Organization of the Yeast Seipin Complex Reveals Differential Recruitment of Regulatory Proteins

  1. Yoel A. Klug
  2. Pedro Carvalho

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Abstract

Lipid droplets (LDs) are neutral lipid storage organelles that emerge from the endoplasmic reticulum (ER). Their assembly occurs in ER regions enriched with seipin which, through its homooligomeric ring-like structure, facilitates neutral lipid nucleation. In yeast, seipin (Sei1) partners with Ldb16, Ldo45 (yeast homologue of human LDAF1) and Ldo16, which regulate LD formation and consumption. How the molecular architecture of the yeast seipin complex and its interaction with regulatory proteins adapt to different metabolic conditions remains poorly understood. Here, we show that multiple Ldb16 regions contribute differently to recruiting Ldo45 and Ldo16 to the seipin complex. Using an in-vivo site-specific photo-crosslinking approach, we further show that Ldo45 resides at the center of the seipin ring both in the absence and presence of neutral lipids. Interestingly, neutral lipid synthesis leads to the recruitment of Ldo45 but not Ldo16 to the complex. Our findings suggest that the seipin complex serves as a pre-assembled scaffold for lipid storage that can be remodeled in response to increased neutral lipid availability.

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    Reply to the reviewers

    __We thank the reviewers for the supportive suggestions and comments. We have addressed all comments underneath the original text in red. As suggested, we added to line numbers to the text and use these numbers to refer to the changes made. __

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript is well written and presents solid data, most of which is statistically analyzed and sound. Given that the author's previous comprehensive publications on seipin organization and interactions, it might be beneficial (particularly in the title and abstract) to emphasize that this manuscript focuses on the metabolic regulation of lipid droplet assembly by Ldb16, to distinguish it from previous work. Perhaps one consideration, potentially interesting, involves changes in lipid droplet formation under the growth conditions used for galactose-mediated gene induction.

    We thank the reviewer for the supportive comments and suggestions.

    Comments: (1) Fig. 3 and 4. The galactose induction of lipid droplet biogenesis in are1∆/2∆ dga1∆ lro1∆ cells though activation of a GAL1 promoter fusion to DGA1 is a sound approach for regulating lipid droplet formation. Although unlikely, carbon sources can impact lipid droplet proliferation and (potentially interesting) metabolic changes under growth in non-fermentable carbon sources may impact lipid droplet biogenesis; in fact, oleate has significant effects (e.g. PMID: 21422231; PMID: 21820081). The GAL1 promoter is a very strong promoter and the overexpression of DGA1 via this heterologous promoter might itself cause unforeseen changes. Affirmation of the results using another induction system might be beneficial.

    We thank the reviewer for these suggestions. In this study we focused on the organisation of the yeast seipin complex during the process of LD formation. We chose to use galactose-based induction of Dga1 because this is a well-established and widely used assay in the field, extensively characterized by many groups over the years. The tight control it provides, enabling synchronous and rapid LD induction, makes it the method of choice for many researchers. Importantly, the LDs formed using this assay are morphologically normal and involve the same components as LDs formed under other conditions.

    Regarding the role of metabolism in LD formation, it is worth noting that galactose is metabolized by yeast primarily through fermentation, following its conversion to UDP-glucose. Therefore, its use does not involve drastic metabolic changes. The impact of metabolism in LD biogenesis is an interesting question but it falls beyond the scope of the current study.

    (2) Fig. 3B. Although only representative images are shown, the panel convincingly shows that lipid droplets do form upon galactose induction of DGA1 in are1∆/2∆ dga1∆ lro1∆ cells. However, it does not show to what extent. Are lipid droplets synthesized at WT levels? How many cells were counted? How many lipid droplets per cell? Is there a statistical difference with respect to WT cells?

    We did not assess these parameters in this study. The aim of the study was to assess the relations between components of the seipin complex with and without lipid droplets. For this purpose, inducing lipid droplet formation over a 4-hour period was sufficient to address that specific question. As mentioned above, LDs formed using this assay are morphologically normal and involve the same components as LDs formed under other conditions. This being said, it is known that prolonged overexpression of Dga1 (> 12hours) can lead to enlarged LDs.

