Emergence of Dip2-mediated Specific DAG-based PKC Signalling Axis in Eukaryotes

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.

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.

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.

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.

(2) Does Dip2 colocalize with Plc1 or Pkc1? Does Dip2 reach the plasma membrane upon Plc activation?

Author response:

While we note that the reviewers find this study novel, interesting and supported with clear and strong data, the eLife assessment has categorised this study as ‘useful’.

We believe that this study has a larger impact on the emerging paradigm of specific acyl chains’ importance in lipid metabolism. While specific DAG based modulation of PKC signalling has been proposed for a long time, this is the first ever demonstration of specific DAG regulation directly impacting the signalling process in vivo. The study also cuts across multiple areas such as lipid metabolism, genetics, signalling, bioinformatics and evolution bringing to light the Dip2-mediated DAG-PKC signalling axis in eukaryotes.

In addition to its contribution to the fundamentals of cell biology, these findings carry immense relevance for medical and biotechnological applications. Dip2 and Pkc1 have not only been reported to be essential for the virulence of several pathogenic fungi but Dip2 has also been identified as a vaccine candidate. Therefore, this work paves the way for future studies aimed at understanding Dip2’s role in fungal virulence and exploring its potential as a target for antifungal therapies and vaccines.

The study also provides a solid platform to test the regulation of PKC by Dip2 and selective DAGs in metazoans, including humans where Dip2 mutations have been linked to multiple neurological disorders like autism and dyslexia. Moreover, dysregulation of PKC has been implicated in multiple cancers and neurodevelopmental disorders in humans. Our study has thus uncovered the regulatory and evolutionary origin of a physiologically and therapeutically important signalling pathway in eukaryotes.

We would therefore request the Editors to kindly reconsider the assessment to a much better and more suitable one, especially for the ‘significance of findings’!