DIP2 is a unique regulator of diacylglycerol lipid homeostasis in eukaryotes

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Chain-length specific subsets of diacylglycerol (DAG) lipids are proposed to regulate differential physiological responses ranging from signal transduction to modulation of the membrane properties. However, the mechanism or molecular players regulating the subsets of DAG species remains unknown. Here, we uncover the role of a conserved eukaryotic protein family, DISCO-interacting protein 2 (DIP2) as a homeostatic regulator of a chemically distinct subset of DAGs using yeast, fly and mouse models. Genetic and chemical screens along with lipidomics analysis in yeast reveal that DIP2 prevents the toxic accumulation of specific DAGs in the logarithmic growth phase, which otherwise leads to endoplasmic reticulum stress. We also show that the fatty acyl-AMP ligase-like domains of DIP2 are essential for the redirection of the flux of DAG subspecies to storage lipid, triacylglycerols. Such modulation of selective DAG abundance by DIP2 is found to be crucial for optimal vacuole-membrane fusion and consequently osmoadaptation in yeast. Thus, the study illuminates an unprecedented DAG metabolism route and provides new insights on how cell fine-tunes DAG subspecies for cellular homeostasis and environmental adaptation.

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  1. Evaluation Summary:

    The DISCO-interacting protein 2 (DIP2) family consists of poorly characterized proteins linked to lipid metabolism, with a previously unclear role in cell physiology. DIP2 proteins contain putative fatty acyl-AMP ligase domains (FAALs), which are thought to influence fatty acid activation and attachment to various metabolites. Here, the authors analyze the role of budding yeast ScDIP2, and propose that it regulates a specific sub-pool of diacylglycerol (DAG) lipids and their conversion into storage triglycerides. While the exact molecular mechanism is not clear yet, this study will be of interest to cell biologists interested in lipids, metabolism, and ER stress.

    (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.)

  2. Reviewer #1 (Public Review):

    The work, mostly performed in yeast S. cerevisiae, shows that the knockout of DIP2 leads to accumulation in cells of some DAG subspecies (36:0 and 36:1), and also a deficit of similar TAG subspecies (something which mostly occurs, as they showed, in early to mid log growth phase). Accordingly, over-expression of DIP2 leads to the opposite outcome (lower DAG and higher TAG subspecies levels). ∆DIP2 cells showed increased ER stress and UPR, which can be counterbalanced by incubating cells with oleic acid. Moreover, the authors show that the absence of DIP2 causes vacuole fusion defects, which they ascribe to a localization of the protein in the vacuole and possibly to the fact that enhanced levels of DAG in the vacuole membrane can promote vacuole fusion. Although it is true that neither of these claims are fully supported by the experimental results, the data that the authors show serves as a starting point for future, more robust studies to test those claims. Finally, the authors show that the DBD1 domain is not necessary and that the two FLD domains are key for the observed lipid metabolism induced by DIP2 expression. Altogether this manuscript presents interesting new data on an uncharacterized protein that seems to be regulating the metabolism of relatively low abundant DAG/TAG subspecies in cells, and by doing so possibly control cell homeostasis.

  3. Reviewer #2 (Public Review):

    In this manuscript the authors study the role of the protein DIP2 which contains two fatty acyl-AMP ligase (FAAL)-like domains in lipid metabolism. They find that deletion of DIP2 in yeast, drosophila and mouse cells results in the increase of specific diacylglycerol species which is often coupled to a reduction in triglycerides. Overexpression studies in yeast corroborate this evidence. The DIP2 KO induced lipid deregulation correlates with a moderate induction of ER stress that can be rescued by promoting diacylglycerol conversion to triglycerides through the administration of oleic acid. The authors also show that DIP2 expression in yeast is maximal in the exponential growth phase where the effects of its KO on lipid metabolism are more sustained. The authors report that DIP2 in yeast is localized at the vacuole and at mitochondria and that manipulation of DIP2 levels impair the normal vacuolar response to osmotic stresses. They conclude by demonstrating that the (FAAL)-like domains of DIP2 are necessary and sufficient to sustain the function of DIP2 in regulating diacylglycerol levels and ER stress, with mutations in specific amino acids possibly required for the FAAL enzymatic activity rendering DIP2 inactive. While the manuscript is compelling in many respects and most of the conclusions drawn by the authors are well supported by their data, this paper does not contain a clear proof of the enzymatic activity of DIP2, nor a molecular explanation for the substrate specificity and mode of action of DIP2.

  4. Reviewer #3 (Public Review):

    This study examines a family of poorly defined enzymes that contain fatty acyl-AMP ligase like domains (FAALs). The study reveals that these DISCO-interacting protein 2 (DIP2) enzymes are required to maintain a specific pool of diacylglycerol (DAG) lipids containing primarily C36 acyl chain lengths in budding yeast. Using primarily yeast, the study shows that deletion of ScDIP2 significantly increases C36 DAG pools while leaving the more abundant C32 and C34 DAG pools generally unaltered. Triglyceride (TAG) is also reduced in this deletion. Conversely, ScDIP2 over-expression promotes C36 inclusion in TAG. The ScDIP2 KO yeast manifests ER stress that can be relieved by the addition of oleic acid, but not other fatty acids. In the last section of the study, ScDIP2 is proposed to localize to the vacuole and mitochondria, where it maintains a specific DAG pool to enable proper vacuole morphology and fusion, as well as proper osmoregulation of the vacuole.

    This is a well executed study that begins to characterize a conserved and generally poorly understood family of enzymes. However, questions still remain about some of the conclusions of the study. There are two general issues with the study. The first is the specificity of the effect of loss of ScDIP2. The study beautifully shows that loss of ScDIP2 (or its over-expression) affects a specific sub-pool of DAG (mainly the C36 species). TAG levels are also somewhat lower. However, how ScDIP2 impacts other lipid precursors to DAG synthesis such as PA and lyso-PA is under-examined, and should be looked at as they can also affect ER stress. Whether the change in DAG/TAG is primarily driven by decreased synthesis versus increased lipolysis also required additional analysis.

    The second issue relates to how ScDIP2 relates to the yeast vacuole. It is proposed that some of the ScDIP2 enzyme is vacuole localized, and influences vacuole morphology. The evidence presented here does not strongly support that model. From imaging at least, it appears that ScDIP2 is primarily mitochondria localized. It is therefore possible that it influences vacuole lipid composition and morphology distally from the mitochondria. Resolving ScDIP2's native subcellular localization would strengthen the manuscript.