    (3) Fig. 2D. It is not clear how standard deviation can be meaningfully applied to two data points, let alone providing a p-value. For some of these experiments, triplicate trials might provide a more robust statistical sampling.

    We thank the reviewer for this suggestion. We have added 2 more repeats to the Co-IP in figure 2.

    Reviewer #1 (Significance (Required)):

    Klug and Carvalho report on the lipid droplet architecture of the yeast seipin complex. Specifically, the mechanism of yeast seipin Sei1 binding to Ldo16 and the subsequent recruitment of Ldb45 is analyzed. These results follow from a recent publication (PMID: 34625558) from the same authors and aims to define a more precise role for the components of the seipin complex. Using photo-crosslinking, Ldo45 and Ldo16 interactions are analyzed in the context of lipid droplet assembly.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary:

    Klug and Carvalho apply a photo-crosslinking approach, which has been extensively used in the Carvalho group, to investigate the subunit interactions of the seipin complex in yeast. The authors apply this approach to further study possible changes within the seipin complex following induction of neutral lipid synthesis and lipid droplet (LD) formation. The authors propose that Ldo45 makes contact with Ldb16 and that the seipin complex subunits assemble even in the absence of LDs.

    Major comments:

    Overall, this is a focused and well-executed study on one of the fundamental structural components of LDs. The study addresses the subunit interactions of the seipin complex but does not look into their functional consequences, for example how the mutations on Ldb16 that affect its interaction with Ldo45, influence LD formation; similarly, the authors make the interesting observation that Ldo16 may be differentially affected by the lack of neutral lipids (Fig. 3A) but this observation is not explored.

    We thank the reviewer for this comment. The Ldb16 mutations analyzed in this study have been previously characterized by us (see Klug et al., 2021 – Figure 3) and exhibit a mild defect in lipid droplet (LD) formation. This phenotype is unlikely to result from impaired Ldo16/45 recruitment, as deletion of Ldo proteins causes only a very mild effect on LD formation (as shown in Teixeira et al., 2018 and Eisenberg-Bord et al., 2018).

    We agree that the differential effect on Ldo proteins by the absence of neutral lipids is particularly interesting. However, its exploration falls outside of the scope of the current study and should be thoroughly investigated in the future.

    For the crosslinking pull-downs (Fig. 1), it seems that the authors significantly overexpress (ADH1 promoter) the Ldb16 subunit that carries the various photoreactive amino acid residues, while keeping the other (tagged) seipin complex members at endogenous levels. Would not this imbalance affect the assembly of the complex and therefore the association of the different subunits with each other?

    We thank the reviewer for this comment. The in vivo site-specific crosslinking is highly sensitive methodology to detect protein-protein interactions in a position-dependent manner. However, one of the caveats of the approach is the low efficiency of amber stop codon suppression and BPA incorporation. To mitigate this limitation, we (and others) induce the expression of the amber-containing protein (in this case Ldb16) from a strong constitutive promoter such as ADH1. Therefore, despite using a strong promoter, the overall levels of LDB16 remain comparable to endogenous levels due to the inherently low efficiency of amber suppression. Moreover, it is known that when not bound to Sei1, Ldb16 is rapidly degraded in a proteasome dependent manner (Wang, C.W. 2014), further preventing its accumulation.

    Although the authors do show delta4 cells with no LDs (Fig. 3B, 0h), galactose-inducible systems in yeast are known to be leaky. Given that the authors' conclusion that the complex is "pre-assembled" irrespective of the addition of galactose, I think it would be important to confirm biochemically that there is no neutral lipid at time point 0. Alternatively, it may be better to simply compare wt vs dga1 lro1 or are1are2 mutants - there is no need for GAL induction since the authors look at one time point only.

