Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis

  1. Yasuyuki Suda
  2. Hiroyuki Tachikawa
  3. Tomomi Suda
  4. Kazuo Kurokawa
  5. Akihiko Nakano
  6. Kenji Irie

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Abstract

Gametogenesis in budding yeast involves a large-scale rearrangement of membrane traffic to allow de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number, presumably accompanying a transient stall in membrane traffic, resulting in the loss of preexisting Golgi apparatus from the cell, was followed by local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, sed4 Δ, a mutant with impaired ERES formation, showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth.

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  1. Version published to 10.1101/2023.07.10.548399v2 on bioRxiv
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    Referee #3

    Evidence, reproducibility and clarity

    This work uses state-of-the art cell imaging and careful image quantification to study early secretory pathway dynamics during budding yeast gametogenesis. The work builds on previous findings of roles for Sec16 in ER exit site formation, Sec-body formation during specific developmental stages, and for Vps13 in lipid homeostasis. The work appears to be carefully conducted and is nicely presented.

    Much of the early work - here relating to meiosis in budding yeast, reflects that from studies of mitosis in other systems. This work therefore adds nicely to our understanding of membrane dynamics during cell division. Figures 1 and 2 are useful additions to the literature in this regard. I would have preferred images to be presented in magenta/green rather than red/green for wider accessibility.

    The advance is therefore not conceptual but functional. It would, in my view, be unfair to dismiss this as incremental.

    Major

    My major comment here relates to the FRAP data - the difference in half-life of recovery is clear but there are also substantial differences in the immobile fraction. It is vital that this is expanded on and discussed - it has direct relevance to the conclusions relating to the ongoing functional activity of ERES and the comparison to Sec bodies. Is it not possible that the immobile element here is a functional "reserve" like with Sec bodies? This might be consistent with multiple pools of COPII proteins acting at different stages to maintain then promote secretory activity. Some consideration needs to be given to expanding this and possibly including these data in the main figure. Further analysis and controls should also be included here, other COPII proteins and other markers that one might predict would not alter dynamics in these conditions.

    The key mechanistic advance in the manuscript relates to the role of Gip1 with clearly defined outcomes showing its role in ERES remodelling in nascent spores, regeneration of the Golgi and PSM elongation. The context of this part of the work is most important. Specifically, the comparison to VPS13 mutant needs to be expanded on and better explained. The analysis in Fig.4 needs a clearer explanation within the figure of how localization to the PSM is defined. The detail in the methods is also insufficient and the "custom R script" should be published with the work (or on a publicly accessible repository such as Git/Zenodo etc).

    The development of this work with the delta-sep mutant gives useful insight and the analysis of Sec16 does indeed support a model where this is an early marker for the process. Despite the link to septins no direct analysis of YSW1 is included (which suppresses the sporulation defect in gip1 ts alleles.

    Minor:

    Figure 3 introduces new data on reticulons and their impact on ER membrane shape. Again, this reflects findings in other systems but does not add much to the specific narrative of this story but is useful for those in the field. Similar to this, the data on Sec4 are of interest to the specialist but add little to the overall story.

    The discussion is quite lengthy and speculative dealing with themes and ideas that are not addressed directly by this work. My comments on the FRAP data relate directly to the models in Figure 8 and this discussion. Given the emphasis on nutrient starvation in the final discussion more detail is needed on the relative experimental conditions used here and in flies/mammals.

    Some relevant prior work should also be cited e.g. on the role of Sec16 on exit from mitosis PMID: 21045114, other work relating to gip1 mutants (PMID: 19465564).

    Consider presenting images as magenta/green.

    Referees cross-commenting

    I agree broadly with the other reviewers comments.

    While there are elements that could be developed much further. I am not familiar with the role of GIP1 in transcriptional regulation - is this from work in yeast or solely Arabidopsis (is GIP1 here - GBF1 interacting protein, a true equivalent?).

    I agree with the comments on the need for further - and well explained - statistical analyses.

    Significance

    Overall, the work is solid and adds nicely to our understanding. It is likely to be of most interest to a quite specialist audience. The work on PSM formation and spore formation is a clear advance with significant sections of the work being of interest to a wider audience working on early secretory pathway (notably COPII dynamics). Deeper mechanistic insight is missing but non-trivial. More depth would be added by studying further deletion mutants but I am not entirely convinced that this will rapidly advance the field further than this current presentation.

