I cannot give a precise answer as it would depend on whether they decide to much more narrowly interpret their findings and temper their major claims (less than a month) or to expand efforts to generalize results (time frame unknown and perhaps not feasible).

    1. As a specific (but not the only) example of over-reaching in generalizing the findings, the abstract ends with the following statement: "Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins." My concern is that although this mechanism might be generalizable, the authors have only presented data for RNF152.
    2. There is a complete reliance on over-expressed, GFP-tagged RNF152. There is no demonstration that the endogenously expressed protein undergoes such high rates of turnover. It is thus possible that the data does not reflect the normal turnover pathway for this protein.
    3. In Figure 2B, why is the loss of full length RNF152-GFP not accompanied by an increase in the signal for free GFP during these pulse-chase experiments?
    4. Figure 2E: Were all of the pairs of Input and IP immunoblots subject to the same exposure and image adjustments?
    5. Figure 3C-E: The RNF152 mutants have slowed but not eliminated degradation. Is this dependent on their association with or ubiquitination by the endogenouslyh expressed RNF152?
    6. Methods section indicates that t-tests were performed for all statistics. However, many experiments contain multiple comparisons and are thus ideally suited to t-tests. The authors should either justify the use of t-tests or provide a more suitable statistical analysis.
    7. Although the model in Figure 7 shows the E3 (RNF152) ubiquitinating other proteins and promoting their ESCRT-dependent sorting into ILVs, this study did not identifying any such clients of RNF152.

    Minor

    Page 3: "Without treatment, almost all types of LSD patients will develop severe neurodegeneration in the central nervous system." This statement is misleading as there are multiple forms of LSDs that do not result in neurodegeneration and it is only these LSDs which can be successfully treated via enzyme replacement therapies. Unfortunately, the neuropathic LSDs remain largely untreatable due largely to issues of blood brain barrier permeability.

    Page 3: "As we age, the lysosome membrane gradually accumulates damaged proteins and loses its activity, which dampens the cell's ability to remove pathogenic protein aggregates and damaged organelles, eventually leading to cell death and inflammation (Carmona-Gutierrez et al., 2016; Cheon et al., 2019; Yambire et al., 2019)." The references provided do not provide sufficient direct support for this broad statement.

    Page 10: STED imaging results (currently "data not shown") should be supported by showing the relevant data.

    Camera and objective information should be provided for microscopy studies.

    Significance

    The identification of a generalizable mechanism for the turnover of mammalian LMPs would represent a significant advance and would raise many interesting questions about mechanisms, regulations and physiological impact. While this studies contributes some interesting new clues to this topic, it falls short of unambiguously establishing how most LMPs are turned over in human cells. The data with respect to RNF152 is intriguing as it supports the idea that a novel form of ESCRT-dependent protein clearance occurs at the limiting membrane of lysosomes. However, it remains very much unclear to what extent this can be generalized to other proteins.

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    Referee #2

    Evidence, reproducibility and clarity

    The mechanisms involved in lysosome membrane protein turnover are not well understood. Weichao et al. used a cycloheximide chase screen and overexpression 30+ lysosome membrane proteins in HEK293 cells to identify LMPs (lysosome membrane proteins) with fast turnover rates. They identified RNF152 as a suitable candidate for study given its high turnover rate and physiological relevance. They showed that RNF152's levels were regulated by ubiquitination by mutating cytosolic lysine residues and RNF152's ring domain and finding that these changes increased RNF152 stability. The researchers found that knocking down ESCRTIII and overexpressing a dominant-negative mutant of VPS4 increased RNF152 levels at steady-state and delayed RNF152 turnover. When expressed in yeast, RNF152 is localized on vacuole membrane and is also subject to regulation by the ESCRT pathway. Early ESCRT pathway members are essential for RNF152 degradation in yeast but not in mammalian cells. Taken together, these findings are important for furthering our understanding of how the levels of lysosome membrane proteins are regulated. A better understanding of ESCRT mediated LMP degradation is important not only for understanding mechanisms involved in controlling lysosomal activities but also for therapeutic development for many diseases involving dysregulation of LMP protein levels.

    However, the following concerns should be addressed before the paper is published:

    1. The authors have found that among 30 LMPs, three LMPs, LAPTM4A, RNF152, and OCA2, have half-lives less than 9 hours. RNF152 is a ubiquitin ligase and the authors showed that auto-ubiquitination is important for the recognition by the ESCRT machinery. Can the authors speculate how the ligase activity of RNF152 is regulated? Also, is similar mechanism involved in LAPTM4A and OCA2 turnover? Are these two proteins also ubiquitinated?
    2. The authors should at least demonstrate that endogenous RNF152 levels and turnover are also regulated by ESCRT III and VPS4, using the stable cell lines the authors have already made. All of the mammalian cell experiments are performed using overexpression of RNF152, and an endogenous experiment would inspire confidence that the author's findings are not an artifact of over-expression.
    3. While the authors showed that the K->R and C->S mutants of RNF152 have increased stability, it would be more compelling if they could perform an IP using HA-ubiquitin to prove this effect is due to a loss/reduction of RNF152 ubiquitination and not due to other changes in the protein. Another concern is whether mutating 8 lysine or 4 cysteine residues simultaneously would affect the folding of the protein, leading to abnormal aggregation in the cell.
    4. For some of the data, statistical analysis is missing: a. All of the cycloheximide chase experiments. b. statistical significance for the puncta vs membrane GFP signal data shown in figure 6f c. The flow cytometry data
    5. Fig. 4A and Fig. S2A, why MG132 treatment affects the levels of free GFP if it's inside of the lysosome?
    6. Make sure that the figures are properly referenced in the text, there is one instance where the authors referenced figure 2d, when they clearly meant to reference figure 2e, and figure 2e where the authors meant to reference figure 2f etc.