    Among the various regulable promoters, GAL1 shows a superior level of control. For example, expression of essential genes from GAL1 promoter frequently leads to cell death in glucose containing media, a condition that represses GAL1 promoter. Having said this, we cannot exclude that minute amounts of DGA1 are expressed prior galactose induction. However, if this is the case, the resulting levels of TAG are insufficient to be detected by sensitive lipid dyes and to induce LDs, as noted by the reviewer. Therefore, we believe our conclusions remain valid. This is consistent that we use in the text, where we refer to LD formation rather than complete loss of neutral lipids. To make this absolutely clear we replaced the word “presence” to “abundance” in line 236.

    Lastly, we do not agree with the reviewer that using double mutants (are1/2 or dga1/lro1 mutants) would be sufficient since these mutations are not sufficient to abolish LD formation – a key aspect of this study. The GAL1 system allows us to monitor 2 time points in the same cells –no LDs (time 0h) and with LDs (Time 4h). The system proposed by the reviewer would only allow a snap shot of steady state levels in different cells rather than within the same cell culture.

    Some methodological issues could be better detailed. For example, which of the three delta4 strains was used to induce neutral lipid in Fig. 4B? How exactly were the quantifications in Fig. 4D performed (I assume they were done under non-saturating band intensity conditions, as for some residues it is difficult to conclude whether the blot aligns with the quantification results).

    We thank the reviewer for these comments. We have clarified the strain number in the figure legend of figure 4B (strain yPC12630).

    We have also added the following text in rows 437-441 in the methods section: “Reactive bands were detected by ECL (Western Lightning ECL Pro, Perkin Elmer #NEL121001EA), and visualized using an Amersham Imager 600 (GE Healthcare Life Sciences). Data quantification was performed using Image Studio software (Li-Cor) to measure line intensity under non saturating conditions.”

    "our findings support the notion that Ldo45 is important for early steps of LD formation as previously proposed" I find this statement confusing given that the authors claim that Ldo45 is already bound to the complex before LD formation.

    We thank the reviewer for raising this important point. We believe that our findings support previous hypotheses on the role of Ldo45. It has been suggested that Ldo45 is important for the early stages of lipid droplet (LD) formation (Teixeira et al., 2018; Eisenberg-Bord et al., 2018). As such, Ldo45 would need to be recruited to the seipin complex before or at the onset of LD formation. The observation that Ldo45 is present at the complex prior to LD formation provides strong support for its role in the initial steps of this process.

    To clarify this idea in the manuscript, we have revised the sentence on line 310 as follows:

    “Irrespective of the mechanism, our findings support the notion that Ldo45 plays a role in the early steps of LD formation, as previously proposed…”

    The model in Fig. 5 is essentially the same as the one shown in Fig. 1G.

    To aid the reader and avoid confusion, we intentionally used a similar color scheme throughout the manuscript. This may contribute to the perception that the figures are very similar. However, there are clear distinctions between them. In Figure 1G, we summarize our findings regarding the positioning of Ldo45 within the complex and note that we do not yet have data on Ldo16. Building upon these findings, in Figure 5 we speculate where Ldo16 might interact with Ldb16 and highlight that recruitment of both Ldo16 and Ldo45 increases with neutral lipid availability.

    Therefore, we believe that both figures serve distinct and complementary purposes, and that each is useful for communicating our overall message.

    Minor comments

    In the pull-downs in Fig. 2C, it seems that full-length Ldb16 is not enriched after the FLAG IP. What is the reason of this?

    We thank the reviewer for raising this interesting aspect. We do not know why this occurs, but it is clear that full length Ldb16 is not efficiently pulled down. We could speculate that this has to do with access to the FLAG moiety at the C terminus that may become inaccessible due to interactions or folding in the long unstructured C-terminus of Ldb16. This might explain why when we truncate the C terminus in the 1-133 mutant we achieve a more efficient IP.

    At the blots at Fig. 2C and 3A, the anti-Dpm1 Ab seems to recognize in the IP fractions a band labelled as non-specific, however this band is absent from the input.

