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Brief summary of the study:

The main findings of the preprint regarding GPI-anchored proteins (GPI-APs) and clathrin-mediated endocytosis are as follows:

● Impact of Unremodeled GPI-APs: The study investigates how the accumulation of GPI-APs, specifically those with phosphoethanolamine (EtNP) not removed from mannose-2 (Man2) of the GPI moiety, affects the plasma membrane of yeast cells. It was found that the persistence of EtNP on Man2 prevents the cleavage of certain GPI-APs, leading to their accumulation at the plasma membrane.

● Increased Lipid Disorder: The presence of uncleaved, unremodeled GPI-APs increases lipid disorder within the membrane, which is indicative of stress responses in the cell.

● Activation of Stress Responses: The study reports that unremodeled GPI-APs trigger stress responses, including the activation of the cell wall integrity pathway and non-canonical activation of the spindle assembly checkpoint (SAC).

● Aberrant Endocytosis: The accumulation of these unremodeled GPI-APs leads to abnormal clathrin-mediated endocytosis of certain membrane proteins. This process results in the formation of numerous small vacuoles within the cells.

● Physiological Consequences: The findings suggest that defects in GPI-AP remodeling and cleavage can disrupt plasma membrane homeostasis, potentially leading to broader physiological consequences in eukaryotic cells.Overall, the study highlights the critical importance of GPI-AP Man2 remodeling for maintaining plasma membrane integrity and homeostasis, and it identifies a novel mechanism by which plasma membrane stress signals are transmitted into the cell.

Major comments:

The authors have rigorously characterized the impact of unmodeled Man2 EtNP in GPI-anchored proteins. While the results and figures are largely compelling, further experiments would strengthen their observations. Additionally, there is a lack of clarity in some experimental strategies and the corresponding results, and the flow of information throughout the manuscript should be more lucid.

1. In the results section for Figure 1, the authors state, "Based on these DRM extraction experiments, it appears that Gas1p (and by inference other GPI-APs) in which EtNP is added to Man2 but not removed may also have altered biochemical properties. However, incompletely remodeled Gas1p did not impact the partitioning of at least one other DRM resident protein (i.e., Pma1p, Figure 1C and 1D)." If the authors intend to suggest that this is a general mechanism, it would be beneficial to demonstrate this with additional GPI-APs, rather than relying solely on Gas1p, or they should avoid overgeneralization.

2. Regarding the results section of Figure 2A, where the authors state, "This observation is consistent with the data from the DRM experiments (Figure 1D) and suggests that in IPEM2 cells, Gas1p cannot be cleaved and cross-linked to the cell wall, and thus the protein is free to diffuse in the plasma membrane (Figure 2A)," it appears from the images and the fractionation experiment (Figure 1D) that Gas1p localizes not only at the plasma membrane but also in intracellular compartments. The authors should either provide additional images to support their claim or include a discussion to address this observation.

3. In the results section of Figure 2B, the authors claim, "These observations support the idea that the EtNP modification on Man2 impairs the cleavage and cross-linking of Gas1p to the cell wall." To strengthen this claim, it would be useful to include a positive control of a protein that localizes in the cell wall, and intensity band calculations should be performed relative to this control.

4. Image quantifications for Figure 2C, specifically regarding the fluorescence intensity of SNAP-Gas1p at the surface in mother and daughter cells, should be improved to better support the authors' conclusions. Also, Figure 2D could be incorporated as part of Figure 2C as a positive control. The corresponding explanations in the results section can be adjusted accordingly.

5. In Figures 2E and 2F, the authors note the presence of a single large vacuole. To confirm that the FM 4-64 signal is indeed on the vacuole membrane, it would be beneficial to include a PM staining. Without this, the FM 4-64 signal might be mistaken for PM membrane localization. Additionally, quantification of the number and size of vacuoles would make this point stronger, as the presence or absence of small vacuoles can be difficult to assess from images alone.

6. In conclusion to Figure 2, the authors state, "Based on these data, we concluded that the rate of FM4-64 uptake into IPEM2-GR cells was indistinguishable from that in mNeon-Vph1 expressing (and otherwise wild type) cells. Presumably, the increased FM4-64 fluorescence intensity observed in IPEM2-GR cells reflects some alteration to vacuoles in this strain." The authors should provide a clearer explanation of how they arrived at this conclusion, as the data suggest that IPEM2-GR cells may have enhanced endocytosis.