    My expertise is in early secretory pathway function.

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    Referee #2

    Evidence, reproducibility and clarity

    Suda et al. conducted an in-depth investigation of gametogenesis in budding yeast, focusing on the formation of the prospore membrane (PSM) through membrane trafficking rearrangement. They made an interesting observation that the number of endoplasmic reticulum exit sites (ERES) fluctuates during PSM formation, transitioning from decreasing to increasing. The study proposed that ERES regeneration, facilitated by protein phosphatase-1 and its specific subunit, Gip1, plays a crucial role in this process. However, the mechanism by which Gip1 regulates ERES numbers remains unclear, and the authors primarily used Gip1 mutants that may affect transcriptional regulation through Glc7, raising concerns about potential indirect effects. It is essential for the authors to experimentally validate the key mechanisms underlying their findings to strengthen their conclusions.

    Major Points:

    1. The conclusion that the loss of ERES causes a transient stall in membrane trafficking and leads to Golgi loss is based on the phenotype of GIP1 KO and SED4 KO cells. However, how Gip1 regulates ERES numbers remains unclear. The authors need to define whether Gip1 mediates this regulation through Glc7 dephosphorylation or via transcriptional regulation.
    2. The claim of ERES fluctuation during gametogenesis lacks statistical validation (Figure 1D). Since the difference is very small, the authors should perform a statistical analysis to determine if there is a significant difference in ERES numbers during different stages of gametogenesis.
    3. The conclusion regarding the loss and regeneration of the Golgi apparatus is based on qualitative observations of Mnn9, Sys1, and Sec7 signals. A quantitative analysis is necessary to strengthen these findings, as some cells may retain these signals despite their disappearance in representative images.
    4. Based on phenotypic similarity between GIP1 KO and SED4 KO cells, they concluded that Gip1 regulates the ERES number required for PSM expansion. They demonstrated that the number of ERES and Golgi dramatically decreased in GIP1 KO cells. The authors also need to do this experiment in SED4 KO cells? Since Sed4 affects ER function in general, the authors should demonstrate that SED4 KO cells are appropriate to make a conclusion about ERES regulation and PSM expansion.

    Referees cross-commenting

    Consistent with the other two reviewers, we feel our comments should be addressed prior to publication of this manuscript.

    Significance

    Overall, the study presents a high-quality imaging analysis of gametogenesis in budding yeast. However, the authors should experimentally validate the mechanisms underlying ERES regulation by Gip1 and conduct rigorous statistical analyses to support their observations. Additionally, since gametogenesis and Gip1 are yeast specific, the significance of this study might be limited.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: Yeast gametogenesis requires major membrane reorganization to ensure proper spore formation for survival during starvation, but many questions remain for how this process occurs. This current manuscript by Suda et al. uses fluorescence live imaging to visualize the dynamics of secretory pathway components which are critical for contributing lipids to the prospore membrane (PSM) in the developing spore. The authors find that ER exit sites (ERES) initially decrease and then gradually increase, coinciding with their appearance inside the PSM, suggesting that new lipids for PSM growth are trafficked through the secretory pathway from within the PSM. By screening through known genetic mutants that cause meiosis defects, the authors identify Gip1p, an adaptor for protein phosphatase 1, as a master upstream factor for prospore-associated ERES formation. Interestingly, the authors additionally identify a non-essential component of ERES in vegetative cells, sed4, to be important for sporulation and ERES PSM localization.

    Overall, the biological question is interesting and the imaging quality is appropriate. The general conclusion that ERES foci localize inside developing PSMs in a gip1- and sed4- dependent manner is supported by the data. However, the manuscript is purely descriptive; not much molecular insight is gleaned into how secretory pathway components localize to the inside of the PSM, nor is it clear how important this localization is in contributing new lipids to the PSM. Additionally, there are multiple points within the writing and presentation of the results, some specified below, that require clarification; more details in the quantifications also need to be included to ascertain whether the data robustly support the authors' current conclusions.