    We thank the reviewer for raising this. This non-specific band is the light chain of the antibody used in the pull down that detaches from the matrix during elution – thus not found in the input. This is a common non-specific band that appears in Co-IP blots.

    Reviewer #2 (Significance (Required)):

    Regulation of seipin function is essential for proper LD biogenesis in eukaryotes, so this study addresses a fundamental question in the field. As stated above some functional analysis that goes beyond the biochemistry would be beneficial. There is some overlap with a recently published paper from the Wang group that also examines the assembly of seipin in yeast.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Klug and Carvalho investigates the interaction of the yeast seipin complex (Sei1 and Ldb16) with Ldo45 and Ldo16. Using a site-specific photocrosslinking approach, the authors map some residues of the seipin complex in contact Ldo45, demonstrating that Ldo45 likely binds to Ldb16 in the center of the Sei1-Ldb16 complex. They find that both Ldo45 and Ldo16 copurify with Ldb16. Complex assembly is demonstrated to occur independently of the presence of neutral lipids. An Ldb16 mutant, harbouring the transmembrane domain (1-133) but lacking the cytosolic region (previously shown to allow normal LD formation and still bind to Sei1) showed photocrosslinks with Ldo45, but not Ldo16. No crosslinks between Sei1 and either Ldo45 or Ldo16 were detected.

    Major:

    1. Figure 2 shows CoIPs using different Ldb16 mutants/truncations to test for binding of Ldo45 and Ldo16. Both Ldo16 and Ldo45 copurify with full length Ldb16. Loss of the cytosolic part of Ldb16 strongly reduced binding of both Ldo45 and Ldo16, indicating that the TM-Helix-TM domain of Ldb16 (1-133) alone is not sufficient for proper binding of Ldo45 or Ldo16. The quantifications (2D and 2E) presented for this CoIP represent a n=2 with mean, standard deviation and statistics. To be a meaningful statistical analysis, the authors need to increase their n to at least n=3. In addition, they refer to the statistics they use here as "two-sided Fischer's T-test" in the respective Figure legend. To my knowledge, there is no such test, either it is Student's T-test or Fischer's exact test? Can the authors please clarify?

    We thank the reviewer for this comment and suggestions. We have now included 2 additional repeats for this experiment and the results essentially support our conclusion.

    The two-sided Fischer’s T-test is the name of the test in Graphpad- Prism. We wanted to acknowledge the test name so that the reader can trace the exact test we used in the program.

    1. Figure 2E shows the same data as 2D with different normalization to highlight the differences between binding to the domain 1-133 per se and binding to this domain when the linker helix is mutated. These mutations seem to cause a further decrease in binding of both Ldo45 and Ldo16. Still, effects are rather small, and the n=2 does not allow any meaningful statistical tests. To make this point, the authors should increase their sample number (at least n=3) to show that this difference is indeed meaningful and to allow statistical analysis.

    We thank the reviewer for this comment and suggestions. We have now included 2 additional repeats for this experiment and the results essentially support our conclusion.

    For Ldo16, no crosslinks were detected with Ldb16 TM-HelixTM domain (Figure 1). In line, CoIP demonstrated that the interaction between Ldo16 and Ldb16 was strongly reduced when the Ldb16 domain 1-133 was used for IP. Still, additional mutation of the linker helix in this 1-133 domain further reduced this interaction (to a similar extend as for Ldo45). Could the authors please clarify why the additional mutations in the linker helix region also decreased the binding of Ldo16, though the authors conclude from their crosslinking approach in Fig. 1 that Ldo16 does not interact with this region?

    We thank the reviewer for raising this point. Our negative crosslinking results for Ldo16 do not exclude the possibility of binding to that region; rather, they indicate that we were unable to detect Ldo16 there. Additionally, mutations in the linker helix may influence how Ldb16 interacts with seipin, including its positioning within the seipin ring and the membrane bilayer. These structural changes could, in turn, affect Ldo16 recruitment in ways that we do not fully understand.

