Author response:
Reviewer #1 (Public review):
Summary:
The study dissects distinct pools of diacylglycerol (DAG), continuing a line of research on the central concept that there is a major lipid metabolism DAG pool in cells, but also a smaller signaling DAG pool. It tests the hypothesis that the second pool is regulated by Dip2, which influences Pkc1 signaling. The group shows that stressed yeast increase specific DAG species C36:0 and 36:1, and propose this promotes Pkc1 activation via Pck1 binding 36:0. The study also examines how perturbing the lipid metabolism DAG pool via various deletions such as lro1, dga1, and pah1 deletion impacts DAG and stress signaling. Overall this is an interesting study that adds new data to how different DAG pools influence cellular signaling.
Strengths:
The study nicely combined lipidomic profiling with stress signaling biochemistry and yeast growth assays.
We thank the reviewer for finding this study of interest and appreciating our multi-pronged approach to prove our hypothesis that a distinct pool of Dip2 regulated by DAGs activate PKC signalling.
Weaknesses:
One suggestion to improve the study is to examine the spatial organization of Dip2 within cells, and how this impacts its ability to modulate DAG pools. Dip2 has previously been proposed to function at mitochondria-vacuole contacts (Mondal 2022). Examining how Dip2 localization is impacted when different DAG pools are manipulated such as by deletion Pah1 (also suggested to work at yeast contact sites such as the nucleus-vacuole junction), or with Lro1 or Dga1 deletion would broaden the scope of the study.
We thank the reviewer for the valuable suggestions regarding the spatial organization of Dip2 in cells under the influence of different DAG pools. As suggested, we will probe the localization of Dip2 in the absence of Pah1. We would also trace the localization of Dip2 in LRO1 and DGA1 deletion where the bulk DAGs are accumulated and present the data in the revised manuscript.
Reviewer #2 (Public review):
Summary:
The authors use yeast genetics, lipidomic and biochemical approaches to demonstrate the DAG isoforms (36:0 and 36:1) can specifically activate PKC. Further, these DAG isoforms originate from PI and PI(4,5)P2. The authors propose that the Psi1-Plc1-Dip2 functions to maintain a normal level of specific DAG species to modulate PKC signalling.
Strengths:
Data from yeast genetics are clear and strong. The concept is potentially interesting and novel.
We would like to thank the reviewer for the positive comments on our work. We are happy to know that the reviewer finds the study novel and interesting.
Weaknesses:
More evidence is needed to support the central hypothesis. The authors may consider the following:
(1) Figure 2: the authors should show/examine C36:1 DAG. Also, some structural evidence would be highly useful here. What is the structural basis for the assertion that the PKC C1 domain can only be activated by C36:0/1 DAG but not other DAGs? This is a critical conclusion of this work and clear evidence is needed.
We agree with the reviewer that PKC activated by C36:0 and C36:1 DAGs is a critical conclusion of our work. While we understand that there is no obvious structural explanation as to how the DAG binding C1 domain of PKC attains the acyl chain specificity for DAGs, our conclusion that yeast Pkc1 is selective for C36:0 and C36:1 DAGs is supported by a combination of robust in vitro and in vivo data
In Vitro Evidence: The liposome binding assays demonstrate that the Pkc1 C1 domain only binds the selective DAG and does not interact with bulk DAGs.
In Vivo Evidence: Lipidomic analyses of wild-type cells subjected to cell wall stress reveal increased levels of C36:0 and C36:1 DAGs, while levels of bulk DAGs remain unaffected. This clearly parallels the Dip2 knockout scenario in which the levels of the same set of DAGs go up and Pkc1 gets hyperactivated.
These findings collectively indicate that Pkc1 neither binds nor is activated by bulk DAGs, reinforcing its specificity for C36:0 and C36:1 DAGs. It is also further corroborated by DGA1 and LRO1 knockouts wherein the increase of the bulk DAGs does not result in a significant increase in Pkc1 signalling.
Moreover, elucidating the structural basis of this selectivity would require a specific DAG-bound C1 domain structure of Pkc1, which is difficult owing to the flexibility of the longer acyl chains present in C36:0 and C36:1 DAGs. Furthermore, capturing the full-length Pkc1 structure that might provide deeper insights has been challenging for several other groups for a long time. Additionally, we believe that the DAG selectivity by Pkc1 is more of a membrane-associated phenomenon wherein these DAGs might create a specific microdomain or a particular curvature which are required for Pkc1’s ability to bind DAG followed by activation. Investigating this would require extensive structural and biophysical studies, which are beyond the scope of the current work but are planned for future research.
(2) Does Dip2 colocalize with Plc1 or Pkc1? Does Dip2 reach the plasma membrane upon Plc activation?
Thank you for your questions regarding the colocalization and potential translocation of Dip2 upon Plc1 or Pkc1 activation.
In the wild-type scenario, Dip2 does not colocalize with Pkc1. Dip2 predominantly localizes to the mitochondria and mitochondria-vacuole contact sites, while Pkc1 is found in the cytosol, plasma membrane and bud site. Moreover, the localization of Plc1 has not yet been studied in yeast and therefore we currently lack data on the colocalisation of Dip2 and Plc1.
However, to investigate whether Dip2 translocates to the plasma membrane under conditions requiring Plc1 or Pkc1 activation, we plan to probe the localization of Dip2 under cell wall stress condition. This would provide a better understanding of the spatial crosstalk between Dip2 and Pkc1. We will include the results in the revised manuscript.
