Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast

  1. Takuro Tojima
  2. Yasuyuki Suda
  3. Natsuko Jin
  4. Kazuo Kurokawa
  5. Akihiko Nakano

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Abstract

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis -cisternae, the earliest Golgi sub-compartment, is generated and how the Golgi matures into the trans -Golgi network (TGN). Here, we use high-speed and high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term ‘yeast ERGIC’. It occasionally exhibits approach and contact behavior toward the ER exit sites and gradually matures into the cis -Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis - and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins to better understand the Golgi-TGN transition. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the entire cisternal maturation process from the ERGIC to the Golgi and further to the TGN.

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  1. Version published to 10.7554/elife.92900 on eLife
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The present manuscript presents a thorough description of the relative localization (in space and time) of a number of proteins of the early secretory pathway. To that aim, the authors used by their custom-made 3D live cell super-resolution microscope (SCLIM) and the yeast S. cerevisiae as a model system. The main claim of these data is that the early secretory pathway in S. cerevisiae is organized by maturation from a newly proposed yeast ERGIC compartment all the way to the trans-Golgi network (TGN).

    Major comments:

    I have two major comments regarding this manuscript:

    1. It is not clear to me how the presented data shows the existence of an ERGIC in the yeast S. cerevisiae. I understand, and appreciate from this text too, that a clear definition of ERGIC, even in a mammalian system, is unclear. For this reason, I would first suggest that the authors provide a clear definition of what ERGIC means to them. Next, the experiments herein presented are all based on a very careful, thorough and nicely organized spatio-temporal mapping of a large number of early secretory pathway proteins, including the ERGIC53 yeast "counterpart" Emp46 (it would help to add, even as a supplementary figure, an alignment/sequence comparison between the human ERGIC53 and the S. cerevisiae emp46). However, the data presented here does not clearly indicate to me that there is a bona fide ERGIC in yeast. Couldn't it just be that what the authors call ERGIC is a cis-Golgi cisterna? I understand that the BFA experiments show a different behavior for some proteins, which fits with what the authors previously names GECCO in plants, so why not calling this GECCO? Again, it will be important to provide definitions of these compartments for the audience. Next, my main concern here is that this is all based on SCLIM, which is a very nice technique, but the resolution is limited in both space and time (by the way, it would be nice to explicitly measure of quantify the spatial resolution in x-y-z). Hence, it is not possible to discern whether an "independent" ERGIC is formed as compared to cis-Golgi cisterna. Electron microscopy (possibly CLEM) could help somehow resolve that and massively increase the strength of the claims, but I do understand this might be difficult for this group and very time consuming, so it might be important to clearly state the limitation of the herein presented data. A possible alternative to test if protein that are seen segregated are within the same membrane (as claimed here) would be to do trapping experiments where a reagent induces dimerization between the two proteins (when tagged with specific tags, such as FKBP/FRB).
    2. I could not find any details (maybe I have missed them) about how many times experiments were replicated and the statistical significance of the findings herein reported. In most figures, examples of microscopy images/videos are shown, and selected lines profiles are presented. However, it is not clear how robust these experiments are. Some ideas:

    2.1.) The major source of quantification is the peak-to-peak time distance between two proteins. In Table S1 some stdev is presented, but not clear how it is find (it is the sted of all n number of puncta? or of the mean duration per cell? or of the mean duration per experiment? I would suggest that the authors provide the results shown in Table S1 plotted as a histogram or superplot (see e.g. https://rupress.org/jcb/article/219/6/e202001064/151717/SuperPlots-Communicating-reproducibility-and) and clearly explain how statistics is performed.

    2.2) Also, the time-lapse movies are acquired with a 5s gap between time points. How is this included in the incertainty of the peak-to-peak duration in Table S1?

    2.3) In pg. 7 the authors write "Although experimental variation was high, the two zones appeared to be spatially segregated". Can the authors provide quantitative and statistical support of this claim?