    Similarly, also in 4D, a quantification with n=2 is presented, showing that some of the crosslinks are more prominently detectable when LD biogenesis is induced. The findings of this manuscript are completely based on results obtained with CoIP and photocrosslinking, and quantification of a sufficient n to allow statistical analysis will be essential.

    While we agree that additional experiments are useful for the Co-IP because of variability between experiments, this is less of a concern for the photocrosslinking experiments. In the case of photocrosslinking, we typically see much less variability and normally, for a given position, the effects are much more “black and white”- either there is a crosslink or not.

    Why is there nowhere a blot with crosslinked Ldb16 bands shown (but only non-crosslinked Ldb16, e.g. Fig. 1C)?

    We thank the reviewer for this comment. In all cases the amount of crosslinked product is very minor. This is particularly obvious in the case of Ldb16, where the non-crosslinked species dominates in the blots (as can be observed in figure S1B).

    Figure 3: The authors conclude that galactose-induced expression of either Dga1, Lro1 or Are1 in cells lacking all four enzymes for neutral lipid synthesis (quadruple deletion mutant) increases the levels of Ldb16. However, I do not see any difference on the FLAG-Ldb16 blot when comparing Ldb16 levels in the quadruple deletion mutant with or without Dga1, Lro1 or Are1, and no quantification is presented that might reveal very subtle differences not visible on the blot.

    We agree with the reviewer and modified the text to more accurately describe our results.

    OPTIONAL: Have the authors considered to assess which sites/domains of Ldo45 and Ldo16 are employed to bind to Ldb16?

    This is a logical next step that will be undertaken in a future study.

    Minor:

    1. Page numbers would have been helpful to refer to specific text sections.

    Page numbers have been added

    1. Figure 3C: Unclear to me why the authors label a part of their immunoblot where they detected HA with OSW5?

    This was a mistake and has been corrected

    1. Figure 4D and corresponding figure legend could be improved in respect to labeling to clarify.

    we have added an X axis label and made extra clarifications in the legend

    1. Please correct his sentence: "These variants we expressed in cells where the other subunits of the Sei1 complex were epitope tagged to facilitate detection and expressed their endogenous loci."

    This sentence has been corrected

    Reviewer #3 (Significance (Required)):

    This is a short and interesting study completely based on UV-induced site-specific photocrosslinking and CoIPs that provides some new insights into the interaction surface between the Seipin complex and Ldo45 and the interaction between Ldo16 and Ldb16. Though in parts still premature, these findings will likely be of interest to the large community interested in lipid metabolism, expanding the role of Ldb16 from neutral lipid binding to regulator recruitment.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Klug and Carvalho investigates the interaction of the yeast seipin complex (Sei1 and Ldb16) with Ldo45 and Ldo16. Using a site-specific photocrosslinking approach, the authors map some residues of the seipin complex in contact Ldo45, demonstrating that Ldo45 likely binds to Ldb16 in the center of the Sei1-Ldb16 complex. They find that both Ldo45 and Ldo16 copurify with Ldb16. Complex assembly is demonstrated to occur independently of the presence of neutral lipids. An Ldb16 mutant, harbouring the transmembrane domain (1-133) but lacking the cytosolic region (previously shown to allow normal LD formation and still bind to Sei1) showed photocrosslinks with Ldo45, but not Ldo16. No crosslinks between Sei1 and either Ldo45 or Ldo16 were detected.

    Major:

