High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae

  1. Robert M Gingras
  2. Abigail M Sulpizio
  3. Joelle Park
  4. Anthony Bretscher

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    The process of secretory vesicle formation, transport, and fusion in yeast has mainly been characterized through biochemical and genetic means. Only limited information was available about the detailed timeline and order of events. This study fills the gap with a high-resolution temporal analysis, which provides new insights into when key components arrive and depart and how they promote vesicle tethering and fusion. The work is experimentally strong, and improvements to the presentation will ensure that the findings are communicated effectively.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Most of the components in the yeast secretory pathway have been studied, yet a high-resolution temporal timeline of their participation is lacking. Here, we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4’s GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5 s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 s prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.

Article activity feed

  1. Version published to 10.7554/elife.78750 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    I'm curious about whether the microscopy provided any information about when secretory vesicles leave the TGN. Do they leave throughout the lifetime of a TGN structure, or do they leave in a burst when a TGN structure disperses as marked by loss of Sec7? This information might take us a step closer to understanding how secretory vesicles are made.

    Given the limitations of our current imaging set-up with regards to high-speed 3D two-color microscopy, we were unable to capture a large number of these events and therefore cannot make concrete statements about this, however, the quantified events did not appear to be preceded or followed by additional events, suggesting some temporal separation.

    Reviewer #2 (Public Review):

    The authors are encouraged to integrate their data together better with published biochemistry and structural work into more complete mechanisms for vesicle trafficking, tethering and fusion. The manuscript would be improved by a clearer model(s) of how these factors come together to carry out exocytosis.

    This suggestion has been addressed by the addition of a new model figure (Figure 9).

    Moreover, many conclusions (especially as they appear in the Results and Figures) are written as if they are well supported by the data (or others' data), when they are often speculative, or reasonable alternative explanations exist. The authors should be clear about which conclusions are well supported, and which are hypotheses. (e.g. Fig 6I, which is a terrific figure, but some of the "conclusions/statements" are speculations).

    We have made textual changes to make clearer distinctions between conclusions that are supported by the data, and which are more speculative.

    The mechanistic and experimental definitions for the start/end of "tethering" and "fusion" are not clearly stated in the main text, which leads to confusion when examining the arrival of different factors (and seems to lead to circular arguments about what is defining what). Are these definitions well supported by the previously published and current data? E.g. is the disappearance of GFP-Sec4 really equal to the fusion event? Without data showing membrane-merger or content delivery, this needs to be described as an assumption that is being made.

    Early in the results, we now define precisely what we interpret as the start of tethering and time of fusion. Unfortunately, thus far, all attempts at designing a cargo marker suitable for defining membrane fusion have not succeeded, however, we believe the observations in Figure 4 strongly support assumption that loss of GFP-Sec4 signal coincides with fusion.

    The Sro7 results and conclusions are complicated, and not always carefully supported, for several reasons: there is a functionally redundant paralog Sro77, and data shows Sro7 can bind to Sec4, Sec9 and Exo84 in exocyst (Brennwald, Novick and Guo labs). The authors should be clearer, as they seem to pick and choose which interactions they think are relevant for different observations.

    We did not intend to “pick-and-choose” relevant interactions and now more clearly state what our Sro7 results mean.

    The assumption that yeast Sec1 behaves similarly to other Sec1/Munc18 proteins for "templating" SNARE complex assembly, e.g. Vps33 in Baker et al, is unlikely, given the binding studies from a number of labs (Carr, McNew, Jantti). Furthermore, the evidence for Sec1 interaction with exocyst suggests that they may work together (Novick, Munson labs). Previous data from the Guo lab (Yue et al 2017) and new BioRxiv data from the Munson/Yoon labs suggest that exocyst may play key roles in SNARE complex assembly and fusion.