    2.4) It is not clear to me how the puncta for analysis are selected. For example, in Fig. 1C, the punctum shown already shows some initial co-localization (it could be e.g. that a peak value was prior or after the duration of the time lapse movie, thereby biassing the computation of the peak-to-peak duration). So, if one would consider those spots e.g., positive for Emp46 that do not contain Mnn9 signal, how often do you see conversion (that is, appearance of Mnn9 signal)? Along the same lines, in pg. 8 the authors write "... signal appeared first and then mnn9-mCherry came up". Details on how this quantification is done and statistical analysis would be needed, to my opinion, to support the claim.

    Minor comments:

    1. The color code for the 3 color microscopy images is nice, however, the use of green and red for the 2 color images is a bit unfortunate for some people (like myself) who suffer from color blindness. I'd suggest to use green and magenta instead.
    2. Pg. 8: have the authors tested Rer1 vs Emp46?
    3. Pg. 8: I was of the impression that GRASP65 (GORASP1) is considered to be a cis-Golgi protein (see e.g., Tie et al eLife 2018). Then, what the authors call "ERGIC" couldn't it simply be a cis-Golgi cisterna?
    4. pg. 13: "propose to define Grh1, Rer1, and Sed5 as yeast ERGIC/GECCO...". What about Emp46?
    5. The first part of the manuscript (up to mid page 13) is clearly focused on defining ERGIC in yeast, then the paper appears as a set of experiments aimed at adding more components in their spatio-temporal mapping. This is ok, but is should be clearly motivated and explained in the Title, abstract and intro.
    6. The visualization of colocalization according to the opacity (as said in the methods) is somehow confusing to me. Are the 3D images projections or 3D renderings (no axes are seen)? In e.g. Fig. 6G or 8L, regions where green and magenta (or green and red) are colocalized do not appear white (or yellow), which visually suggests to the inattentive reader that there is no colocalization, when there is.
    7. I have not understood what this sentence in pg. 18 means: Similar segregation patterns are also observed during the Golgi-TGN maturation process (Tojima et al., 2019). "We propose that the ERGIC, Golgi, and TGN can coexist as structurally and functionally distinct zones within a single, maturing cisterna." Are they referring to ERGIC, Golgi, and TGN steady state components (proteins) or the structures themselves?
    8. The introduction of new data (mammalian data) in the discussion is odd. It might be ok, but I would frame it within a results section and use it later in the discussion.
    9. Fig.9: the arrows should go from protein to protein (some seem to go from in between proteins, such as the bottom-most arrow with 87.8 s time duration. Also in panel B, bottom part, some proteins are missing (Erd2 ad Chs5).
    10. Fig. 1 and many other: in the line profiles the distance in the x axis has no units or labels. Please add this and the direction of the line profile (an arrowhead would suffice).

    Significance

    General assessment:

    The experiments herein presented are based on a very careful, thorough and nicely organized spatio-temporal mapping of a large number of early secretory pathway proteins, including the ERGIC53 yeast "counterpart" Emp46. However, the data presented here does not convincingly show that there is a bona fide ERGIC in yeast. A major limitation is that the experiments are all based on a state-of-the-art, but still with a limited resolution, fluorescence microscopy technique. Ultrastructural data (e.g., CLEM) would massively help support or revisit the claims presented in this manuscript regarding the existence of an ERGIC compartment in yeast. Also, adding the information about the number of biological replicates and proper statistical analyses on the presented results would be needed to further support the claims.

    Advance:

    This manuscript builds on the authors' custom build 4D super-resolution microscope (SCLIM) and on previous results (e.g., Tojima et al., J. Cell Sci. 2019). The main novelty is in the study of a number of new early secretory pathway proteins and in the proposal of the existence of a non-stable, maturing ERGIC compartment in S. cerevisiae.

    Audience:

    This paper might be attractive for a broad audience of cell biologists, especially those interested in membrane biology, cell compartmentalization, and intracellular trafficking and secretion.

    Describe your expertise:

    I am an expert in membrane trafficking.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Tojima and colleagues present a very exciting 4D SCLIM analysis of 20 key-proteins controlling or occupying different stages of Golgi-mediated protein trafficking, taking the reader on a trip from the ER export sites to the ERGIC/GECCO, cis-, medial- and trans-Golgi towards the TGN/recycling endosome. The choice of molecular markers to be patiently time-resolved in 3D allowed the authors to assemble a temporal roadmap for the molecular players studied. This is most impressive.