    Minor Comments:

    1. In figure 1A, at CHX 3h, there's ~40% reduction of GFP-RNF152, however, in the rest of the figures, such as figure 2B,at CHX 2h, there's ~70-80% reduction of GFP-RNF152. How to explain the difference in the kinetics?
    2. In figure 2F, it is hard to differentiate when the underline for input ends and the underline for IP begins unless the reader zooms in, please separate them a bit more.
    3. Fig. 4F, it's very hard to see the red and green signals, maybe get rid of the DAPI channel increase the intensity for both green and red channels, and zoom in?
    4. Scale bars are missing in the insert images in figure 1C, figure 4G and figure 6E.
    5. In figure S1, the labels do not match with the blot for GFP-TMEM106B time points.

    Significance

    These findings are important for furthering our understanding of how the levels of lysosome membrane proteins are regulated. A better understanding of ESCRT mediated LMP degradation is important not only for understanding mechanisms involved in controlling lysosomal activities but also for therapeutic development for many diseases involving dysregulation of LMP protein levels.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    Lysosomes play key roles in cellular homeostasis by functioning as a signaling hub for growth control and acting as a terminal catabolic station. Deregulation of lysosomes are now linked to multiple human diseases including cancer, neurodegeneration and etc. An emerging topic of interests in lysosomal biology is the regulation of lysosomal proteostasis and how it impacts the overall fitness and functionality of the lysosome per se. Zhang et al presents here a case study of quality control of lysosomal membrane proteins, with a focus on the turnover of a lysosomal anchor E3 ubiquitin ligase RNF152. They showed that RNF152 is rapidly degraded through an ESCRT-dependent fashion and that this mechanism is also conserved in yeast.

    Major comments:

    1. The writing of the manuscript including the abstract could be further polished. The manuscript in its present form appears to be a technical report that does not sufficiently convey the significance of this study.
    2. Cyclohexamide is commonly used in studying the half-lives of proteins of interests. This is not a new method authors developed in the first place.
    3. The data of protein turnover was presented by plotting the relative level of proteins as a function of time. But the use of degradation kinetics was all over the place in the manuscript, which is inappropriate scientifically. The authors should first generate fit to first order decay to acquire a degradation rate constant, k (min-1) and calculate half-life (T1/2) from there.
    4. What are the functional consequences of RNF152 degradation? What are the biological impacts at both lysosomal and cellular levels in RNF152-depleted cells?
    5. Given the rapid turnover of RNF152 at basal state, one can predict that this protein may become functionally important under specific circumstances, for example, certain stress. This aspect is worth exploring.
    6. The authors chose RNF152 over OCA2, a melanosome-specific protein. However, OCA2 was shown to colocalize with LAMP2 much better than RNF152.

    Minor comments:

    1. Mislabeling and typo errors detected in the text: a. Page 7 "As expected, the full-length GFP-RNF152 and other lysosomal proteins such as LAMP2 and cathepsin D (CTSD) were enriched by Lyso-IP. In contrast, PDI (ER), Golgin160 (Golgi), EEA1 (endosomes), and GAPDH (cytosol) were not enriched (Figure 2D)." - should be Figure 2E instead. b. Page 7 "Our result confirmed that the lysosome population of GFP-RNF152 is quickly turned over, while LAMP2 is very stable on the lysosome (Figure 2E)." - should be Figure 2F instead. c. Page 14 "knocking down either TSG101 or both TSG101 and RNF152 only had a minor impact on the degradation kinetics of GFP-RNF152 (Figure S3A-B)." - should be ALIX instead of RNF152.
    2. Stable cells expressing GFP-RNF152 or 3xFLAG-RNF152 were primarily used in this study. It will be useful to perform some experiments by examining the endogenous counterpart using antibodies against RNF152. For example, Figure 2D and 2E.
    3. For all the flow cytometry analysis, the value of GFP intensity in respective graphs should be indicated.
    4. Statistics analysis was not performed on Figure 5D.
    5. In Figure 6D and J, what are the reasons for the appearance of multiple peaks, particularly, by the red line?
    6. In Figure 3A, the question marks should be removed to avoid confusion. "Predicted" can be used instead if there is no direct evidence from mass spec analysis.
    7. In Figure 3C, the authors identified two mutants including KR and CS that are refractory to degradation. It will be more insightful by showing the ubiquitination of these two mutants as in Figure 3B.

    Significance

    Multiple mechanisms including ESCRT complex have been reported to regulate the quality control of lysosomes. Understanding the roles of each mechanisms and selection of their substrates in maintenance of lysosomal integrity is of great interest in cell biology. Zhang and colleagues showed a case study of RNF152, a substrate of ESCRT-dependent degradation, but did not further pursue the biological functions of RNF152. This somewhat limits the conceptual advance of the study.

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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