    We did not mean to imply that the exocyst does not play a meaningful and critical role in SNARE complex assembly and fusion. This was an unintentional omission, which we have now addressed in the text. Our interpretation of the published meaning of SM-protein “templating” is that SM’s facilitate the alignment of the critical zero-layer ionic residues in the SNARE motifs, which may be possible regardless of affinity to single SNARE motifs. Indeed, for Sec1 specifically, it may be possible that this exact function is of lower importance relative to, perhaps, the stabilization and protection of trans-SNARE complexes prior to membrane fusion. Future studies may clarify this.

    There is concern that the number of molecules of each of the factors measured is accurate, and how the authors really know that they are visualizing single vesicle events (especially with data showing that "hot-spots" may exist). For example, why is the number of molecules of exocyst is ~double or more than that previously observed (Picco et al; Ahmed et al with mammalian exocyst).

    Estimating the numbers of molecules is subject to some variation due to fluorescent tags used and to some extent where the protein is tagged. Since different tags were used in the earlier studies, being within a factor of two is not that surprising.

    For puncta of exocyst subunits in the mother or moving towards the plasma membrane, what is the evidence that they are actually on vesicles? The clearest argument seems to be the velocity at which they move, but this could be due to the direct interaction of exocyst with the myosin (which is a tighter interaction in vitro than exocyst-Sec4 binding), rather than being on vesicles. Furthermore, do all the exocyst complexes in the cell show this behavior, or could these be newly synthesized/assembled complexes?

    Transport of the exocyst by myosin alone without a vesicle seems very unlikely, as this myosin V needs to be activated by binding vesicle-associated Sec4 (Donovan et al., 2012, 2015). Moreover, transport of just two exocyst complexes by a myosin dimer would be very hard to detect. Nonetheless, we have added an additional supplementary figure (Figure 1 Supplement 5C) illustrating a clear example of exocyst complex colocalization with a secretory vesicle in the mother cell which we hope will quell fears that the exocyst complex is indeed on secretory vesicles, albeit in small numbers, during this stage of transport.

    With regard to the exocyst octamer leaving at the time of "fusion," the authors should discuss Ahmed et al.'s finding of Sec3 leaving prematurely in mammalian cells, as well as data from the Toomre lab.

    We did reference this earlier work in mammalian cells and indicate that it differs from the situation in yeast. We don't have anything insightful to be drawn from these differences.

    Reviewer #3 (Public Review):

    In this context, it is notable that dual-channel imaging appears to be made by sequential, not simultaneous, acquisition, which deserves a currently missing comment. Moreover, given the weight that image acquisition plays in this project, it might be described and justified better.

    As noted above, we have expanded our description of the microscopy. We took two-color images sequentially as our microscope is not configured with a beam-splitter for simultaneous imaging.

    This referee could not fully understand the routine of image acquisition, specifically, the continuous movement of the stage in the Z-axis as images are streamed (to the RAM or to the disk? the latter takes time, line 177); does it mean that Z-stepping is solely governed by the exposure time? The CCD camera penalizes pixel size (16 µm) at the expense of achieving outstanding quantum efficiency. The optical path includes a 100x objective and a 2x magnification lens to compensate for the large camera pixel size, thereby achieving 0.085 µm/pixel, but these lenses 'waste' part of the fluorescent signal. One wonders if the CMOS camera (6.5 µm pixel size) coupled with a 63x objective wouldn't be appropriate? A brief discussion on this choice would be helpful for readers.

    We now discuss the microscopy in more detail and why we use an EMCCD rather than aCMOS camera.

    It is remarkable that Sec2 and Sec4 are recruited to membranes even before a vesicle is formed (Fig 6I). I find somewhat weak the evidence that RAB11s 'mark' the TGN, and disturbing the fact that RAB11 reaches the PM (does GFP tagging prevent GAP accession?). I should like to recommend strongly that the authors integrate into the introduction/discussion information on the late steps of exocytosis available for Aspergillus nidulans, another ascomycete that is particularly well suited for studying this process. Here RAB11 is not a late Golgi resident but is transiently (20 s) recruited to TGN cisternae in the late stages of their 120 s maturation cycle to drive the transition between Golgi and post-Golgi (Pantazopoulou MBoC, 2014). Recruitment of RAB11 to the TGN is preceded by the arrival of its TRAPPII GEF (Pinar, PNAS 2015; Pinar PLOS Gen 2019), a huge complex that is incorporated en bloc to the TGN (Pinar JoCS, 2020). Upon RAB11 acquisition RAB11 membranes engage molecular motors (Penalva, MBoC 2017) to undertake a several-micron journey that transports them to a vesicle supply center located underneath the apex (review, Pinar & Penalva, 2021). Here is where Sec4 is located, strongly indicating that there is a division of work between two Rabs each mediating one of the two stages between the TGN and the membrane (Pantazopoulou, 2014, MBoC).