    1. Figure 2 shows CoIPs using different Ldb16 mutants/truncations to test for binding of Ldo45 and Ldo16. Both Ldo16 and Ldo45 copurify with full length Ldb16. Loss of the cytosolic part of Ldb16 strongly reduced binding of both Ldo45 and Ldo16, indicating that the TM-Helix-TM domain of Ldb16 (1-133) alone is not sufficient for proper binding of Ldo45 or Ldo16. The quantifications (2D and 2E) presented for this CoIP represent a n=2 with mean, standard deviation and statistics. To be a meaningful statistical analysis, the authors need to increase their n to at least n=3. In addition, they refer to the statistics they use here as "two-sided Fischer's T-test" in the respective Figure legend. To my knowledge, there is no such test, either it is Student's T-test or Fischer's exact test? Can the authors please clarify?
    2. Figure 2E shows the same data as 2D with different normalization to highlight the differences between binding to the domain 1-133 per se and binding to this domain when the linker helix is mutated. These mutations seem to cause a further decrease in binding of both Ldo45 and Ldo16. Still, effects are rather small, and the n=2 does not allow any meaningful statistical tests. To make this point, the authors should increase their sample number (at least n=3) to show that this difference is indeed meaningful and to allow statistical analysis.
    3. For Ldo16, no crosslinks were detected with Ldb16 TM-HelixTM domain (Figure 1). In line, CoIP demonstrated that the interaction between Ldo16 and Ldb16 was strongly reduced when the Ldb16 domain 1-133 was used for IP. Still, additional mutation of the linker helix in this 1-133 domain further reduced this interaction (to a similar extend as for Ldo45). Could the authors please clarify why the additional mutations in the linker helix region also decreased the binding of Ldo16, though the authors conclude from their crosslinking approach in Fig. 1 that Ldo16 does not interact with this region?
    4. Similarly, also in 4D, a quantification with n=2 is presented, showing that some of the crosslinks are more prominently detectable when LD biogenesis is induced. The findings of this manuscript are completely based on results obtained with CoIP and photocrosslinking, and quantification of a sufficient n to allow statistical analysis will be essential.
    5. Why is there nowhere a blot with crosslinked Ldb16 bands shown (but only non-crosslinked Ldb16, e.g. Fig. 1C)?
    6. Figure 3: The authors conclude that galactose-induced expression of either Dga1, Lro1 or Are1 in cells lacking all four enzymes for neutral lipid synthesis (quadruple deletion mutant) increases the levels of Ldb16. However, I do not see any difference on the FLAG-Ldb16 blot when comparing Ldb16 levels in the quadruple deletion mutant with or without Dga1, Lro1 or Are1, and no quantification is presented that might reveal very subtle differences not visible on the blot.

    OPTIONAL: Have the authors considered to assess which sites/domains of Ldo45 and Ldo16 are employed to bind to Ldb16?

    Minor:

    1. Page numbers would have been helpful to refer to specific text sections.
    2. Figure 3C: Unclear to me why the authors label a part of their immunoblot where they detected HA with OSW5?
    3. Figure 4D and corresponding figure legend could be improved in respect to labeling to clarify.
    4. Please correct his sentence: "These variants we expressed in cells where the other subunits of the Sei1 complex were epitope tagged to facilitate detection and expressed their endogenous loci."

    Significance

    This is a short and interesting study completely based on UV-induced site-specific photocrosslinking and CoIPs that provides some new insights into the interaction surface between the Seipin complex and Ldo45 and the interaction between Ldo16 and Ldb16. Though in parts still premature, these findings will likely be of interest to the large community interested in lipid metabolism, expanding the role of Ldb16 from neutral lipid binding to regulator recruitment.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Klug and Carvalho apply a photo-crosslinking approach, which has been extensively used in the Carvalho group, to investigate the subunit interactions of the seipin complex in yeast. The authors apply this approach to further study possible changes within the seipin complex following induction of neutral lipid synthesis and lipid droplet (LD) formation. The authors propose that Ldo45 makes contact with Ldb16 and that the seipin complex subunits assemble even in the absence of LDs.

    Major comments:

    Overall, this is a focused and well-executed study on one of the fundamental structural components of LDs. The study addresses the subunit interactions of the seipin complex but does not look into their functional consequences, for example how the mutations on Ldb16 that affect its interaction with Ldo45, influence LD formation; similarly, the authors make the interesting observation that Ldo16 may be differentially affected by the lack of neutral lipids (Fig. 3A) but this observation is not explored.