    Major comments:

    • The loss of gip1 affects multiple aspects of sporulation and leads to an early termination of spore formation, giving little insight into how ERES are established inside the PSM. The most intriguing result is that loss of sed4, a nonessential paralog of the membrane-bound Sar1 GEF, leads not only to sporulation defects, but also affects the localization of Sec13/ERES to the spores. Given that some spores still form in the sed4 cells, more experiments detailing ERES and golgi localization within the forming spores could be done. Does the golgi no longer localize within the PSM in sed4 cells? Is there are a PSM size difference between those that do and do not have ERES foci in this genetic background? Where does Sed4 localize in gip1 cells?
    • While Vps13 is introduced as an additional pathway for supplying lipids, this manuscript does not address the relative contribution of vesicular trafficking versus vps13 lipid transport in PSM formation. Where does Vsp13 localize in the sed4 cells? Are they enriched around/within those spores that do form?
    • The clarity of writing in the results and discussion section could be improved, some of which I point out below. The discussion could also be shortened.

    Specific comments:

    • For all quantifications, more information is necessary, including sample sizes, mean/median values, and number of biological replicates. It may be helpful to include these values in a separate supplemental table.
    • Relatedly, for 1D, 3C, and 4C graphs: It is difficult to judge whether the changes of ERES # are significantly different across the various genetic backgrounds as displayed, and given the large spread and small changes, statistical analyses are required to make such conclusions. Could the authors comment on why there is a minor yet noticeable percent of cells with very high ERES numbers?
    • To make specific conclusions that ERES 'regenerate' inside PSMs, more detailed quantifications of ERES foci # inside the developing prospores should be included, with appropriate statistical analysis.
    • Figure 6A, B: The localization of Glc7 does not look different to me, as claimed. The septin-like cable localization presumably occurs during elongation, as seen in 6A, and gip1D cells do not enter this phase, then it should be expected that there would be no septin-like localization. In 6B, the lower panels seem to show mature, closed PSMs; can the authors label the phases and explain why this is?
    • Figure 8, Top panels, indicate the purple coverage is PSM. It is unclear why the authors suddenly say that ERES are 'transiently inactivated' here and in the discussion to describe the lower # of ERES foci, whereas the appearance of PSM-associated ERES foci is considered 'regenerated' (which implies de novo assembly). In general, from the present data, one cannot conclude inactivation vs. formation/regeneration, so some caution in terminology is warranted.

    Minor comments:

    • A schematic showing the different stages of meiosis and of PSM formation would be useful.
    • Scale bar dimensions are missing for most of the figures.
    • It may be helpful to use an alternate color combination for merged images (i.e. cyan/yellow, red/cyan, or magenta/green), to accommodate colorblind readers.
    • For Figure 1C, authors should show orthogonal views along the z plane at timepoint 8 to show that ERES foci are indeed inside the PSM.
    • Figure 1E legend, define closed arrowhead; additionally, include an explanation in the main text of what the Spo20(51-91) marker is.
    • For kymograph displays (Figures 1E, 5A, 7E), please include time points in each frame.
    • Figure 4D, 4E legend, the multiple terms describing PSM circumference length is confusing: 'cell perimeter, 'PSM perimeter' and 'PSM length'. Please choose one term and describe this fully in the text.
    • Fig 5: The images in Fig 5 are dim and the gain should be adjusted accordingly. Figure 5B, is this an intensity trace of one punctum, or punctae from multiple cells as implied in the text?
    • More details, not just references, in methods for sporulation induction and image analysis should be included.

    Referees cross-commenting

    I also agree that our comments should be addressed prior to publication. Of the existing data, the need for further statistical analysis is a high priority.

    Significance

    The data and conclusions presented here are for a specialized, basic audience interested in yeast meiosis, especially focused on how membranes and the secretory pathway are remodeled during this process. The paper's results have some implications for the reproductive aging field, but this area is not directly investigated in this current manuscript. The paper uses mostly established organelle markers and gene mutants previously known to be involved. The finding of Sed4's involvement in sporulation is, to my knowledge, novel and intriguing.

    Reviewer Expertise: organelle morphology, the secretory pathway, protein aggregation, stress responses, aging, fluorescence microscopy, yeast, C. elegans, mammalian cells, biochemistry

  6. Version published to 10.1101/2023.07.10.548399v1 on bioRxiv