    In the general comments above, we discuss the possible artifact of tagged Ypt31 on the PM. In the Discussion, we now compare our results in S. cerevisiae with the findingss in A. nidulans.

  3. eLife

    Evaluation Summary:

    The process of secretory vesicle formation, transport, and fusion in yeast has mainly been characterized through biochemical and genetic means. Only limited information was available about the detailed timeline and order of events. This study fills the gap with a high-resolution temporal analysis, which provides new insights into when key components arrive and depart and how they promote vesicle tethering and fusion. The work is experimentally strong, and improvements to the presentation will ensure that the findings are communicated effectively.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This analysis of yeast secretory vesicles centers around the Rab GTPase Sec4. The authors previously imaged Sec4-labeled secretory vesicles, but not at a speed that would allow the processes of secretory vesicle biogenesis and exocytosis to be dissected. Various Rab4 interactors engage at different times. The data presented here indicate that Sec2, the GEF for Sec4, binds to the TGN, followed quickly by Sec4, followed by the type V myosin Myo2. The multisubunit exocyst tether is a Sec4 effector that is recruited during secretory vesicle transport. Next comes the Rho3 GTPase at the plasma membrane. Then the loss of Sec2 and Myo2 is followed by recruitment of the SNARE regulator Sro7, followed by recruitment of the SM protein Sec1 and the lipid-interacting protein Mso1, which together trigger the final SNARE-dependent fusion event.

    In my opinion, this is an impressive study that takes the analysis of secretory vesicles to a new level. The authors describe rigorous quantitative microscopy that leads to a plausible mechanistic timeline of vesicle tethering and fusion. Among the insights described is evidence that the exocyst progressively associates with secretory vesicles during their transport, and that Sec3 is a stable component of the heterooctameric exocyst rather than a separable "landmark" subunit. Additional data imply that loss of Sec4 from the vesicle is needed to progress to fusion, that Sro7 and Sec1 associate with secretory vesicles only after tethering, and that Mso1 helps to concentrate Sec1 locally at sites of vesicle fusion. Overall, this approach gives a new perspective on the choreographed events that take place during the lifetime of a secretory vesicle.

    As the authors note in the Discussion, when we consider the first stage in the lifetime of a secretory vesicle, the biogenesis mechanism "is still shrouded in mystery." I'm curious about whether the microscopy provided any information about when secretory vesicles leave the TGN. Do they leave throughout the lifetime of a TGN structure, or do they leave in a burst when a TGN structure disperses as marked by loss of Sec7? This information might take us a step closer to understanding how secretory vesicles are made.

    I have no significant concerns with the data or the presentation.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript presents a tour de force of imaging of budding yeast exocytosis factors and their arrival and disappearance from "fusion sites" at the plasma membrane. This work builds on previous data from their lab and others, with substantial technological advances that improve the temporal and spatial resolution of various factors, plus data for additional factors. The presented data fills detailed gaps in the field and may lead to (or support) new mechanistic hypotheses.

    The authors use substantially improved microscopy techniques and new tagged constructs, to image their dynamics and co-localization with increased resolution compared to previous studies. These data provide an improved picture of how these factors engage temporally and spatially in live yeast cells during exocytosis.

    Overall, these studies are of high importance in the field. However, the manuscript itself is difficult for a non-specialist reader to follow, very little introduction and explanations are given for most of the numerous components. The authors are encouraged to integrate their data together better with published biochemistry and structural work into more complete mechanisms for vesicle trafficking, tethering and fusion. The manuscript would be improved by a clearer model(s) of how these factors come together to carry out exocytosis.

