Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity

• Published in: Traffic (Copenhagen, Denmark) Denmark), 2017-05, Vol.18 (5), p.315-329
• Main Authors: Miner, Gregory E., Starr, Matthew L., Hurst, Logan R., Fratti, Rutilio A.
• Format: Article
• Language: English
• Subjects: Dgk1; diacylglycerol; Diacylglycerol kinase; Diacylglycerol Kinase - metabolism; Diglycerides; Endosomes - metabolism; Fluidity; Kinases; Lipid bilayers; lipin‐1; Membrane fluidity; Membrane Fluidity - physiology; membrane fusion; Membrane Fusion - physiology; Mon1‐Ccz1; Pah1; Phosphatase; Phosphatidate Phosphatase - metabolism; Phosphatidic acid; Phosphatidylinositol 4,5-diphosphate; Phosphatidylinositol Phosphates - metabolism; Phospholipase C; Protein Binding - physiology; rab GTP-Binding Proteins - metabolism; Rab7; Repressor Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; SNAP receptors; SNARE; SNARE Proteins - metabolism; Vacuoles; Vam7; Vesicular Transport Proteins - metabolism; Yck3; Yeast; Ypt7
• ISSN: 1398-9219; 1600-0854
• DOI: 10.1111/tra.12479

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity. In this study we found that the yeast diacylglycerol kinase Dgk1 acts as a negative regulator of vacuole fusion. Deletion of DGK1 leads to the accumulation of diacylglycerol (DAG) on isolated vacuoles resulting in augmented fusion efficiency. This was thought to be caused by the destabilization of lipid bilayers and increased fluidity and membrane curvature that lowers the energy barrier needed for SNARE‐triggered fusion. In addition, there was increased Ypt7 activity and vesicle tethering.