    Major comments

    There is an enormous amount of patient systematic analysis packed in this paper, following a punctate organelle as it emerges from the dark, evolves over time (following a combination of 2 markers) with fluorescence peaks at specific time points, after which the signal disappears again. I am certain other cell biologists will be impressed, as I was, viewing the individual images and graphs presented, culminating ultimately in figure 9 that could go straight into a textbook to form a starting point for anybody who wishes to study a particular protein of interest and chose the most appropriate markers to compare it with. The authors propose that the ERGIC/GECCO/Golgi-remnants compartment is an evolutionary conserved structure even though it has a different subcellular distribution/morphology in different classes of eukaryotes. The data presented here and in earlier work seem to support this notion. In particular, the authors demonstrate that the yeast GRASP 65 homologue Grh1 is the earliest to appear closely followed by Ypt1 and Emp46. The fact that RER1 and ERD2 come slightly later is in line with a proposed gate-keeper function, because if they were instead to recycle continuously they should appear first in line. I agree with the authors that the simple model of ER-derived COPII vesicles fusing with each other and thus creating an ERGIC/GECCO de novo is probably too simplistic. The idea of a more permanent structure, pulsating between cargo-loading and cargo-releasing events, possibly associated with creating zones/subdomains within a single cisterna seems very attractive given the data shown here. This work is descriptive, but it is of very high importance to anybody engaged with experimental approaches to study protein sorting from the Golgi-apparatus back to the ER, or on to the plasma membrane or the lytic compartments. The 5 functional stages proposed for Golgi-maturation is an attractive starting point for future research, and I very much like the notion that ERGIC and cis-Golgi cisternae may start as zones/subdomains within a single cisternae, possibly formed via phase separations involving both protein-protein and protein-lipid interactions.

    Minor comments

    The title strongly focusses on the ERGIC and therefore the earliest sorting steps in the ER-Golgi system, but this manuscripts offers so much more. I was fascinated to learn that Ypt1 appears twice during cisternal maturation in yeast. This may be a yeast-specific phenomenon but it is very interesting. The same can be said about the proposal that Gea1 and Gea2 have different roles in the Golgi and act in different cisternae, and the localisation of AP-3 at the trans-Golgi rather than the TGN. The functional distinction between trans-Golgi and TGN and the differences in their origin are important points and it will be a shame if readers don't realise that this manuscript offers further insight into later steps in Golgi-mediated transport. There may be a case to add something to the abstract and/or modify the title accordingly, but then I also feel that long titles are not ideal and the ERGIC/GECCO portion is the more important take-home message. This is a case for the editorial team and the authors to make the most of the findings.

    Given the importance of ERD2 in sorting soluble proteins to be returned back to the ER, the authors may consider using the biological active XFP-TM-ERD2 fusion instead of ERD2-GFP, but this may be kept for future work. In plants, ERD2-GFP is mainly at the Golgi when overexpressed, its erroneous leakage to the ER is only observed at low expression or when K/HDEL proteins are co-expressed. The XFP-TM-ERD2 construct may be better confined to the ERGIC-GECCO and may have a different temporal pattern.
    The authors may consider citing Stornaiuolo et al., Mol Biol Cell 2003 Mar;14(3):889-902 who compared the trafficking of KDEL and KKXX pathways and concluded that KDEL proteins are retrieved prior to KKXX proteins....as this fits nicely into the current findings showing that ERD2 and RER1 appear sooner than COPI markers.

    Significance

    The significance of the work is high because it basically allows to add facts to models. We use models to explain an elusive process because it escapes direct observation. Once we can observe directly, a model can become fact. In simple terms, the authors allow us to see things that we could only speculate about in the past. Therefore, the results present a very significant advance and will be highly relevant to the entire cell biology community. This paper is an important landmark and will help the field to formulate new experimental approaches and models to understand the origins of the Golgi apparatus, the core of the secretory pathway that defines being a eukaryote.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    It is generally believed that budding yeast does not have the ER-Golgi intermediate compartment (ERGIC). In this study, the authors attempt to prove the existence of the ERGIC. They measured the kinetics of a few Golgi markers involved in the early secretory pathway in live cell imaging and observed the recruitment of Grh1 precedes that of Mnn9, suggesting the presence of pre-early cisternae. The authors propose that the Grh1-positive cisternae, assembling at the ER exit site (ERES) and progressing to become the early Golgi cisternae, represent the equivalent of the mammalian ERGIC in yeast.