    Moreover, many conclusions (especially as they appear in the Results and Figures) are written as if they are well supported by the data (or others' data), when they are often speculative, or reasonable alternative explanations exist. The authors should be clear about which conclusions are well supported, and which are hypotheses. (e.g. Fig 6I, which is a terrific figure, but some of the "conclusions/statements" are speculations).

    The mechanistic and experimental definitions for the start/end of "tethering" and "fusion" are not clearly stated in the main text, which leads to confusion when examining the arrival of different factors (and seems to lead to circular arguments about what is defining what). Are these definitions well supported by the previously published and current data? E.g. is the disappearance of GFP-Sec4 really equal to the fusion event? Without data showing membrane-merger or content delivery, this needs to be described as an assumption that is being made.

    The Sro7 results and conclusions are complicated, and not always carefully supported, for several reasons: there is a functionally redundant paralog Sro77, and data shows Sro7 can bind to Sec4, Sec9 and Exo84 in exocyst (Brennwald, Novick and Guo labs). The authors should be clearer, as they seem to pick and choose which interactions they think are relevant for different observations.

    The assumption that yeast Sec1 behaves similarly to other Sec1/Munc18 proteins for "templating" SNARE complex assembly, e.g. Vps33 in Baker et al, is unlikely, given the binding studies from a number of labs (Carr, McNew, Jantti). Furthermore, the evidence for Sec1 interaction with exocyst suggests that they may work together (Novick, Munson labs). Previous data from the Guo lab (Yue et al 2017) and new BioRxiv data from the Munson/Yoon labs suggest that exocyst may play key roles in SNARE complex assembly and fusion.

    There is concern that the number of molecules of each of the factors measured is accurate, and how the authors really know that they are visualizing single vesicle events (especially with data showing that "hot-spots" may exist). For example, why is the number of molecules of exocyst is ~double or more than that previously observed (Picco et al; Ahmed et al with mammalian exocyst).

    For puncta of exocyst subunits in the mother or moving towards the plasma membrane, what is the evidence that they are actually on vesicles? The clearest argument seems to be the velocity at which they move, but this could be due to the direct interaction of exocyst with the myosin (which is a tighter interaction in vitro than exocyst-Sec4 binding), rather than being on vesicles. Furthermore, do all the exocyst complexes in the cell show this behavior, or could these be newly synthesized/assembled complexes?

    With regard to the exocyst octamer leaving at the time of "fusion," the authors should discuss Ahmed et al.'s finding of Sec3 leaving prematurely in mammalian cells, as well as data from the Toomre lab.

  6. eLife

    Reviewer #3 (Public Review):

    Four decades after the seminal work of the Schekman's lab on the genetic identification of the core eukaryotic secretory machinery the molecular roles of the individual components have been largely characterized. Yet our understanding of how these components are organized to define processes is wanting, with notable controversies still hovering over at several levels of the secretory pathway, including the events that take place in the ER/Golgi interface, the transit across the Golgi, the biogenesis of secretory vesicles and the delivery, tethering and docking of these vesicles to the membrane. This manuscript mostly addresses the latest steps of this chain of events and makes some incursions into the biogenesis of vesicles at the TGN. It represents a serious and honest attempt to define the timeline of events that, driven by key components such as the Sec4 ras-in-brain (Rab) GTPase, its effectors myosin-5, Sro7 and the exocyst, its GEF, Sec2 and the prototypic Sec/Munc protein Sec1, a regulator of trans-SNARE complex formation, ultimately result in the tethering, docking and fusion of vesicles with the membrane of the polarized bud of the ascomycete yeast Saccharomyces cerevisiae. Tethering, as defined by light microscopy appears to be a robust process reproducibly lasting for five seconds, before fusion, as defined by the loss of vesicle components, takes place. Important evidence is provided that the exocyst is incorporated as an holo-complex to secretory vesicles. Overall, even though this work will likely suffer modifications and amendments as knowledge and technology progress, it will nevertheless become the reference blueprint around which any future work in the field will pivot.