7. In Figure 3A, the authors state, "In wild-type cells, Snc1p cycles between the Golgi, the plasma membrane, and the endosome; but at steady state, the protein is predominantly localized to the plasma membrane and to nascent buds." However, the data provided do not offer strong evidence that Snc1p has differential PM localization between wild-type and IPEM2-GR cells. More convincing data should be presented, including a PM marker in the microscopy images, to clarify whether Snc1p is reaching the PM or if it is localized in a separate compartment from the cell wall.

8. In Figure 3, there is a possibility of clathrin-independent trafficking given that free GFP in IPEM2 clc1D and sla1D does not reach the level observed in IPEM2. This, or the presence of small and many vacuoles (which seem to be absent in IPEM2 clc1D and sla1D, although this is not clear from the figures), could influence the relative free GFP in IPEM2. The authors should consider commenting on the potential for a clathrin-independent mechanism to coexist. Additionally, the conclusion that GFP-Snc1p did not reach the vacuole is not sufficiently strong; in fact, there is partial colocalization with the FM 4-64 dye and a small band of free GFP in the immunoblot.

9. Higher quality images for Figures 3D and 3E are needed to draw any meaningful conclusions about EmC-tagged proteins. Including an arrow pointing to the bud neck in the image could also help readers. Additionally, it would be valuable if the authors could comment on a potential mechanism that might explain why certain PM proteins are sensitive to endocytosis while others are not.

10. For all the immunoblots shown in this study, a loading control is necessary, such as the expression of a housekeeping gene/protein or an image of the Ponceau stain of the blot. Additionally, incorporating quantifications of immunoblot results, fluorescence images, and the morphology of cellular structures where changes are observed would greatly benefit the readers and further strengthen the authors' claims.

Minor comments:

1. The manuscript requires thorough proofreading to address typographical errors, case sensitivity issues, symbols, and grammatical mistakes. Complex sentences should be simplified for clarity. For example, the description of each distinct strain used in the study within the results section is quite complicated, although the schematic provided is helpful. It would be better to simplify and clearly describe each strain to avoid confusion. The explanation for Figures 1C and 1D could be improved, among other areas.

2. The Abstract lacks a clear statement on the reason behind conducting the study and its potential significance. Additionally, the sentence structure could be simplified to enhance readability.

3. In the Introduction, the transition between the first and second paragraphs is abrupt. A smoother connection between the two is needed to improve the flow of information.

4.The Figure legends and Result titles should be more concise and to the point. Additional details could be moved to the Methods section or expanded upon in the Results section, if not already stated, to avoid redundancy.

5. Suitable citations should be provided for the statement in the Results section related to Figure 1: "Importantly, although permanent EtNP attachment to Man2 generates a lethal phenotype eventually." If this statement references an earlier study by the same group, it needs to be properly cited.

6. Some parts of the Results section are overly detailed with methodological content, which sometimes overshadows the key findings. It would be beneficial to carefully consider which parts should be moved to the Methods section and which need to remain in the Results to ensure clarity.

7. Several terminologies used in the Results section, such as "Prototypical GPI-AP" and the significance of high/low GP index, have not been introduced earlier in the manuscript. These terms should be explained in the Methods section to provide context for the study.

8. In several instances within the Results section, the term "Immunostaining" is used when the authors are actually referring to an Immuno-blot. Technical errors like this should be addressed throughout the manuscript.

9. The explanation for fractionation controls in Figure 1 is really appreciated but it should be explained before the Gas1p results to ensure proper understanding by the readers.

10. The dye excitation wavelength values and ranges used in the experiments are inconsistently stated between the Results and Methods sections. This discrepancy needs to be corrected for consistency.

11. In Figure 2A, it would be helpful if the authors clarify how they distinguish between daughter cells and explain the relevance of the relative distribution of newly synthesized proteins. It is also important to specify the exact conditions under which the GAL1/10 promoter is active. Additionally, the figure is somewhat confusing as the asterisk is placed in the mNeon channel, while the text discusses the distribution of mScarlet-Gas1p. To avoid confusion, the authors might consider omitting the asterisks in the mNeon-Gas1p channel, as they obscure the localized distribution of Gas1p.

12. Generalized statements that are not supported by data from this study or the literature should either be removed or clearly presented as hypotheses.

13. The inclusion of antibody information in the structured methods is a valuable addition, but the actual immunoblotting technique is not clearly described. The presentation of immunoblots as cut-ups rather than whole blots, along with the use of probes targeting proteins with similar molecular weights, suggests that a "strip and re-probe" approach may have been employed. However, this is not explicitly mentioned in the manuscript, and it would be helpful to clarify this to avoid any potential confusion or misinterpretation related to repeated experimentation under the same conditions.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.