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

Abstract

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 pah1Δ 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 pah1Δ 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.

Phosphatidic acid in membrane rearrangements

“…Phosphatidic acid, LPA and DAG are involved in many membrane fusion and membrane fission events, such as fission of Golgi membranes , fission during endocytic transport , vacuole fission and fusion , mitochondrial dynamics , exocytosis , vesicle‐vesicle fusion , myoblast fusion , osteoclast fusion , membrane fusion during sporulation , membrane fusion during fertilization . Fusion of PA‐containing vesicles was studied in Refs .…”

“…Phosphatidic acid, LPA and DAG are involved in many membrane fusion and membrane fission events, such as fission of Golgi membranes [140,162,163,[165][166][167][168][169], fission during endocytic transport [170,171], vacuole fission [172] and fusion [132,147,[173][174][175], mitochondrial dynamics [17,40,95,[176][177][178][179][180][181][182][183][184][185], exocytosis [30,40,76,79,125,[186][187][188][189][190][191][192][193][194], vesicle-vesicle fusion [164], myoblast fusion [195,196], osteoclast fusion [197], membrane fusion during sporulation [198][199][200][201], membrane fusion during fertilization [202,203]. Fusion of PA-containing vesicles was studied in Refs [87,…”

Metabolic control of cytosolic‐facing pools of diacylglycerol in budding yeast

“…Furthermore, localization of Dgk1 has not been clearly defined. Dgk1 is an integral membrane protein initially proposed to localize to the ER, although it has recently been found associated with vacuoles …”

“…Dgk1 is an integral membrane protein initially proposed to localize to the ER, 29 although it has recently been found associated with vacuoles. 30 DAG is a minor component of eukaryotic membranes. Lipidomic studies of wild-type yeast grown in standard defined medium conditions revealed that DAG represented 4-6 mol% of total lipids.…”

Copper blocks V‐ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion

“…SNARE complex isolation was performed as described previously using GST‐Vam7 . Briefly, 6x fusion reactions were incubated with 85 μg/mL anti‐Sec17 IgG to block priming.…”