    1. For the crosslinking pull-downs (Fig. 1), it seems that the authors significantly overexpress (ADH1 promoter) the Ldb16 subunit that carries the various photoreactive amino acid residues, while keeping the other (tagged) seipin complex members at endogenous levels. Would not this imbalance affect the assembly of the complex and therefore the association of the different subunits with each other?
    2. Although the authors do show delta4 cells with no LDs (Fig. 3B, 0h), galactose-inducible systems in yeast are known to be leaky. Given that the authors' conclusion that the complex is "pre-assembled" irrespective of the addition of galactose, I think it would be important to confirm biochemically that there is no neutral lipid at time point 0. Alternatively, it may be better to simply compare wt vs dga1 lro1 or are1are2 mutants - there is no need for GAL induction since the authors look at one time point only.
    3. Some methodological issues could be better detailed. For example, which of the three delta4 strains was used to induce neutral lipid in Fig. 4B? How exactly were the quantifications in Fig. 4D performed (I assume they were done under non-saturating band intensity conditions, as for some residues it is difficult to conclude whether the blot aligns with the quantification results).
    4. "our findings support the notion that Ldo45 is important for early steps of LD formation as previously proposed" I find this statement confusing given that the authors claim that Ldo45 is already bound to the complex before LD formation.
    5. The model in Fig. 5 is essentially the same as the one shown in Fig. 1G.

    Minor comments

    In the pull-downs in Fig. 2C, it seems that full-length Ldb16 is not enriched after the FLAG IP. What is the reason of this?

    At the blots at Fig. 2C and 3A, the anti-Dpm1 Ab seems to recognize in the IP fractions a band labelled as non-specific, however this band is absent from the input.

    Significance

    Regulation of seipin function is essential for proper LD biogenesis in eukaryotes, so this study addresses a fundamental question in the field. As stated above some functional analysis that goes beyond the biochemistry would be beneficial. There is some overlap with a recently published paper from the Wang group that also examines the assembly of seipin in yeast.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The manuscript is well written and presents solid data, most of which is statistically analyzed and sound. Given that the author's previous comprehensive publications on seipin organization and interactions, it might be beneficial (particularly in the title and abstract) to emphasize that this manuscript focuses on the metabolic regulation of lipid droplet assembly by Ldb16, to distinguish it from previous work. Perhaps one consideration, potentially interesting, involves changes in lipid droplet formation under the growth conditions used for galactose-mediated gene induction.

    Comments:

    1. Fig. 3 and 4. The galactose induction of lipid droplet biogenesis in are1∆/2∆ dga1∆ lro1∆ cells though activation of a GAL1 promoter fusion to DGA1 is a sound approach for regulating lipid droplet formation. Although unlikely, carbon sources can impact lipid droplet proliferation and (potentially interesting) metabolic changes under growth in non-fermentable carbon sources may impact lipid droplet biogenesis; in fact, oleate has significant effects (e.g. PMID: 21422231; PMID: 21820081). The GAL1 promoter is a very strong promoter and the overexpression of DGA1 via this heterologous promoter might itself cause unforeseen changes. Affirmation of the results using another induction system might be beneficial.
    2. Fig. 3B. Although only representative images are shown, the panel convincingly shows that lipid droplets do form upon galactose induction of DGA1 in are1∆/2∆ dga1∆ lro1∆ cells. However, it does not show to what extent. Are lipid droplets synthesized at WT levels? How many cells were counted? How many lipid droplets per cell? Is there a statistical difference with respect to WT cells?
    3. Fig. 2D. It is not clear how standard deviation can be meaningfully applied to two data points, let alone providing a p-value. For some of these experiments, triplicate trials might provide a more robust statistical sampling.

    Significance

    Klug and Carvalho report on the lipid droplet architecture of the yeast seipin complex. Specifically, the mechanism of yeast seipin Sei1 binding to Ldo16 and the subsequent recruitment of Ldb45 is analyzed. These results follow from a recent publication (PMID: 34625558) from the same authors and aims to define a more precise role for the components of the seipin complex. Using photo-crosslinking, Ldo45 and Ldo16 interactions are analyzed in the context of lipid droplet assembly.

  5. Version published to 10.1101/2025.03.14.642698 on bioRxiv