    This work represents a very substantial advance in the field of exocytosis. Besides reporting with unmatched time resolution the tethering of vesicles with the membrane, it describes a herculean effort to gain mechanistic understanding of the process by using a score of genetic perturbations and fluorescent reporters. I feel that evidence that Sec3 travels with the exocyst rather than contributing a milestone for exocyst landing will be disputed, but this referee finds it as convincing as appealing. Nearly as important is the timing of Sec1 action in the fusion step. However, it is the delineation of a timeline that will make this paper a reference in the field.

    Understanding the technology for image acquisition is critical to appreciating the strengths of this MS (333 ms/Z-stack time point may be considered super-resolution - in the time dimension. Therefore, its description requires clarification in places. The experimental work is almost exclusively based on live microscopy using fluorescent proteins tagged by allelic replacement. The microscopy routine for single fluorophore analysis provides time series with a resolution of 3-5 fps that enables authors to resolve, using robust statistical tests, events separated by seconds. In this context, it is notable that dual-channel imaging appears to be made by sequential, not simultaneous, acquisition, which deserves a currently missing comment. Moreover, given the weight that image acquisition plays in this project, it might be described and justified better. The Materials and methods lack detail, for example, the laser lines & power used for excitation. This referee could not fully understand the routine of image acquisition, specifically, the continuous movement of the stage in the Z-axis as images are streamed (to the RAM or to the disk? the latter takes time, line 177); does it mean that Z-stepping is solely governed by the exposure time? The CCD camera penalizes pixel size (16 µm) at the expense of achieving outstanding quantum efficiency. The optical path includes a 100x objective and a 2x magnification lens to compensate for the large camera pixel size, thereby achieving 0.085 µm/pixel, but these lenses 'waste' part of the fluorescent signal. One wonders if the CMOS camera (6.5 µm pixel size) coupled with a 63x objective wouldn't be appropriate? A brief discussion on this choice would be helpful for readers.

    There is an elephant in the room of in vivo microscopy that no one dares to comment on: reporter proteins are mutant versions carrying a heavy and potentially oligomerising rucksack - the fluorescent protein tag. The authors take the honest approach of acknowledging that some of the tagged proteins such as Sec4 are disfunctional and that certain reporters are incompatible with each other as they give rise to synthetic negative effects. In the end, they conclude that using diploids carrying the GFP-tagged allele in heterozygosis with the wt represents the most physiological approach to track proteins until less intrusive fluorescent tags are developed.

    It is remarkable that Sec2 and Sec4 are recruited to membranes even before a vesicle is formed (Fig 6I). I find somewhat weak the evidence that RAB11s 'mark' the TGN, and disturbing the fact that RAB11 reaches the PM (does GFP tagging prevent GAP accession?). I should like to recommend strongly that the authors integrate into the introduction/discussion information on the late steps of exocytosis available for Aspergillus nidulans, another ascomycete that is particularly well suited for studying this process. Here RAB11 is not a late Golgi resident but is transiently (20 s) recruited to TGN cisternae in the late stages of their 120 s maturation cycle to drive the transition between Golgi and post-Golgi (Pantazopoulou MBoC, 2014). Recruitment of RAB11 to the TGN is preceded by the arrival of its TRAPPII GEF (Pinar, PNAS 2015; Pinar PLOS Gen 2019), a huge complex that is incorporated en bloc to the TGN (Pinar JoCS, 2020). Upon RAB11 acquisition RAB11 membranes engage molecular motors (Penalva, MBoC 2017) to undertake a several-micron journey that transports them to a vesicle supply center located underneath the apex (review, Pinar & Penalva, 2021). Here is where Sec4 is located, strongly indicating that there is a division of work between two Rabs each mediating one of the two stages between the TGN and the membrane (Pantazopoulou, 2014, MBoC).

  7. Version published to 10.1101/2022.03.17.484778v1 on bioRxiv