    Major comments:

    The concept of the ERGIC in mammalian cells was initially proposed based on the protein ERGIC-53 in the 1990s. However, recent nanoscopy imaging data from the Lippincott-Schwartz lab challenges the conventional view of ERGIC by revealing the "ERGIC" is a membrane domain of the ERES (Wegel et al., Cell, 2021; PMID: 33852913), suggesting it might not be appropriate to adopt this concept.

    The authors' observations could be interpreted differently. Since the ERGIC is not molecularly defined in their study, the authors cannot prove its existence in yeast unequivocally. Their data indicate the presence of Golgi cisternae, characterized by Grh1, that precede the earliest known cisternae. Although the authors refer to these Grh1-positive cisternae as the "ERGIC", they are essentially "pre-early" cisternae that progress to become the early Golgi cisternae. Nevertheless, their findings could extend the budding yeast Golgi cisternal progression unit further upstream to include the ERES as the starting point for Golgi cisternal maturation. To further explore this, it would be interesting to investigate the kinetics of COPII subunits in cisternal progression along with Grh1 or Mnn9 and to plot COPII components in the Figure 9 map.

    The second half of the manuscript appears to deviate from the main focus on identifying the ERGIC. This section primarily presents the Golgi localization of four Golgi proteins (Ypt1, Gea1, Gea2, and Alp6) deduced from kinetics. However, it lacks functional studies to substantiate the authors' claims on their cellular functions. As a result, this part of the study remains purely speculative and might not support the authors' claims. Given that Figure 9 provides a highly informative summary of all kinetics and localization data, I recommend the authors keep but significantly abridge this section.

    The manuscript also has a few major concerns.

    1. The analysis of only one fluorescent particle or Golgi cisternal punctate structure is insufficient for a Golgi marker, considering the substantial variation of Golgi cisternae. To improve statistical robustness, the authors should select multiple fluorescent particles from multiple cells, displaying plots with averaged intensities, error bars, and sample sizes (n).
    2. In Fig. 5A, the BFA-induced lumps positive for Grh1, Rer1, and Sed5 may potentially represent the ERES, as observed in mammalian cells (Ward et al., JCB, 2002; PMID: 11706049). To verify this, the authors should co-label these lumps with COPII subunits.
    3. The authors previously reported the "hug-and-kiss" model for cargo transport from the ERES to the early Golgi cisternae. As the current study is highly relevant to the "hug-and-kiss" model, it is disappointing that the authors did not provide further data and comment on it. The "hug-and-kiss" and "ERGIC" transport modes are two distinct ways for secretory cargo transport from the ERES to the early Golgi cisternae. The authors should verify the "hug-and-kiss" transport and report the relative frequency of the two transport modes.
    4. The current version has minimal background knowledge of ERGIC in mammalian and yeast cells. Therefore, the authors should provide a comprehensive introduction to ERGIC.

    Significance

    General assessment:

    The data presented in the manuscript is novel and appears to be convincing. However, one of the major concerns is the lack of statistical robustness, which requires to be addressed. Furthermore, the manuscript's data could be interpreted differently, as elaborated in the major comments.

    Advance:

    While the interpretation of the manuscript's data requires reconsideration, it can contribute to our understanding of secretory trafficking at the Golgi. The manuscript could fill a crucial gap in our knowledge in this field by addressing the major comments.

    Audience:

    Cell biologists in the membrane trafficking field, particularly those working on the Golgi, would find the manuscript interesting.

    My expertise:

    My research focuses on membrane trafficking at the ER, Golgi, and endosome.

  6. Version published to 10.1101/2023.08.03.551802v1 on bioRxiv